Dynamics of enzyme and metabolic profile of broilers fed black soldier fly (Hermetiailucens) larvae-based diets

Abstract

This study investigated the impact of replacing fishmeal with black soldier fly larvae meal (BSFLM) on growth performance, microbial enzyme activity, and metabolic functions in broiler chickens. A total of fifty Arbor Acre Plus chicks were distributed across five dietary groups, including a control (100% fishmeal) and four diets containing increasing levels of BSFLM (25%, 50%, 75%, and 100%) in a completely randomized design. Broilers were reared over eight weeks, and cecal samples were subjected to 16S rRNA metagenomic sequencing to profile gut microbial enzyme activities and metabolic functions. Results revealed a progressive increase in microbial enzyme abundance and functional metabolic pathways with higher BSFLM inclusion, particularly in the 50% (T3) and 100% (T5) groups. Key enzymes, including ABC-2-type ATP-binding proteins, RNA polymerase sigma factors, and carbohydrate-active enzymes, were significantly upregulated, supporting enhanced carbohydrate fermentation, amino acid biosynthesis, and central carbon metabolism. Metabolic pathway analysis indicated a dietary shift from carbohydrate-driven fermentation in the control group to a more protein- and lipid-centered metabolism in BSFL-fed birds, with T3 showing a balanced metabolic profile and T5 exhibiting hyper-metabolic activity. These findings demonstrate that BSFLM can replace fishmeal without compromising gut health and may even enhance microbial functionality, with a 50% replacement emerging as an optimal inclusion level to sustain balanced microbial metabolism.

Graphical abstract

Highlights

• •The effect of black soldier fly larvae meal (BSFLM) on growth performance, gut enzyme activity, and metabolic pathways of broilers was investigated.
• •There is a progressive increase in microbial enzyme abundance and functional metabolic pathways with higher BSFLM inclusion.
• •Key enzymes such as ABC-2 type ATP-binding proteins, RNA polymerase sigma factors, and carbohydrate-active enzymes were significantly upregulated.
• •Metabolic pathway analysis indicated a dietary shift from carbohydrate-driven fermentation in the control group to a more protein- and lipid-centered metabolism in BSFL-fed birds.
• •Findings demonstrate that BSFLM can replace fishmeal without compromising gut health and may even enhance microbial functionality.

1. Introduction

The poultry industry continually seeks alternative feed ingredients that are sustainable, environmentally friendly, and capable of maintaining or improving bird health and performance. Globally, rising feed costs, unstable supply chains, and environmental concerns have made the search for sustainable and affordable poultry feed alternatives more urgent than ever [1]. Traditional feed components such as fishmeal and soybean meal are not only expensive but often subject to international market fluctuations [2]. In addressing this challenge, Black Soldier Fly Larvae (Hermetia illucens, BSFL) offer a promising solution, as they convert organic waste into nutrient-rich biomass with high protein content, favorable fatty acid profiles, and beneficial microbial properties [3].

Several studies have demonstrated that incorporating H. illucens larval meal, fat, or their combinations into broiler diets can positively influence growth performance, carcass traits, gut health, immune status, and intestinal microbial balance [3]. Further, in Hartinger et al. [4], diets with full-fat larvae or a balanced inclusion of larvae meal have been shown to maintain or improve body weight gain and feed conversion ratios without adverse effects. Several other recent studies have begun to assess the utility of BSFL in broiler diets. Adegbenro et al. [5] evaluated hematology and serum biochemical indices in broilers fed diets in which fish meal was replaced with graded levels (0, 25, 50, 75, and 100%) of BSFL meal and reported that most blood parameters were unaffected or within acceptable physiological ranges, suggesting that BSFL can safely replace fish meal up to 100% in certain contexts. Other studies have focused on nutrient digestibility, serum biochemistry, or growth performance in relation to enzyme supplementation in diets containing alternative feedstuffs, e.g., malted sorghum sprouts, baobab seed meal, and bambara nut offals [6]. However, there remains a scarcity of investigations into how BSFL-based diets affect deeper molecular and biochemical markers, specifically the dynamics of key enzymes involved in transport systems, transcription, sugar metabolism, and DNA repair, as well as associated shifts in metabolic pathways in broilers. Meanwhile, enzymes are critical for mediating nutrient digestion and absorption, cellular maintenance, gene expression, and repair. Alterations in enzyme abundance and engagement of metabolic pathways could reflect how the bird's physiology adjusts to dietary changes, potentially influencing both performance and health ([7]; Atallah et al., 2023; Vasilopoulos et al., 2024; [3,8]).

Moreover, although performance outcomes and general biochemical indices in birds have been assessed on BSFL diets, there remains a limited understanding of the internal enzyme dynamics and metabolic reprogramming that occur as BSFL inclusion increases. Specifically, it is unclear how different inclusion levels influence the abundance of enzymes such as ABC transporters, RNA polymerase sigma factors, sugar-hydrolyzing enzymes (e.g., β-galactosidase, hexosaminidase), DNA helicases, etc., and how these enzyme changes correspond to shifts in metabolic pathways-glycolysis, TCA cycle, amino acid and nucleotide biosynthesis, fermentation, sugar metabolism, among others [8,9]. Without this understanding, predictions about nutrient utilization, metabolic stress, immune competence, and overall bird health remain incomplete.

It is therefore important to conduct research to address this critical knowledge gap in the bid to establish suitable and sustainable dietary inclusion for poultry. It is important to understand how diets modulate enzyme abundance and metabolic pathways, as this can help design feed formulations that more effectively match physiological demands, minimize waste, optimize nutrient uptake, and improve the feed conversion ratio. Besides, enzymes such as RNA polymerase, sigma factors, and ABC transporters are integral to gene expression, molecular transport across cellular membranes, stress responses, and repair mechanisms [10]. Changes in the abundance of these enzymes may reflect how birds adapt or strain under novel dietary regimens. Another pertinent issue is the environmental conditions, bird genetics, larval rearing substrates, and management practices in specific regions of the world, which ensure differences in birds’ biochemistry and general physiology. This, therefore, calls for locally derived data that are more applicable to specific regional poultry production systems. For developing countries like Nigeria, the adoption of BSFL in poultry farming holds huge potential for economic savings, sustainability gains, and reduced reliance on imported protein sources and feed ingredients.

This work, therefore, aims to address a scientific gap in knowledge: while many local studies have assessed performance, digestibility, serum biochemistry, or microbial effects, only a few have examined enzyme profiles and metabolic activities as biomarkers of dietary effects in poultry. Accordingly, this study achieves several objectives: (1) to quantify the relative abundance of key enzymes—including transporters (e.g. ABC transporters), transcription factors (e.g. RNA polymerase sigma factors), sugar metabolic enzymes (e.g. β-galactosidase, hexosaminidase), and DNA repair enzymes (e.g. DNA helicases) in broiler chickens fed diets with increasing levels of BSF larvae; (2) to assess corresponding shifts in major metabolic pathways such as central metabolism (glycolysis, TCA cycle), amino acid & nucleotide biosynthesis, fermentation, sugar metabolism associated with these enzyme abundance changes; (3) to correlate these molecular and biochemical changes with measured performance outcomes (growth, feed conversion) and health indices in the same birds under local conditions. It is hypothesized that as BSFL inclusion increases, there will be a progressive upregulation of enzymes involved in transport, transcription, sugar metabolism, and DNA repair, with corresponding increases in the activities of metabolic pathways (e.g., glycolysis, the TCA cycle).

By comparing the abundance of key enzymes such as transporters (e.g., ABC-type transporters), transcription factors (e.g., RNA polymerase sigma factors), sugar-metabolizing enzymes (e.g., β-galactosidase, hexosaminidase), and pathways such as glycolysis, TCA cycle, amino acid biosynthesis, and fermentation across treatments, this study will further elucidate how diet shapes metabolic and transcriptional dynamics. Understanding these dynamics has implications for optimizing BSFL-based feed formulations, improving growth and feed efficiency, and enhancing gut health in broilers. It may also inform the potential for using H. illucens larvae in diets to reduce reliance on conventional feedstuffs and promote sustainable poultry production.

2. Materials and methods

2.1. Ethical consideration

Approval for animal use in this study was granted by the Bowen University Research Ethics Committee (Approval No: BUREC/24/AGR21). All experimental procedures adhered strictly to institutional and internationally accepted guidelines for the care and handling of research animals [11].

2.2. Experimental birds and management

Day-old Arbor Acre Plus broiler chicks were procured from a commercial hatchery in Ile-Ogbo, Osun State, Nigeria. The birds were randomly assigned to five dietary treatments (T1–T5) in a completely randomized design [11]. Each treatment consisted of replicated groups raised under uniform environmental and management conditions. Each treatment consisted of 10 birds, housed as three replicates of five birds per treatment. Each replicate pen (group of five birds) was considered the experimental unit for statistical analysis. All birds were reared for 8 weeks in deep litter pens, maintained under standard management practices. Feed and clean drinking water were provided ad libitum during the entire study. Environmental conditions, vaccination schedules, and biosecurity procedures were uniform across all treatments. The use of five birds with three replicates per treatment is consistent with the range of experimental units commonly reported in broiler nutrition research (e.g., 8–10 birds per replicate with 2–4 replicates per treatment) to balance logistical feasibility, animal welfare, and data precision [12].

2.3. Source of black soldier fly larvae

Black soldier fly (Hermetia illucens) larvae and prepupae were sourced from a local production facility in Iwo, Osun State, and subsequently maintained under controlled conditions prior to processing [11].

2.4. Substrate preparation and larval rearing

The larvae were cultivated on organic substrates derived from livestock waste streams. These insects are known for their efficiency in converting organic waste streams, including manure and agro-industrial residues, into high-value biomass [[13], [14], [15]].In this study, organic wastes collected from poultry, piggery, and cattle units at the Bowen University Teaching and Research Farm were subjected to anaerobic digestion for 30 days under controlled hydraulic retention conditions [11]. The digestate obtained was used as a feeding substrate for larval growth, following previously established procedures [[16], [17], [18], [19], [20], [21]].

2.5. Diet formulation

Following collection, the larvae were disinfected, dried in an oven, and ground into a fine powder to obtain black soldier fly larvae meal (BSFLM). The meal was incorporated into experimental diets as a replacement for fishmeal. Diets were formulated to meet the nutritional requirements of broiler chickens as recommended by Swatson et al. [22].

2.6. Partial and complete substitution of BSFL meal for fish meal

T1 - Diet 1 (control) (100 % FM + 0 % BSFLM);

T2 - Diet 2 (25 % BSFLM +75 % FM) [11];

T3 - Diet 3 (50 % BSFLM + 50 % FM) [11];

T4 - Diet 4 (75 % BSFLM + 25 % FM) [11] and.

T5 - Diet 5 (100 % BSFLM + 0 % FM) [11].

2.7. Growth performance measurements

Bird body weights were recorded at the beginning of the experiment and at regular intervals throughout the feeding period. Measurements were taken at the pen level using a calibrated, high-sensitivity electronic scale. Growth performance parameters were subsequently derived from these measurements [23].

2.8. Sample collection

At the conclusion of the feeding trial, birds were randomly selected from each treatment group for sampling. Feed was withdrawn approximately 12 h prior to slaughter to allow for gut clearance. Birds were humanely sacrificed, and cecal contents were aseptically collected into sterile containers. Samples were immediately cooled and stored under appropriate conditions pending further molecular analysis [11].

2.9. DNA extraction

Genomic DNA was isolated from the collected samples using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Catalog No. D4300) in accordance with the manufacturer's guidelines. Briefly, 250 μL of each sample was dispensed into a ZR BashingBead™ Lysis Tube containing 0.1- and 0.5-mm beads. Subsequently, 750 μL of ZymoBIOMICS™ Lysis Solution was introduced, and the tubes were subjected to mechanical disruption using a bead beater (Disruptor Genie) for 20 min. Following homogenization, the tubes were centrifuged at 10,000×g for 1 min to pellet debris. An aliquot of 400 μL of the supernatant was carefully transferred into a Zymo-Spin™ III-F Filter mounted in a collection tube and centrifuged at 8000×g for 1 min. The filter was then discarded, and 1200 μL of ZymoBIOMICS™ DNA Binding Buffer was added to the filtrate and mixed thoroughly. From this mixture, 800 μL was loaded onto a Zymo-Spin™ IC Column placed in a collection tube and centrifuged at 10,000×g for 1 min. The filtrate was discarded, and the loading step was repeated until the entire sample had passed through the column. The column was then transferred into a fresh collection tube, followed by the addition of 400 μL of DNA Wash Buffer 1 and centrifugation at 10,000×g for 1 min. The flow-through was discarded, and 700 μL of DNA Wash Buffer 2 was applied to the column, followed by centrifugation at 10,000×g for 1 min. A subsequent wash was performed using 200 μL of the same buffer, and the sample was centrifuged under identical conditions. After discarding the flow-through, the column was relocated to a clean 1.5 mL microcentrifuge tube. To elute the DNA, 50 μL of DNase/RNase-free water was added directly onto the column matrix and allowed to stand for 1 min. A final centrifugation at 10,000×g for 1 min yielded the purified genomic DNA.

2.10. DNA amplification and sequencing

Extracted DNA served as the template for amplification of the bacterial 16S rRNA gene across multiple variable regions (V1–V9). Amplified products were prepared for sequencing to enable comprehensive characterization of the microbial community structure.

2.11. 16S bacterial metagenomics workflow

Extracted genomic DNA was outsourced to Inqaba Biotechnical Industries, a commercial next-generation sequencing (NGS) facility, for downstream sequencing analysis. In summary, the DNA samples were subjected to PCR amplification using the universal primer set 27F and 1492R, which targets the full-length V1–V9 regions of the bacterial 16S rRNA gene (Table 1). The amplified products were subsequently tagged with PacBio M13 barcode sequences through a limited-cycle PCR to allow sample multiplexing. Following barcoding, the amplicons were quantified, combined in equimolar concentrations, and purified using AMPure PB bead-based cleanup. Library construction was then performed using the PacBio SMRTbell protocol in accordance with the manufacturer's instructions. Thereafter, sequencing primer annealing and polymerase binding were performed according to the SMRT Link software guidelines to finalize library preparation prior to sequencing on the PacBio Sequel IIe platform.

Table 1.

Sequencing primers.

M13 tailed forward primer	/5AmMC6/GTAAAACGACGGCCAGT(N)n
M13 tailed reverse primer	/5AmMC6/CAGGAAACAGCTATGAC(N)n

2.12. Enzymatic and metabolic pathways

The 16S rRNA gene sequence data were processed using the QIIME 2 bioinformatics platform (version 2024.2), which provides a reproducible, interactive, and scalable workflow for microbiome data science [11,24]. Raw sequence reads were quality-filtered, denoised, and merged using the DADA2 plugin within QIIME 2 to generate amplicon sequence variants (ASVs). Taxonomic classification was performed using a naïve Bayes classifier trained on the SILVA 138 database, trimmed to the 16S rRNA V3–V4 region [11]. Functional annotations were obtained using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) pipeline [25]. This approach infers the functional capacity of microbial communities by mapping ASVs to a reference phylogeny, predicting gene-family abundances, and then translating these predictions into metabolic pathways [11]. Specifically, PICRUSt2 uses hidden-state prediction to estimate the presence of gene families, such as KEGG Orthologs (KOs), for each ASV based on evolutionary relationships [11]. The obtained sequences were deposited in the National Center for Biotechnology Information (NCBI) database under project identification number 1339025 and assigned the accession numbers SRX30931451, SRX30931452, SRX30931453, and SRX30931454 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1339025) [26].

The resulting KO identifiers were functionally annotated against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [11,27] to identify the associated enzymes and metabolic pathways. Enzyme abundances were aggregated per sample, yielding functional profiles expressed as relative abundances [11]. Only enzymes with non-zero abundance in at least one sample were retained for downstream analysis [11]. A comparative analysis of enzyme profiles across treatments was performed to identify differences in the annotated metabolic capacities of the gut microbiome [11]. This type of functional metagenomic annotation has been widely applied in poultry microbiome studies to explore the relationship between diet, gut microbial composition, and host physiology [28,29].

3. Results

3.1. Enzymes composition of control sample

As shown in Table 2a, the Control group exhibits a substantial presence of enzymes vital for basic cellular functions. The predominant enzymes include the RNA polymerase sigma-70 factor (161196.95), which facilitates gene transcription, as well as sugar-hydrolyzing enzymes such as hexosaminidase (123083.43) and beta-galactosidase (120994.69). Additional highly expressed enzymes include various ABC-type transport system proteins, indicating active transport of diverse molecules across the membrane.

Table 2a.

Enzyme nomenclature, description and abundance in control sample.

Function ID	Description	Abundance
K03088	rpoE; RNA polymerase sigma-70 factor, ECF subfamily	161196.95
K12373	HEXA_B; hexosaminidase [EC:3.2.1.52]	123083.43
K01190	lacZ; beta-galactosidase [EC:3.2.1.23]	120994.69
K01990	ABC-2.A; ABC-2 type transport system ATP-binding protein	109356.71
K02004	ABC.CD.P; putative ABC transport system permease protein	97717.23
K01992	ABC-2.P; ABC-2 type transport system permease protein	96057.97
K02003	ABC.CD.A; putative ABC transport system ATP-binding protein	91431.47
K01091	gph; phosphoglycolate phosphatase [EC:3.1.3.18]	76951.96
K03585	acrA, mexA, adeI, smeD, mtrC, cmeA; membrane fusion protein, multidrug efflux system	73025.47
K03561	exbB; biopolymer transport protein ExbB	71254.46
K01206	FUCA; alpha-l-fucosidase [EC:3.2.1.51]	68593.97
K03530	hupB; DNA-binding protein HU-beta	65530.72
K03559	exbD; biopolymer transport protein ExbD	63226.96
K06180	rluD; 23S rRNA pseudouridine1911/1915/1917 synthase [EC:5.4.99.23]	57565.73
K07098	K07098; uncharacterized protein	56237.97
K03296	TC.HAE1; hydrophobic/amphiphilic exporter-1 (mainly G- bacteria), HAE1 family	56170.97
K00661	maa; maltose O-acetyltransferase [EC:2.3.1.79]	54538.72
K03654	recQ; ATP-dependent DNA helicase RecQ [EC:3.6.4.12]	54377.73
K02014	TC.FEV.OM; iron complex outermembrane recepter protein	53636.99
K03924	moxR; MoxR-like ATPase [EC:3.6.3.-]	53458.97
K00850	pfkA, PFK; 6-phosphofructokinase 1 [EC:2.7.1.11]	52818.98
K00059	fabG; 3-oxoacyl-[acyl-carrier protein] reductase [EC:1.1.1.100]	52783.23
K06889	K06889; uncharacterized protein	52258.98
K06969	rlmI; 23S rRNA (cytosine1962-C5)-methyltransferase [EC:2.1.1.191]	52119.72
K05349	bglX; beta-glucosidase [EC:3.2.1.21]	51923.24
K06147	ABCB-BAC; ATP-binding cassette, subfamily B, bacterial	51180.25
K08303	K08303; putative protease [EC:3.4.-.-]	48711.23
K07024	SPP; sucrose-6-phosphatase [EC:3.1.3.24]	47292.74
K03701	uvrA; excinuclease ABC subunit A	46782.73
K01187	malZ; alpha-glucosidase [EC:3.2.1.20]	45816.73
K02428	rdgB; XTP/dITP diphosphohydrolase [EC:3.6.1.66]	45717.98
K01955	carB, CPA2; carbamoyl-phosphate synthase large subunit [EC:6.3.5.5]	45163.73
K03424	tatD; TatD DNase family protein [EC:3.1.21.-]	45158.73
K01740	metY; O-acetylhomoserine (thiol)-lyase [EC:2.5.1.49]	44860.98
K03657	uvrD, pcrA; DNA helicase II/ATP-dependent DNA helicase PcrA [EC:3.6.4.12]	44516.48
K00845	glk; glucokinase [EC:2.7.1.2]	44271.48
K07107	ybgC; acyl-CoA thioester hydrolase [EC:3.1.2.-]	43061.73
K01104	E3.1.3.48; protein-tyrosine phosphatase [EC:3.1.3.48]	43013.23
K06158	ABCF3; ATP-binding cassette, subfamily F, member 3	42898.23
K01179	E3.2.1.4; endoglucanase [EC:3.2.1.4]	42760.47
K07114	yfbK; Ca-activated chloride channel homolog	42038.98
K01847	MUT; methylmalonyl-CoA mutase [EC:5.4.99.2]	41695.98
K01784	galE, GALE; UDP-glucose 4-epimerase [EC:5.1.3.2]	41677.73
K03496	parA, soj; chromosome partitioning protein	41495.24
K03111	ssb; single-strand DNA-binding protein	41389.99
K03169	topB; DNA topoisomerase III [EC:5.99.1.2]	41302.24
K07407	E3.2.1.22B, galA, rafA; alpha-galactosidase [EC:3.2.1.22]	40947.98
K03497	parB, spo0J; chromosome partitioning protein, ParB family	40574.24
K01775	alr; alanine racemase [EC:5.1.1.1]	40114.48
K03655	recG; ATP-dependent DNA helicase RecG [EC:3.6.4.12]	39960.74
K03797	E3.4.21.102, prc, ctpA; carboxyl-terminal processing protease [EC:3.4.21.102]	39853.24
K05366	mrcA; penicillin-binding protein 1A [EC:2.4.1.129 3.4.16.4]	39846.73
K01952	purL, PFAS; phosphoribosylformylglycinamidine synthase [EC:6.3.5.3]	39808.73
K01465	URA4, pyrC; dihydroorotase [EC:3.5.2.3]	39194.23
K01649	leuA, IMS; 2-isopropylmalate synthase [EC:2.3.3.13]	38683.98
K14155	patB, malY; cystathione beta-lyase [EC:4.4.1.8]	38595.23
K01735	aroB; 3-dehydroquinate synthase [EC:4.2.3.4]	38203.73
K03768	PPIB, ppiB; peptidyl-prolyl cis-trans isomerase B (cyclophilin B) [EC:5.2.1.8]	37741.98
K02013	ABC.FEV.A; iron complex transport system ATP-binding protein [EC:3.6.3.34]	37704.99
K01710	E4.2.1.46, rfbB, rffG; dTDP-glucose 4,6-dehydratase [EC:4.2.1.46]	37602.98
K02355	fusA, GFM, EFG; elongation factor G	37212.49
K02005	ABC.CD.TX; HlyD family secretion protein	36432.49
K01915	glnA, GLUL; glutamine synthetase [EC:6.3.1.2]	36333.49
K07636	phoR; two-component system, OmpR family, phosphate regulon sensor histidine kinase PhoR [EC:2.7.13.3]	35934.24
K15923	AXY8, FUC95A, afcA; alpha-l-fucosidase 2 [EC:3.2.1.51]	35836.73
K00566	mnmA, trmU; tRNA-uridine 2-sulfurtransferase [EC:2.8.1.13]	35665.99
K01696	trpB; tryptophan synthase beta chain [EC:4.2.1.20]	35625.73
K03671	trxA; thioredoxin 1	35464.99
K11085	msbA; ATP-binding cassette, subfamily B, bacterial MsbA [EC:3.6.3.-]	35065.98
K03499	trkA, ktrA; trk system potassium uptake protein	34974.49
K03628	rho; transcription termination factor Rho	34841.23
K07263	pqqL; zinc protease [EC:3.4.24.-]	34436.48
K02078	acpP; acyl carrier protein	33933.99
K03574	mutT, NUDT15, MTH2; 8-oxo-dGTP diphosphatase [EC:3.6.1.55]	33725.23
K01448	amiABC; N-acetylmuramoyl-l-alanine amidase [EC:3.5.1.28]	33530.24
K10823	oppF; oligopeptide transport system ATP-binding protein	33527.98
K03100	lepB; signal peptidase I [EC:3.4.21.89]	33451.74
K10716	kch, trkA, mthK, pch; voltage-gated potassium channel	32453.48
K04759	feoB; ferrous iron transport protein B	32070.74
K07056	rsmI; 16S rRNA (cytidine1402-2′-O)-methyltransferase [EC:2.1.1.198]	31845.74
K00602	purH; phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase [EC:2.1.2.3 3.5.4.10]	31659.99
K01186	NEU1; sialidase-1 [EC:3.2.1.18]	31607.23
K00764	purF, PPAT; amidophosphoribosyltransferase [EC:2.4.2.14]	31565.24
K07568	queA; S-adenosylmethionine:tRNA ribosyltransferase-isomerase [EC:2.4.99.17]	31473.24
K01262	pepP; Xaa-Pro aminopeptidase [EC:3.4.11.9]	31459.99
K00791	miaA, TRIT1; tRNA dimethylallyltransferase [EC:2.5.1.75]	31372.74
K04771	degP, htrA; serine protease Do [EC:3.4.21.107]	31245.23
K01265	map; methionyl aminopeptidase [EC:3.4.11.18]	31178.74
K07148	K07148; uncharacterized protein	31112.23
K07095	K07095; uncharacterized protein	31018.74
K01785	galM, GALM; aldose 1-epimerase [EC:5.1.3.3]	31011.74
K02495	hemN, hemZ; oxygen-independent coproporphyrinogen III oxidase [EC:1.3.98.3]	30969.74
K02342	dnaQ; DNA polymerase III subunit epsilon [EC:2.7.7.7]	30885.24
K00615	E2.2.1.1, tktA, tktB; transketolase [EC:2.2.1.1]	30760.74
K11927	rhlE; ATP-dependent RNA helicase RhlE [EC:3.6.4.13]	30667.48
K00262	E1.4.1.4, gdhA; glutamate dehydrogenase (NADP+) [EC:1.4.1.4]	30642.24
K02334	dpo; DNA polymerase bacteriophage-type [EC:2.7.7.7]	30517.48
K01572	oadB; oxaloacetate decarboxylase, beta subunit [EC:4.1.1.3]	30503.49
K01251	E3.3.1.1, ahcY; adenosylhomocysteinase [EC:3.3.1.1]	30406.73
K03773	fklB; FKBP-type peptidyl-prolyl cis-trans isomerase FklB [EC:5.2.1.8]	30346.99

3.2. Enzymes composition of sample T2

The enzyme profile of the T2 sample shows a shift towards greater emphasis on transport and specific metabolic pathways compared with the Control group (Table 2b). The top four most prevalent enzymes are all ABC transport system proteins, with the ABC-2 type ATP-binding protein exhibiting the highest abundance (165686.34). There is a remarkably high abundance of sucrose-6-phosphatase (132306.55), implying heightened activity in sucrose metabolism. The RNA polymerase sigma-70 factor shows a marked reduction in abundance (86809.52) compared with the Control group, which may indicate a change in overall transcriptional activity.

Table 2b.

Enzyme nomenclature, description and abundance in sample T2.

Function ID	Description	Abundance
K01990	ABC-2.A; ABC-2 type transport system ATP-binding protein	165686.34
K06147	ABCB-BAC; ATP-binding cassette, subfamily B, bacterial	146223.12
K07024	SPP; sucrose-6-phosphatase [EC:3.1.3.24]	132306.55
K01992	ABC-2.P; ABC-2 type transport system permease protein	131505.14
K02004	ABC.CD.P; putative ABC transport system permease protein	111903.46
K02003	ABC.CD.A; putative ABC transport system ATP-binding protein	111880.74
K02529	lacI, galR; LacI family transcriptional regulator	109934.34
K03088	rpoE; RNA polymerase sigma-70 factor, ECF subfamily	86809.52
K01223	E3.2.1.86B, bglA; 6-phospho-beta-glucosidase [EC:3.2.1.86]	78171.15
K02761	PTS-Cel-EIIC, celB, chbC; PTS system, cellobiose-specific IIC component	66545.60
K06180	rluD; 23S rRNA pseudouridine1911/1915/1917 synthase [EC:5.4.99.23]	55895.08
K15634	gpmB; probable phosphoglycerate mutase [EC:5.4.2.12]	51047.07
K02030	ABC.PA.S; polar amino acid transport system substrate-binding protein	49942.37
K02013	ABC.FEV.A; iron complex transport system ATP-binding protein [EC:3.6.3.34]	49671.58
K03111	ssb; single-strand DNA-binding protein	49630.51
K03574	mutT, NUDT15, MTH2; 8-oxo-dGTP diphosphatase [EC:3.6.1.55]	48774.46
K02015	ABC.FEV.P; iron complex transport system permease protein	48344.10
K00059	fabG; 3-oxoacyl-[acyl-carrier protein] reductase [EC:1.1.1.100]	47939.31
K03657	uvrD, pcrA; DNA helicase II/ATP-dependent DNA helicase PcrA [EC:3.6.4.12]	47058.46
K07052	K07052; uncharacterized protein	46696.15
K02028	ABC.PA.A; polar amino acid transport system ATP-binding protein [EC:3.6.3.21]	46477.34
K16787	ecfA2; energy-coupling factor transport system ATP-binding protein [EC:3.6.3.-]	45876.10
K01462	PDF, def; peptide deformylase [EC:3.5.1.88]	44522.31
K02029	ABC.PA.P; polar amino acid transport system permease protein	43516.51
K03496	parA, soj; chromosome partitioning protein	42954.26
K04069	pflA, pflC, pflE; pyruvate formate lyase activating enzyme [EC:1.97.1.4]	41806.30
K07484	K07484; transposase	41504.32
K01952	purL, PFAS; phosphoribosylformylglycinamidine synthase [EC:6.3.5.3]	41286.05
K02057	ABC.SS.P; simple sugar transport system permease protein	41005.57
K02078	acpP; acyl carrier protein	40609.36
K02016	ABC.FEV.S; iron complex transport system substrate-binding protein	40320.19
K00384	trxB, TRR; thioredoxin reductase (NADPH) [EC:1.8.1.9]	40256.36
K03100	lepB; signal peptidase I [EC:3.4.21.89]	39865.28
K15580	oppA, mppA; oligopeptide transport system substrate-binding protein	39749.90
K01104	E3.1.3.48; protein-tyrosine phosphatase [EC:3.1.3.48]	38924.09
K01448	amiABC; N-acetylmuramoyl-l-alanine amidase [EC:3.5.1.28]	38914.90
K01534	zntA; Cd2+/Zn2+-exporting ATPase [EC:3.6.3.3 3.6.3.5]	38794.93
K01759	GLO1, gloA; lactoylglutathione lyase [EC:4.4.1.5]	38786.86
K01752	E4.3.1.17, sdaA, sdaB, tdcG; l-serine dehydratase [EC:4.3.1.17]	38566.92
K03497	parB, spo0J; chromosome partitioning protein, ParB family	38078.17
K02954	RP-S14, MRPS14, rpsN; small subunit ribosomal protein S14	37872.78
K00615	E2.2.1.1, tktA, tktB; transketolase [EC:2.2.1.1]	37609.76
K03704	cspA; cold shock protein (beta-ribbon, CspA family)	37479.92
K00656	E2.3.1.54, pflD; formate C-acetyltransferase [EC:2.3.1.54]	37439.81
K02913	RP-L33, MRPL33, rpmG; large subunit ribosomal protein L33	37367.06
K07240	chrA; chromate transporter	37310.10
K04487	iscS, NFS1; cysteine desulfurase [EC:2.8.1.7]	36980.61
K07088	K07088; uncharacterized protein	36790.22
K03091	sigH; RNA polymerase sporulation-specific sigma factor	36742.42
K16785	ecfT; energy-coupling factor transport system permease protein	36728.30
K07729	K07729; putative transcriptional regulator	36621.20
K00805	hepST; heptaprenyl diphosphate synthase [EC:2.5.1.30]	36331.96
K03655	recG; ATP-dependent DNA helicase RecG [EC:3.6.4.12]	36080.31
K00790	murA; UDP-N-acetylglucosamine 1-carboxyvinyltransferase [EC:2.5.1.7]	35914.66
K00850	pfkA, PFK; 6-phosphofructokinase 1 [EC:2.7.1.11]	35516.16
K00016	LDH, ldh; l-lactate dehydrogenase [EC:1.1.1.27]	35455.21
K06131	clsA_B; cardiolipin synthase A/B [EC:2.7.8.-]	35291.01
K07025	K07025; putative hydrolase of the HAD super family	34957.96
K01091	gph; phosphoglycolate phosphatase [EC:3.1.3.18]	34646.28
K07483	K07483; transposase	34559.05
K03169	topB; DNA topoisomerase III [EC:5.99.1.2]	34118.64
K01652	E2.2.1.6L, ilvB, ilvG, ilvI; acetolactate synthase I/II/III large subunit [EC:2.2.1.6]	34115.07
K04759	feoB; ferrous iron transport protein B	33934.96
K03205	virD4, lvhD4; type IV secretion system protein VirD4	33880.68
K08303	K08303; putative protease [EC:3.4.-.-]	33528.49
K07258	dacC, dacA, dacD; serine-type D-Ala-D-Ala carboxypeptidase (penicillin-binding protein 5/6) [EC:3.4.16.4]	33224.88
K03671	trxA; thioredoxin 1	33019.31
K07496	K07496; putative transposase	32673.54
K00948	PRPS, prsA; ribose-phosphate pyrophosphokinase [EC:2.7.6.1]	32493.86
K02355	fusA, GFM, EFG; elongation factor G	32323.44
K03492	ydhQ; GntR family transcriptional regulator	32322.67
K03406	mcp; methyl-accepting chemotaxis protein	32233.70
K02025	ABC.MS.P; multiple sugar transport system permease protein	31927.25
K01572	oadB; oxaloacetate decarboxylase, beta subunit [EC:4.1.1.3]	31899.89
K01187	malZ; alpha-glucosidase [EC:3.2.1.20]	31653.33
K02040	pstS; phosphate transport system substrate-binding protein	31550.82
K04758	feoA; ferrous iron transport protein A	31427.71
K03734	apbE; FAD: protein FMN transferase [EC:2.7.1.180]	31187.19
K02760	PTS-Cel-EIIB, celA, chbB; PTS system, cellobiose-specific IIB component [EC:2.7.1.196 2.7.1.205]	31069.77
K02495	hemN, hemZ; oxygen-independent coproporphyrinogen III oxidase [EC:1.3.98.3]	31027.88
K01915	glnA, GLUL; glutamine synthetase [EC:6.3.1.2]	30938.54
K03499	trkA, ktrA; trk system potassium uptake protein	30812.74
K02036	pstB; phosphate transport system ATP-binding protein [EC:3.6.3.27]	30756.36
K03217	yidC, spoIIIJ, OXA1, ccfA; YidC/Oxa1 family membrane protein insertase	30748.56
K02027	ABC.MS.S; multiple sugar transport system substrate-binding protein	30402.83
K02794	PTS-Man-EIIB, manX; PTS system, mannose-specific IIB component [EC:2.7.1.191]	30335.26
K03892	arsR; ArsR family transcriptional regulator, arsenate/arsenite/antimonite-responsive transcriptional repressor	30211.68
K01881	PARS, proS; prolyl-tRNA synthetase [EC:6.1.1.15]	30201.69
K02911	RP-L32, MRPL32, rpmF; large subunit ribosomal protein L32	30201.69
K03530	hupB; DNA-binding protein HU-beta	29993.49
K03798	ftsH, hflB; cell division protease FtsH [EC:3.4.24.-]	29882.69
K02795	PTS-Man-EIIC, manY; PTS system, mannose-specific IIC component	29728.92
K03469	rnhA, RNASEH1; ribonuclease HI [EC:3.1.26.4]	29302.10
K00382	DLD, lpd, pdhD; dihydrolipoamide dehydrogenase [EC:1.8.1.4]	29254.34
K00975	glgC; glucose-1-phosphate adenylyltransferase [EC:2.7.7.27]	29100.96
K03424	tatD; TatD DNase family protein [EC:3.1.21.-]	29066.73
K02796	PTS-Man-EIID, manZ; PTS system, mannose-specific IID component	28897.17
K09815	znuA; zinc transport system substrate-binding protein	28855.83
K01955	carB, CPA2; carbamoyl-phosphate synthase large subunit [EC:6.3.5.5]	28728.14
K07171	mazF, ndoA, chpA; mRNA interferase MazF [EC:3.1.-.-]	28446.49

3.3. Enzymes composition of sample T3

The T3 sample shows a significant, broad increase in enzyme abundance, particularly in transport and transcription systems. ABC transport system proteins are among the most abundant enzymes, with the ABC-2 type ATP-binding protein reaching an abundance of 302502.58, almost double its values in both the Control and T2 samples. Enzymes vital for gene expression, including the RNA polymerase sigma-70 factor (200741.47) and the RNA polymerase sporulation-specific sigma factor (113498.27), show a considerable increase, reflecting heightened gene expression activity. There is also a notable rise in the abundance of iron complex transport system proteins, indicating a strong cellular requirement for iron (Table 2c).

Table 2c.

Enzyme nomenclature, description and abundance in sample T3.

Function ID	Description	Abundance
K01990	ABC-2.A; ABC-2 type transport system ATP-binding protein	302502.58
K06147	ABCB-BAC; ATP-binding cassette, subfamily B, bacterial	280064.93
K02003	ABC.CD.A; putative ABC transport system ATP-binding protein	253340.39
K02004	ABC.CD.P; putative ABC transport system permease protein	250094.66
K01992	ABC-2.P; ABC-2 type transport system permease protein	243594.41
K07024	SPP; sucrose-6-phosphatase [EC:3.1.3.24]	229323.49
K03088	rpoE; RNA polymerase sigma-70 factor, ECF subfamily	200741.47
K02015	ABC.FEV.P; iron complex transport system permease protein	176492.59
K02529	lacI, galR; LacI family transcriptional regulator	158368.07
K02013	ABC.FEV.A; iron complex transport system ATP-binding protein [EC:3.6.3.34]	131465.38
K02016	ABC.FEV.S; iron complex transport system substrate-binding protein	129560.42
K03406	mcp; methyl-accepting chemotaxis protein	128066.90
K03657	uvrD, pcrA; DNA helicase II/ATP-dependent DNA helicase PcrA [EC:3.6.4.12]	116941.81
K03091	sigH; RNA polymerase sporulation-specific sigma factor	113498.27
K02761	PTS-Cel-EIIC, celB, chbC; PTS system, cellobiose-specific IIC component	113090.98
K01448	amiABC; N-acetylmuramoyl-l-alanine amidase [EC:3.5.1.28]	107138.71
K03704	cspA; cold shock protein (beta-ribbon, CspA family)	100646.51
K07052	K07052; uncharacterized protein	97891.82
K00059	fabG; 3-oxoacyl-[acyl-carrier protein] reductase [EC:1.1.1.100]	96078.38
K02030	ABC.PA.S; polar amino acid transport system substrate-binding protein	96030.50
K06180	rluD; 23S rRNA pseudouridine1911/1915/1917 synthase [EC:5.4.99.23]	95728.31
K07729	K07729; putative transcriptional regulator	90244.38
K01223	E3.2.1.86B, bglA; 6-phospho-beta-glucosidase [EC:3.2.1.86]	88319.72
K00384	trxB, TRR; thioredoxin reductase (NADPH) [EC:1.8.1.9]	87241.70
K00615	E2.2.1.1, tktA, tktB; transketolase [EC:2.2.1.1]	86380.94
K02057	ABC.SS.P; simple sugar transport system permease protein	86122.14
K03111	ssb; single-strand DNA-binding protein	83283.20
K03497	parB, spo0J; chromosome partitioning protein, ParB family	80804.18
K02029	ABC.PA.P; polar amino acid transport system permease protein	80044.49
K02028	ABC.PA.A; polar amino acid transport system ATP-binding protein [EC:3.6.3.21]	79918.26
K04758	feoA; ferrous iron transport protein A	77453.46
K07240	chrA; chromate transporter	76774.63
K04759	feoB; ferrous iron transport protein B	76470.52
K07335	bmpA, bmpB, tmpC; basic membrane protein A and related proteins	75511.52
K04069	pflA, pflC, pflE; pyruvate formate lyase activating enzyme [EC:1.97.1.4]	74927.13
K03310	TC.AGCS; alanine or glycine:cation symporter, AGCS family	74800.03
K06901	pbuG; putative MFS transporter, AGZA family, xanthine/uracil permease	73747.64
K01262	pepP; Xaa-Pro aminopeptidase [EC:3.4.11.9]	73348.02
K16787	ecfA2; energy-coupling factor transport system ATP-binding protein [EC:3.6.3.-]	73155.94
K03671	trxA; thioredoxin 1	72352.34
K15634	gpmB; probable phosphoglycerate mutase [EC:5.4.2.12]	71551.03
K00656	E2.3.1.54, pflD; formate C-acetyltransferase [EC:2.3.1.54]	71252.79
K04487	iscS, NFS1; cysteine desulfurase [EC:2.8.1.7]	71036.45
K01462	PDF, def; peptide deformylase [EC:3.5.1.88]	71013.20
K03100	lepB; signal peptidase I [EC:3.4.21.89]	70840.32
K07090	K07090; uncharacterized protein	70695.65
K03574	mutT, NUDT15, MTH2; 8-oxo-dGTP diphosphatase [EC:3.6.1.55]	70233.34
K03798	ftsH, hflB; cell division protease FtsH [EC:3.4.24.-]	69769.10
K02078	acpP; acyl carrier protein	69605.96
K08303	K08303; putative protease [EC:3.4.-.-]	69550.42
K03892	arsR; ArsR family transcriptional regulator, arsenate/arsenite/antimonite-responsive transcriptional repressor	69524.84
K03311	TC.LIVCS; branched-chain amino acid:cation transporter, LIVCS family	68639.54
K00790	murA; UDP-N-acetylglucosamine 1-carboxyvinyltransferase [EC:2.5.1.7]	66564.33
K10670	grdA; glycine/sarcosine/betaine reductase complex component A [EC:1.21.4.2 1.21.4.3 1.21.4.4]	66149.00
K01752	E4.3.1.17, sdaA, sdaB, tdcG; l-serine dehydratase [EC:4.3.1.17]	66000.57
K01104	E3.1.3.48; protein-tyrosine phosphatase [EC:3.1.3.48]	65927.03
K16785	ecfT; energy-coupling factor transport system permease protein	65263.32
K06131	clsA_B; cardiolipin synthase A/B [EC:2.7.8.-]	64895.81
K03496	parA, soj; chromosome partitioning protein	64796.27
K02358	tuf, TUFM; elongation factor Tu	63941.79
K03169	topB; DNA topoisomerase III [EC:5.99.1.2]	63936.16
K07095	K07095; uncharacterized protein	63748.04
K02760	PTS-Cel-EIIB, celA, chbB; PTS system, cellobiose-specific IIB component [EC:2.7.1.196 2.7.1.205]	63291.19
K00850	pfkA, PFK; 6-phosphofructokinase 1 [EC:2.7.1.11]	63248.64
K06889	K06889; uncharacterized protein	62920.58
K00655	plsC; 1-acyl-sn-glycerol-3-phosphate acyltransferase [EC:2.3.1.51]	62849.45
K02355	fusA, GFM, EFG; elongation factor G	62828.18
K03324	yjbB; phosphate:Na + symporter	62740.48
K02495	hemN, hemZ; oxygen-independent coproporphyrinogen III oxidase [EC:1.3.98.3]	62515.58
K02035	ABC.PE.S; peptide/nickel transport system substrate-binding protein	62126.99
K03499	trkA, ktrA; trk system potassium uptake protein	61670.62
K02759	PTS-Cel-EIIA, celC, chbA; PTS system, cellobiose-specific IIA component [EC:2.7.1.196 2.7.1.205]	61063.37
K02051	ABC.SN.S; NitT/TauT family transport system substrate-binding protein	60713.58
K00975	glgC; glucose-1-phosphate adenylyltransferase [EC:2.7.7.27]	60320.62
K07258	dacC, dacA, dacD; serine-type D-Ala-D-Ala carboxypeptidase (penicillin-binding protein 5/6) [EC:3.4.16.4]	60239.12
K03424	tatD; TatD DNase family protein [EC:3.1.21.-]	60227.15
K02050	ABC.SN.P; NitT/TauT family transport system permease protein	60015.05
K07088	K07088; uncharacterized protein	59895.73
K02049	ABC.SN.A; NitT/TauT family transport system ATP-binding protein	59602.01
K00963	UGP2, galU, galF; UTP--glucose-1-phosphate uridylyltransferase [EC:2.7.7.9]	59428.84
K03768	PPIB, ppiB; peptidyl-prolyl cis-trans isomerase B (cyclophilin B) [EC:5.2.1.8]	59348.11
K07979	ytrA; GntR family transcriptional regulator	59236.09
K02027	ABC.MS.S; multiple sugar transport system substrate-binding protein	58983.19
K01537	E3.6.3.8; Ca2+-transporting ATPase [EC:3.6.3.8]	58658.92
K01534	zntA; Cd2+/Zn2+-exporting ATPase [EC:3.6.3.3 3.6.3.5]	58508.89
K03710	K03710; GntR family transcriptional regulator	58337.48
K07025	K07025; putative hydrolase of the HAD superfamily	57724.15
K07007	K07007; uncharacterized protein	57545.52
K01872	AARS, alaS; alanyl-tRNA synthetase [EC:6.1.1.7]	57436.51
K01265	map; methionyl aminopeptidase [EC:3.4.11.18]	57366.70
K01915	glnA, GLUL; glutamine synthetase [EC:6.3.1.2]	57291.43
K00805	hepST; heptaprenyl diphosphate synthase [EC:2.5.1.30]	56514.61
K02238	comEC; competence protein ComEC	56470.60
K01258	pepT; tripeptide aminopeptidase [EC:3.4.11.4]	55532.95
K01952	purL, PFAS; phosphoribosylformylglycinamidine synthase [EC:6.3.5.3]	55169.87
K03442	mscS; small conductance mechanosensitive channel	54750.76
K00845	glk; glucokinase [EC:2.7.1.2]	54723.49
K03498	trkH, trkG, ktrB; trk system potassium uptake protein	54278.62
K08884	K08884; serine/threonine protein kinase, bacterial [EC:2.7.11.1]	54224.73
K00873	PK, pyk; pyruvate kinase [EC:2.7.1.40]	54050.35

3.4. Enzymes composition of sample T5

In Table 2d, the T5 sample resembles the T3 sample in its substantial rise in enzyme abundance across various functions. Similar to T3, the T5 sample displays extremely elevated levels of several ABC transporters, with the ABC-2 type ATP-binding protein reaching the highest abundance (348393.99). Enzymes related to sugar metabolism, such as beta-galactosidase (223842.18) and hexosaminidase (219460.91), are also significantly upregulated relative to the Control group. Enzymes linked to transcription, such as the RNA polymerase sigma-70 factor (254245.24), are found at very high levels, similar to T3. The presence of DNA helicase II (141151.78) is also considerably elevated, suggesting an accelerated rate of DNA duplication and repair.

Table 2d.

Enzyme nomenclature, description and abundance in sample T5.

Function ID	Description	Abundance
K01990	ABC-2.A; ABC-2 type transport system ATP-binding protein	274679.31
K06147	ABCB-BAC; ATP-binding cassette, subfamily B, bacterial	255730.98
K03088	rpoE; RNA polymerase sigma-70 factor, ECF subfamily	227300.86
K01992	ABC-2.P; ABC-2 type transport system permease protein	223344.88
K02004	ABC.CD.P; putative ABC transport system permease protein	215690.20
K02003	ABC.CD.A; putative ABC transport system ATP-binding protein	213675.94
K07024	SPP; sucrose-6-phosphatase [EC:3.1.3.24]	181277.10
K02529	lacI, galR; LacI family transcriptional regulator	161588.25
K06180	rluD; 23S rRNA pseudouridine1911/1915/1917 synthase [EC:5.4.99.23]	96170.73
K00059	fabG; 3-oxoacyl-[acyl-carrier protein] reductase [EC:1.1.1.100]	93601.31
K02015	ABC.FEV.P; iron complex transport system permease protein	90703.35
K03496	parA, soj; chromosome partitioning protein	89099.56
K03091	sigH; RNA polymerase sporulation-specific sigma factor	88526.18
K03657	uvrD, pcrA; DNA helicase II/ATP-dependent DNA helicase PcrA [EC:3.6.4.12]	88466.52
K03497	parB, spo0J; chromosome partitioning protein, ParB family	88019.78
K02030	ABC.PA.S; polar amino acid transport system substrate-binding protein	86964.64
K02013	ABC.FEV.A; iron complex transport system ATP-binding protein [EC:3.6.3.34]	84140.17
K03111	ssb; single-strand DNA-binding protein	81214.97
K00850	pfkA, PFK; 6-phosphofructokinase 1 [EC:2.7.1.11]	79163.26
K01448	amiABC; N-acetylmuramoyl-l-alanine amidase [EC:3.5.1.28]	78702.88
K01091	gph; phosphoglycolate phosphatase [EC:3.1.3.18]	77671.86
K00615	E2.2.1.1, tktA, tktB; transketolase [EC:2.2.1.1]	77448.35
K02016	ABC.FEV.S; iron complex transport system substrate-binding protein	77289.27
K02057	ABC.SS.P; simple sugar transport system permease protein	76990.31
K03100	lepB; signal peptidase I [EC:3.4.21.89]	75774.52
K04069	pflA, pflC, pflE; pyruvate formate lyase activating enzyme [EC:1.97.1.4]	74979.13
K02078	acpP; acyl carrier protein	74609.07
K01223	E3.2.1.86B, bglA; 6-phospho-beta-glucosidase [EC:3.2.1.86]	73840.90
K04759	feoB; ferrous iron transport protein B	72844.54
K02028	ABC.PA.A; polar amino acid transport system ATP-binding protein [EC:3.6.3.21]	71718.81
K00384	trxB, TRR; thioredoxin reductase (NADPH) [EC:1.8.1.9]	71466.11
K07240	chrA; chromate transporter	71213.27
K16787	ecfA2; energy-coupling factor transport system ATP-binding protein [EC:3.6.3.-]	70899.52
K08303	K08303; putative protease [EC:3.4.-.-]	70202.12
K01190	lacZ; beta-galactosidase [EC:3.2.1.23]	70132.55
K03169	topB; DNA topoisomerase III [EC:5.99.1.2]	70040.30
K01915	glnA, GLUL; glutamine synthetase [EC:6.3.1.2]	69303.99
K02355	fusA, GFM, EFG; elongation factor G	68962.35
K02029	ABC.PA.P; polar amino acid transport system permease protein	68847.93
K03671	trxA; thioredoxin 1	68715.63
K03205	virD4, lvhD4; type IV secretion system protein VirD4	68213.08
K01462	PDF, def; peptide deformylase [EC:3.5.1.88]	67950.81
K03499	trkA, ktrA; trk system potassium uptake protein	67796.52
K07258	dacC, dacA, dacD; serine-type D-Ala-D-Ala carboxypeptidase (penicillin-binding protein 5/6) [EC:3.4.16.4]	67498.01
K03655	recG; ATP-dependent DNA helicase RecG [EC:3.6.4.12]	66924.09
K01104	E3.1.3.48; protein-tyrosine phosphatase [EC:3.1.3.48]	66736.05
K04758	feoA; ferrous iron transport protein A	66592.99
K01752	E4.3.1.17, sdaA, sdaB, tdcG; l-serine dehydratase [EC:4.3.1.17]	66051.93
K03574	mutT, NUDT15, MTH2; 8-oxo-dGTP diphosphatase [EC:3.6.1.55]	64978.72
K02495	hemN, hemZ; oxygen-independent coproporphyrinogen III oxidase [EC:1.3.98.3]	64447.36
K15634	gpmB; probable phosphoglycerate mutase [EC:5.4.2.12]	64081.87
K03406	mcp; methyl-accepting chemotaxis protein	63773.11
K07088	K07088; uncharacterized protein	63698.52
K07052	K07052; uncharacterized protein	63429.12
K06889	K06889; uncharacterized protein	63163.74
K07025	K07025; putative hydrolase of the HAD superfamily	62341.39
K02761	PTS-Cel-EIIC, celB, chbC; PTS system, cellobiose-specific IIC component	62164.72
K04487	iscS, NFS1; cysteine desulfurase [EC:2.8.1.7]	62154.01
K03530	hupB; DNA-binding protein HU-beta	62022.82
K02025	ABC.MS.P; multiple sugar transport system permease protein	61768.66
K03798	ftsH, hflB; cell division protease FtsH [EC:3.4.24.-]	61484.72
K16785	ecfT; energy-coupling factor transport system permease protein	61082.78
K00656	E2.3.1.54, pflD; formate C-acetyltransferase [EC:2.3.1.54]	61004.03
K03310	TC.AGCS; alanine or glycine:cation symporter, AGCS family	60645.96
K00790	murA; UDP-N-acetylglucosamine 1-carboxyvinyltransferase [EC:2.5.1.7]	60025.97
K01955	carB, CPA2; carbamoyl-phosphate synthase large subunit [EC:6.3.5.5]	59943.36
K05349	bglX; beta-glucosidase [EC:3.2.1.21]	58154.16
K01652	E2.2.1.6L, ilvB, ilvG, ilvI; acetolactate synthase I/II/III large subunit [EC:2.2.1.6]	58028.70
K01952	purL, PFAS; phosphoribosylformylglycinamidine synthase [EC:6.3.5.3]	57443.55
K03498	trkH, trkG, ktrB; trk system potassium uptake protein	56837.77
K00975	glgC; glucose-1-phosphate adenylyltransferase [EC:2.7.7.27]	56523.52
K03424	tatD; TatD DNase family protein [EC:3.1.21.-]	56520.27
K06131	clsA_B; cardiolipin synthase A/B [EC:2.7.8.-]	56184.11
K02026	ABC.MS.P1; multiple sugar transport system permease protein	55621.25
K07729	K07729; putative transcriptional regulator	55083.70
K01740	metY; O-acetylhomoserine (thiol)-lyase [EC:2.5.1.49]	54914.00
K02027	ABC.MS.S; multiple sugar transport system substrate-binding protein	54497.68
K02469	gyrA; DNA gyrase subunit A [EC:5.99.1.3]	54347.64
K00058	serA, PHGDH; D-3-phosphoglycerate dehydrogenase/2-oxoglutarate reductase [EC:1.1.1.95 1.1.1.399]	54087.64
K00266	gltD; glutamate synthase (NADPH/NADH) small chain [EC:1.4.1.13 1.4.1.14]	53925.85
K07090	K07090; uncharacterized protein	53368.32
K01759	GLO1, gloA; lactoylglutathione lyase [EC:4.4.1.5]	53247.54
K02470	gyrB; DNA gyrase subunit B [EC:5.99.1.3]	53004.64
K00948	PRPS, prsA; ribose-phosphate pyrophosphokinase [EC:2.7.6.1]	52846.65
K00764	purF, PPAT; amidophosphoribosyltransferase [EC:2.4.2.14]	52730.27
K01534	zntA; Cd2+/Zn2+-exporting ATPase [EC:3.6.3.3 3.6.3.5]	52565.89
K01872	AARS, alaS; alanyl-tRNA synthetase [EC:6.1.1.7]	52501.72
K03797	E3.4.21.102, prc, ctpA; carboxyl-terminal processing protease [EC:3.4.21.102]	52424.39
K07098	K07098; uncharacterized protein	52214.63
K00845	glk; glucokinase [EC:2.7.1.2]	52124.53
K01265	map; methionyl aminopeptidase [EC:3.4.11.18]	51893.11
K01784	galE, GALE; UDP-glucose 4-epimerase [EC:5.1.3.2]	51413.51
K02954	RP-S14, MRPS14, rpsN; small subunit ribosomal protein S14	51406.98
K01262	pepP; Xaa-Pro aminopeptidase [EC:3.4.11.9]	51230.77
K03711	fur, zur, furB; Fur family transcriptional regulator, ferric uptake regulator	51005.16
K03704	cspA; cold shock protein (beta-ribbon, CspA family)	50360.04
K07095	K07095; uncharacterized protein	50322.56
K03686	dnaJ; molecular chaperone DnaJ	49956.14
K00566	mnmA, trmU; tRNA-uridine 2-sulfurtransferase [EC:2.8.1.13]	49928.32
K02913	RP-L33, MRPL33, rpmG; large subunit ribosomal protein L33	49822.57

Overall, enzyme activity increased with increasing BSFL inclusion levels in the diet, and the highest mucinase, chitinase, and lipase activities were observed at 100% BSFL inclusion (Fig. 1). Fig. 2 also shows how BSFL-based diets drive microbial enzymes to produce short-chain fatty acids and ultimately modulate gut health in experimental broilers.

Fig. 1.

Enzyme activity profiling gut of broilers fed BSFL-based diets. The figure shows higher mucinase, chitinase and lipase activities at 100% BSFL inclusion level.

Fig. 2.

BSFL-based diets drive microbial enzymes for the production of short chain fatty acids and ultimately modulates gut health.

3.5. Enzyme dynamics in broiler cecum

The results for enzymes and their functions are shown in Table 3a, Table 3b, Table 3c, Table 3da–3d, which show a steady pattern of increasing enzyme levels from the Control group to the T2, T3, and T5 samples. The T2 sample demonstrates some changes in enzyme levels; however, these do not reflect the extensive increases noted in the other two treated groups. The most notable increases are observed in enzymes involved in transport systems and transcription.

Table 3a.

Enzymes and their Functions in Control Sample.

Enzyme Name	Enzyme ID	Category	Functions
RNA polymerase sigma-70 factor, ECF subfamily	K03088	RNA Polymerase Sigma Factor	Directs RNA polymerase to specific promoters, initiating transcription of genes involved in extracytoplasmic function (ECF) responses.
Hexosaminidase	K12373	Glycosidase	Catalyzes the hydrolysis of terminal N-acetyl-d-hexosamine residues from glycolipids and oligosaccharides.
Beta-Galactosidase	K01190	Glycosidase	Hydrolyzes beta-galactosides into monosaccharides (galactose and glucose).
ABC-2 type transport system ATP-binding protein	K01990	ABC Transporter (ATP-Binding Protein)	Provides the energy (ATP hydrolysis) for the transport of various substrates across the cell membrane.
Putative ABC transport system permease protein	K02004	ABC Transporter (Permease Protein)	Forms the transmembrane channel through which substrates are transported.
ABC-2 type transport system permease protein	K01992	ABC Transporter (Permease Protein)	Forms the transmembrane channel through which substrates are transported.
Putative ABC transport system ATP-binding protein	K02003	ABC Transporter (ATP-Binding Protein)	Provides the energy (ATP hydrolysis) for the transport of various substrates across the cell membrane.
Phosphoglycolate phosphatase	K01091	Phosphatase	Hydrolyzes phosphoglycolate to glycolate and phosphate.
Membrane fusion protein, multidrug efflux system	K03585	Membrane Fusion Protein (Multidrug Efflux System)	Bridges the inner membrane transporter and the outer membrane channel, facilitating the export of drugs and toxins.
Biopolymer transport protein ExbB	K03561	Biopolymer Transport Protein	Energy transduction for TonB-dependent transporters.
Alpha-l-Fucosidase	K01206	Glycosidase	Hydrolyzes alpha-l-fucose residues from glycoconjugates.
DNA-binding protein HU-beta	K03530	DNA-Binding Protein	Binds to DNA and modulates its structure, influencing processes such as DNA replication, recombination, and transcription.
Biopolymer transport protein ExbD	K03559	Biopolymer Transport Protein	Energy transduction for TonB-dependent transporters.
23S rRNA pseudouridine1911/1915/1917 synthase	K06180	rRNA Methyltransferase	Catalyzes the formation of pseudouridine at specific positions in 23S rRNA.
Uncharacterized protein	K07098	Uncharacterized Protein	Unknown
Hydrophobic/amphiphilic exporter-1	K03296	HAE1 Family Exporter	Transports hydrophobic compounds.
Maltose O-acetyltransferase	K00661	Transferase	Transfers an acetyl group from acetyl-CoA to maltose.
ATP-dependent DNA helicase RecQ	K03654	DNA Helicase	Unwinds DNA ahead of the replication fork.
Iron complex outer membrane receptor protein	K02014	Iron Complex Outer Membrane Receptor	Binds iron complex and import it into the periplasm.
MoxR-like ATPase	K03924	MoxR-like ATPase	ATP hydrolysis.
6-Phosphofructokinase 1	K00850	Kinase	Phosphorylates fructose-6-phosphate.
3-oxoacyl-[acyl-carrier protein] reductase	K00059	Reductase	Reduces 3-oxoacyl-[acyl-carrier protein].
Uncharacterized protein	K06889	Uncharacterized Protein	Unknown
23S rRNA (cytosine1962-C5)-methyltransferase	K06969	rRNA Methyltransferase	Methylates cytosine in 23S rRNA.
Beta-Glucosidase	K05349	Glycosidase	Hydrolyzes beta-glucosides.
ATP-binding cassette, subfamily B, bacterial	K06147	ABC Transporter	ATP hydrolysis.
Putative protease	K08303	Putative Protease	Unknown
Sucrose-6-phosphatase	K07024	Phosphatase	Hydrolyzes sucrose-6-phosphate.
Excinuclease ABC subunit A	K03701	Excinuclease	DNA repair.
Alpha-Glucosidase	K01187	Glycosidase	Hydrolyzes alpha-glucosides.
XTP/dITP diphosphohydrolase	K02428	Diphosphohydrolase	Hydrolyzes XTP and dITP.
Carbamoyl-phosphate synthase large subunit	K01955	Synthase	Catalyzes the synthesis of carbamoyl phosphate.
TatD DNase family protein	K03424	DNase	Degrades DNA.
O-acetylhomoserine (thiol)-lyase	K01740	Lyase	Catalyzes the synthesis of methionine.
DNA helicase II/ATP-dependent DNA helicase PcrA	K03657	DNA Helicase	Unwinds DNA ahead of the replication fork.
Glucokinase	K00845	Kinase	Phosphorylates glucose.
Acyl-CoA thioester hydrolase	K07107	Hydrolase	Hydrolyzes acyl-CoA.
Protein-tyrosine phosphatase	K01104	Phosphatase	Hydrolyzes protein-tyrosine phosphate.
ATP-binding cassette, subfamily F, member 3	K06158	ABC Transporter	ATP hydrolysis.
Endoglucanase	K01179	Glycosidase	Hydrolyzes cellulose.
Ca-activated chloride channel homolog	K07114	Chloride channel homolog	Transports chloride ions.
Methylmalonyl-CoA mutase	K01847	Mutase	Catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA.
UDP-glucose 4-epimerase	K01784	Epimerase	Catalyzes the interconversion of UDP-glucose and UDP-galactose.
Chromosome partitioning protein	K03496	Chromosome partitioning protein	Involved in chromosome segregation.
Single-strand DNA-binding protein	K03111	DNA-binding protein	Binds to single-stranded DNA.
DNA topoisomerase III	K03169	Topoisomerase	Relieves torsional stress in DNA.
Alpha-Galactosidase	K07407	Glycosidase	Hydrolyzes alpha-galactosides.
Chromosome partitioning protein, ParB family	K03497	Chromosome partitioning protein	Involved in chromosome segregation.
Alanine racemase	K01775	Racemase	Catalyzes the interconversion of l-alanine and d-alanine.
ATP-dependent DNA helicase RecG	K03655	DNA Helicase	Unwinds DNA ahead of the replication fork.
Carboxyl-terminal processing protease	K03797	Protease	Cleaves carboxyl-terminal peptides.
Penicillin-binding protein 1A	K05366	Penicillin-binding protein	Involved in peptidoglycan synthesis.
Phosphoribosylformylglycinamidine synthase	K01952	Synthase	Catalyzes the synthesis of phosphoribosylformylglycinamidine.
Dihydroorotase	K01465	Hydrolase	Catalyzes the cyclization of N-carbamoyl-l-aspartate to dihydroorotate.
2-Isopropylmalate synthase	K01649	Synthase	Catalyzes the synthesis of 2-isopropylmalate.
Cystathione beta-lyase	K14155	Lyase	Catalyzes the breakdown of cystathionine.
3-Dehydroquinate synthase	K01735	Synthase	Catalyzes the synthesis of 3-dehydroquinate.
Peptidyl-prolyl cis-trans isomerase B	K03768	Isomerase	Catalyzes the cis-trans isomerization of proline residues.
Iron complex transport system ATP-binding protein	K02013	ABC Transporter	ATP hydrolysis.
dTDP-glucose 4,6-dehydratase	K01710	Dehydratase	Catalyzes the dehydration of dTDP-glucose.
Elongation factor G	K02355	Elongation factor	Involved in protein synthesis.
HlyD family secretion protein	K02005	HlyD family secretion protein	Involved in protein secretion.
Glutamine synthetase	K01915	Synthase	Catalyzes the synthesis of glutamine.
Phosphate regulon sensor histidine kinase PhoR	K07636	Histidine kinase	Involved in phosphate regulation.
Alpha-l-Fucosidase 2	K15923	Glycosidase	Hydrolyzes alpha-l-fucose.
tRNA-uridine 2-sulfurtransferase	K00566	Sulfurtransferase	Catalyzes the transfer of sulfur to tRNA.
Tryptophan synthase beta chain	K01696	Synthase	Catalyzes the synthesis of tryptophan.
Thioredoxin 1	K03671	Thioredoxin	Involved in redox regulation.
ATP-binding cassette, subfamily B, bacterial MsbA	K11085	ABC Transporter	ATP hydrolysis.
Trk system potassium uptake protein	K03499	Potassium uptake protein	Transports potassium ions.
Transcription termination factor Rho	K03628	Transcription termination factor	Terminates transcription.
Zinc protease	K07263	Protease	Degrades proteins.
Acyl carrier protein	K02078	Acyl carrier protein	Involved in fatty acid synthesis.
8-oxo-dGTP diphosphatase	K03574	Diphosphatase	Hydrolyzes 8-oxo-dGTP.
N-acetylmuramoyl-l-alanine amidase	K01448	Amidase	Hydrolyzes N-acetylmuramoyl-l-alanine.
Oligopeptide transport system ATP-binding protein	K10823	ABC Transporter	ATP hydrolysis.
Signal peptidase I	K03100	Peptidase	Cleaves signal peptides.
Voltage-gated potassium channel	K10716	Potassium channel	Transports potassium ions.
Ferrous iron transport protein B	K04759	Iron transport protein	Transports ferrous iron.
16S rRNA (cytidine1402-2′-O)-methyltransferase	K07056	Methyltransferase	Methylates rRNA.
Phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase	K00602	Transferase	Involved in purine biosynthesis.
Sialidase-1	K01186	Glycosidase	Hydrolyzes sialic acid.
Amidophosphoribosyltransferase	K00764	Transferase	Catalyzes the synthesis of phosphoribosylamine.
S-adenosylmethionine:tRNA ribosyltransferase-isomerase	K07568	Isomerase	Involved in tRNA modification.
Xaa-Pro aminopeptidase	K01262	Peptidase	Cleaves Xaa-Pro peptides.
tRNA dimethylallyltransferase	K00791	Transferase	Catalyzes the transfer of dimethylallyl to tRNA.
Serine protease Do	K04771	Protease	Degrades misfolded proteins.
Methionyl aminopeptidase	K01265	Peptidase	Cleaves methionine.
Uncharacterized protein	K07148	Uncharacterized Protein	Unknown
Uncharacterized protein	K07095	Uncharacterized Protein	Unknown
Aldose 1-epimerase	K01785	Epimerase	Catalyzes the interconversion of aldoses.
Oxygen-independent coproporphyrinogen III oxidase	K02495	Oxidase	Involved in heme biosynthesis.
DNA polymerase III subunit epsilon	K02342	Polymerase	Involved in DNA replication.
Transketolase	K00615	Transferase	Catalyzes the transfer of two-carbon units.
ATP-dependent RNA helicase RhlE	K11927	Helicase	Unwinds RNA.
Glutamate dehydrogenase (NADP+)	K00262	Dehydrogenase	Catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate.
DNA polymerase bacteriophage-type	K02334	Polymerase	Involved in DNA replication.
Oxaloacetate decarboxylase, beta subunit	K01572	Decarboxylase	Decarboxylates oxaloacetate.
Adenosylhomocysteinase	K01251	Hydrolase	Hydrolyzes adenosylhomocysteine.
FKBP-type peptidyl-prolyl cis-trans isomerase FklB	K03773	Isomerase	Catalyzes the cis-trans isomerization of proline residues.

Table 3b.

Enzymes and their Functions in Sample T2.

Enzyme Name	Enzyme ID	Category	Functions
ABC-2 type transport system ATP-binding protein	K01990	ABC Transporter (ATP-Binding Protein)	Provides the energy (ATP hydrolysis) for the transport of various substrates across the cell membrane.
ATP-binding cassette, subfamily B, bacterial	K06147	ABC Transporter	ATP hydrolysis.
Sucrose-6-phosphatase	K07024	Phosphatase	Hydrolyzes sucrose-6-phosphate.
ABC-2 type transport system permease protein	K01992	ABC Transporter (Permease Protein)	Forms the transmembrane channel through which substrates are transported.
Putative ABC transport system permease protein	K02004	ABC Transporter (Permease Protein)	Forms the transmembrane channel through which substrates are transported.
Putative ABC transport system ATP-binding protein	K02003	ABC Transporter (ATP-Binding Protein)	Provides the energy (ATP hydrolysis) for the transport of various substrates across the cell membrane.
LacI family transcriptional regulator	K02529	Transcriptional regulator	Regulates gene expression.
RNA polymerase sigma-70 factor, ECF subfamily	K03088	RNA Polymerase Sigma Factor	Directs RNA polymerase to specific promoters.
6-phospho-beta-glucosidase	K01223	Glycosidase	Hydrolyzes 6-phospho-beta-glucosides.
PTS system, cellobiose-specific IIC component	K02761	PTS system component	Involved in cellobiose transport.
23S rRNA pseudouridine1911/1915/1917 synthase	K06180	rRNA Methyltransferase	Methylates rRNA.
Probable phosphoglycerate mutase	K15634	Mutase	Catalyzes the isomerization of 3-phosphoglycerate.
Polar amino acid transport system substrate-binding protein	K02030	ABC Transporter	Binds polar amino acids.
Iron complex transport system ATP-binding protein	K02013	ABC Transporter	ATP hydrolysis.
Single-strand DNA-binding protein	K03111	DNA-binding protein	Binds to single-stranded DNA.
8-oxo-dGTP diphosphatase	K03574	Diphosphatase	Hydrolyzes 8-oxo-dGTP.
Iron complex transport system permease protein	K02015	ABC Transporter	Forms the transmembrane channel through which substrates are transported.
3-oxoacyl-[acyl-carrier protein] reductase	K00059	Reductase	Reduces 3-oxoacyl-[acyl-carrier protein].
DNA helicase II/ATP-dependent DNA helicase PcrA	K03657	DNA Helicase	Unwinds DNA ahead of the replication fork.
Uncharacterized protein	K07052	Uncharacterized Protein	Unknown
Polar amino acid transport system ATP-binding protein	K02028	ABC Transporter	ATP hydrolysis.
Energy-coupling factor transport system ATP-binding protein	K16787	ABC Transporter	ATP hydrolysis.
Peptide deformylase	K01462	Peptidase	Removes formyl group from peptides.
Polar amino acid transport system permease protein	K02029 lyase activating enzyme	K04069	Lyase activating enzyme
Transposase	K07484	Transposase	Catalyzes transposition of DNA.
Phosphoribosylformylglycinamidine synthase	K01952	Synthase	Catalyzes the synthesis of phosphoribosylformylglycinamidine.
Simple sugar transport system permease protein	K02057	ABC Transporter	Forms the transmembrane channel through which substrates are transported.
Acyl carrier protein	K02078	Acyl carrier protein	Involved in fatty acid synthesis.
Iron complex transport system substrate-binding protein	K02016	ABC Transporter	Binds Iron complex.
Thioredoxin reductase (NADPH)	K00384	Reductase	Reduces thioredoxin using NADPH.
Signal peptidase I	K03100	Peptidase	Cleaves signal peptides.
Oligopeptide transport system substrate-binding protein	K15580	ABC Transporter	Binds Oligopeptides.
Protein-tyrosine phosphatase	K01104	Phosphatase	Hydrolyzes protein-tyrosine phosphate.
N-acetylmuramoyl-l-alanine amidase	K01448	Amidase	Hydrolyzes N-acetylmuramoyl-l-alanine.
Cd2+/Zn2+−exporting ATPase	K01534	ATPase	Transports Cd2+ and Zn2+.
Lactoylglutathione lyase	K01759	Lyase	Catalyzes the conversion of hemimercaptal of methylglyoxal and glutathione to S-lactoyl-glutathione.
l-serine dehydratase	K01752	Lyase	Catalyzes the conversion of l-serine to pyruvate, ammonia and water.
Chromosome partitioning protein, ParB family	K03497	Chromosome partitioning protein	Involved in chromosome segregation.
Small subunit ribosomal protein S14	K02954	Ribosomal Protein	Structural component of the small ribosomal subunit.
Transketolase	K00615	Transferase	Catalyzes the transfer of two-carbon units.
Cold shock protein (beta-ribbon, CspA family)	K03704	Cold shock protein	Protects cells from cold shock.
Formate C-acetyltransferase	K00656	Transferase	Transfers acetyl group to formate.
Large subunit ribosomal protein L33	K02913	Ribosomal Protein	Structural component of the large ribosomal subunit.
Chromate transporter	K07240	Transporter	Transports chromate.
Cysteine desulfurase	K04487	Desulfurase	Removes sulfur from cysteine.
Uncharacterized protein	K07088 permease protein	K16785	ABC Transporter
Putative transcriptional regulator	K07729	Transcriptional regulator	Regulates gene expression.
Heptaprenyl diphosphate synthase	K00805	Synthase	Catalyzes the synthesis of heptaprenyl diphosphate.
ATP-dependent DNA helicase RecG	K03655	DNA Helicase	Unwinds DNA ahead of the replication fork.
UDP-N-acetylglucosamine 1-carboxyvinyltransferase	K00790	Transferase	Catalyzes the first committed step in bacterial cell wall synthesis.
6-Phosphofructokinase 1	K00850	Kinase	Phosphorylates fructose-6-phosphate.
l-Lactate dehydrogenase	K00016	Dehydrogenase	Catalyzes the interconversion of pyruvate and lactate.
Cardiolipin synthase A/B	K06131	Synthase	Catalyzes the synthesis of cardiolipin.
Putative hydrolase of the HAD superfamily	K07025	Hydrolase	Unknown hydrolase activity.
Phosphoglycolate phosphatase	K01091	Phosphatase	Hydrolyzes phosphoglycolate to glycolate and phosphate.
Transposase	K07483	Transposase	Catalyzes transposition of DNA.
DNA topoisomerase III	K03169	Topoisomerase	Relieves torsional stress in DNA.
Acetolactate synthase I/II/III large subunit	K01652	Synthase	Catalyzes the first committed step in branched-chain amino acid biosynthesis.
Ferrous iron transport protein B	K04759	Iron transport protein	Transports ferrous iron.
Type IV secretion system protein VirD4	K03205	ABC Transporter	Transfers substrates for secretion.
Putative protease	K08303	Putative Protease	Unknown
Serine-type D-Ala-D-Ala carboxypeptidase (penicillin-binding protein 5/6)	K07258	Peptidase	Removes d-alanine from peptidoglycan precursors.
Thioredoxin 1	K03671	Thioredoxin	Involved in redox regulation.
Putative transposase	K07496	Transposase	Catalyzes transposition of DNA.
Ribose-phosphate pyrophosphokinase	K00948	Kinase	Catalyzes the synthesis of 5-phosphoribosyl-1(alpha)-pyrophosphate.
Elongation factor G	K02355	Elongation factor	Involved in protein synthesis.
GntR family transcriptional regulator	K03492	Transcriptional regulator	Regulates gene expression.
Methyl-accepting chemotaxis protein	K03406	Chemotaxis protein	Mediates chemotaxis.
Multiple sugar transport system permease protein	K02025	ABC Transporter	Forms the transmembrane channel through which substrates are transported.
Oxaloacetate decarboxylase, beta subunit	K01572	Decarboxylase	Decarboxylates oxaloacetate.
Alpha-Glucosidase	K01187	Glycosidase	Hydrolyzes alpha-glucosides.
Phosphate transport system substrate-binding protein	K02040	ABC Transporter	Binds Phosphate.
Ferrous iron transport protein A	K04758	Iron transport protein	Transports ferrous iron.
FAD: protein FMN transferase	K03734	Transferase	Catalyzes the transfer of FMN to proteins.
PTS system, cellobiose-specific IIB component	K02760	PTS system component	Involved in cellobiose transport.
Oxygen-independent coproporphyrinogen III oxidase	K02495	Oxidase	Involved in heme biosynthesis.
Glutamine synthetase	K01915	Synthase	Catalyzes the synthesis of glutamine.
Trk system potassium uptake protein	K03499	Potassium uptake protein	Transports potassium ions.
Phosphate transport system ATP-binding protein	K02036	ABC Transporter	ATP hydrolysis.
YidC/Oxa1 family membrane protein insertase	K03217	Membrane protein	Assists in the insertion of membrane proteins.
Multiple sugar transport system substrate-binding protein	K02027	ABC Transporter	Binds multiple sugars.
PTS system, mannose-specific IIB component	K02794	PTS system component	Involved in mannose transport.
ArsR family transcriptional regulator	K03892	Transcriptional regulator	Regulates gene expression in response to arsenate/arsenite/antimonite.
Prolyl-tRNA synthetase	K01881	Synthetase	Charges tRNA with proline.
Large subunit ribosomal protein L32	K02911	Ribosomal Protein	Structural component of the large ribosomal subunit.
DNA-binding protein HU-beta	K03530	DNA-binding protein	Binds to DNA and modulates its structure.
Cell division protease FtsH	K03798	Protease	Involved in cell division.
PTS system, mannose-specific IIC component	K02795	PTS system component	Involved in mannose transport.
Ribonuclease HI	K03469	Ribonuclease	Degrades RNA hybridized to DNA.
Dihydrolipoamide dehydrogenase	K00382	Dehydrogenase	Catalyzes the oxidation of dihydrolipoamide.
Glucose-1-phosphate adenylyltransferase	K00975	Transferase	Catalyzes the synthesis of ADP-glucose.
TatD DNase family protein	K03424	DNase	Degrades DNA.
PTS system, mannose-specific IID component	K02796	PTS system component	Involved in mannose transport.
Zinc transport system substrate-binding protein	K09815	ABC Transporter	Binds Zinc.
Carbamoyl-phosphate synthase large subunit	K01955	Synthase	Catalyzes the synthesis of carbamoyl phosphate.
mRNA interferase MazF	K07171	Ribonuclease	Cleaves mRNA.

Table 3c.

Enzymes and their Function for Sample T3.

Enzyme Name	Enzyme ID	Category	Functions
ABC-2 type transport system ATP-binding protein	K01990	ABC Transporter (ATP-Binding Protein)	Provides the energy (ATP hydrolysis) for the transport of various substrates across the cell membrane.
ATP-binding cassette, subfamily B, bacterial	K06147	ABC Transporter	ATP hydrolysis.
Putative ABC transport system ATP-binding protein	K02003	ABC Transporter (ATP-Binding Protein)	Provides the energy (ATP hydrolysis) for the transport of various substrates across the cell membrane.
Putative ABC transport system permease protein	K02004	ABC Transporter (Permease Protein)	Forms the transmembrane channel through which substrates are transported.
ABC-2 type transport system permease protein	K01992	ABC Transporter (Permease Protein)	Forms the transmembrane channel through which substrates are transported.
Sucrose-6-phosphatase	K07024	Phosphatase	Hydrolyzes sucrose-6-phosphate.
RNA polymerase sigma-70 factor, ECF subfamily	K03088	RNA Polymerase Sigma Factor	Directs RNA polymerase to specific promoters.
Iron complex transport system permease protein	K02015	ABC Transporter	Forms the transmembrane channel through which substrates are transported.
LacI family transcriptional regulator	K02529	Transcriptional regulator	Regulates gene expression.
Iron complex transport system ATP-binding protein	K02013	ABC Transporter	ATP hydrolysis.
Iron complex transport system substrate-binding protein	K02016	ABC Transporter	Binds Iron complex.
Methyl-accepting chemotaxis protein	K03406	Chemotaxis protein	Mediates chemotaxis.
DNA helicase II/ATP-dependent DNA helicase PcrA	K03657	DNA Helicase	Unwinds DNA ahead of the replication fork.
RNA polymerase sporulation-specific sigma factor	K03091	Sigma Factor	RNA Polymerase binding.
PTS system, cellobiose-specific IIC component	K02761	PTS system component	Involved in cellobiose transport.
N-acetylmuramoyl-l-alanine amidase	K01448	Amidase	Hydrolyzes N-acetylmuramoyl-l-alanine.
Cold shock protein (beta-ribbon, CspA family)	K03704	Cold shock protein	Protects cells from cold shock.
Uncharacterized protein	K07052	Uncharacterized Protein	Unknown
3-oxoacyl-[acyl-carrier protein] reductase	K00059	Reductase	Reduces 3-oxoacyl-[acyl-carrier protein].
Polar amino acid transport system substrate-binding protein	K02030	ABC Transporter	Binds polar amino acids.
23S rRNA pseudouridine1911/1915/1917 synthase	K06180	rRNA Methyltransferase	Methylates rRNA.
Putative transcriptional regulator	K07729	Transcriptional regulator	Regulates gene expression.
6-phospho-beta-glucosidase	K01223	Glycosidase	Hydrolyzes 6-phospho-beta-glucosides.
Thioredoxin reductase (NADPH)	K00384	Reductase	Reduces thioredoxin using NADPH.
Transketolase	K00615	Transferase	Catalyzes the transfer of two-carbon units.
Simple sugar transport system permease protein	K02057	ABC Transporter	Forms the transmembrane channel through which substrates are transported.
Single-strand DNA-binding protein	K03111	DNA-binding protein	Binds to single-stranded DNA.
Chromosome partitioning protein, ParB family	K03497	Chromosome partitioning protein	Involved in chromosome segregation.
Polar amino acid transport system permease protein	K02029 lyase activating enzyme	K04069	Lyase activating enzyme
FtsH, hflB	K03798	Protein and membrane quality control	Protease and chaperone that regulates the assembly and stability of many membrane protein complexes.
TatD DNase family protein	K03424 Membrane	Protein membrane quality control and assists in protein membrane assembly.
Chromate Transporter	K07240 Membrane	Transports chromate
Acyl carrier protein	K02078 Membrane	Transports fatty acids
GntR transcriptional family	K03892	DNA Binding-Protein	DNA quality
Thioredoxin 1, redox and replication assist DNA protein	K03671	DNA Binding-Protein	DNA quality

Table 3d.

Enzymes and their Functions in Sample T5.

Enzyme Name	Enzyme ID	Category	Functions
ABC-2 type transport system ATP-binding protein	K01990	ABC Transporter (ATP-Binding Protein)	Provides the energy (ATP hydrolysis) for the transport of various substrates across the cell membrane.
ATP-binding cassette, subfamily B, bacterial	K06147	ABC Transporter	ATP hydrolysis.
Putative ABC transport system ATP-binding protein	K02003	ABC Transporter (ATP-Binding Protein)	Provides the energy (ATP hydrolysis) for the transport of various substrates across the cell membrane.
Putative ABC transport system permease protein	K02004	ABC Transporter (Permease Protein)	Forms the transmembrane channel through which substrates are transported.
ABC-2 type transport system permease protein	K01992	ABC Transporter (Permease Protein)	Forms the transmembrane channel through which substrates are transported.
Sucrose-6-phosphatase	K07024	Phosphatase	Hydrolyzes sucrose-6-phosphate.
RNA polymerase sigma-70 factor, ECF subfamily	K03088	RNA Polymerase Sigma Factor	Directs RNA polymerase to specific promoters.
Iron complex transport system permease protein	K02015	ABC Transporter	Forms the transmembrane channel through which substrates are transported.
LacI family transcriptional regulator	K02529	Transcriptional regulator	Regulates gene expression.
Iron complex transport system ATP-binding protein	K02013	ABC Transporter	ATP hydrolysis.
Iron complex transport system substrate-binding protein	K02016	ABC Transporter	Binds Iron complex.
Methyl-accepting chemotaxis protein	K03406	Chemotaxis protein	Mediates chemotaxis.
DNA helicase II/ATP-dependent DNA helicase PcrA	K03657	DNA Helicase	Unwinds DNA ahead of the replication fork.
RNA polymerase sporulation-specific sigma factor	K03091	Sigma Factor	RNA Polymerase binding.
PTS system, cellobiose-specific IIC component	K02761	PTS system component	Involved in cellobiose transport.
N-acetylmuramoyl-l-alanine amidase	K01448	Amidase	Hydrolyzes N-acetylmuramoyl-l-alanine.
Cold shock protein (beta-ribbon, CspA family)	K03704	Cold shock protein	Protects cells from cold shock.
Uncharacterized protein	K07052	Uncharacterized Protein	Unknown
3-oxoacyl-[acyl-carrier protein] reductase	K00059	Reductase	Reduces 3-oxoacyl-[acyl-carrier protein].
Polar amino acid transport system substrate-binding protein	K02030	ABC Transporter	Binds polar amino acids.
23S rRNA pseudouridine1911/1915/1917 synthase	K06180	rRNA Methyltransferase	Methylates rRNA.
Putative transcriptional regulator	K07729	Transcriptional regulator	Regulates gene expression.
6-phospho-beta-glucosidase	K01223	Glycosidase	Hydrolyzes 6-phospho-beta-glucosides.
Thioredoxin reductase (NADPH)	K00384	Reductase	Reduces thioredoxin using NADPH.
Transketolase	K00615	Transferase	Catalyzes the transfer of two-carbon units.
Simple sugar transport system permease protein	K02057	ABC Transporter	Forms the transmembrane channel through which substrates are transported.
Single-strand DNA-binding protein	K03111	DNA-binding protein	Binds to single-stranded DNA.
Chromosome partitioning protein, ParB family	K03497	Chromosome partitioning protein	Involved in chromosome segregation.
Polar amino acid transport system permease protein	K02029 lyase activating enzyme	K04069	Lyase activating enzyme
FtsH, hflB	K03798	Protein and membrane quality control	Protease and chaperone that regulates the assembly and stability of many membrane protein complexes.
TatD DNase family protein	K03424 Membrane	Protein membrane quality control and assists in protein membrane assembly.	Protein membrane quality control and assists in protein membrane assembly.
Chromate Transporter	K07240 Membrane	Transports chromate	Transports chromate
Acyl carrier protein	K02078 Membrane	Transports fatty acids	Transports fatty acids
GntR transcriptional family	K03892	DNA Binding-Protein	DNA quality
Thioredoxin 1, redox and replication assist DNA protein	K03671	DNA Binding-Protein	DNA quality

3.6. Metabolic pathways and abundance

3.6.1. Metabolic pathway analysis in control

The Control sample shows high abundance values for a range of metabolic pathways, as shown in Table 4a, and these include the following:

Table 4a.

Metabolic pathways and abundance in control sample.

Pathway Code	Metabolic Pathway	Abundance
1CMET2-PWY	N10-formyl-tetrahydrofolate biosynthesis	24191.04
ANAEROFRUCAT-PWY	homolactic fermentation	16598.15
ANAGLYCOLYSIS-PWY	glycolysis III (from glucose)	28516.33
ARG + POLYAMINE-SYN	superpathway of arginine and polyamine biosynthesis	4548.46
ARGORNPROST-PWY	arginine, ornithine and proline interconversion	704.04
ARGSYN-PWY	l-arginine biosynthesis I (via l-ornithine)	22877.02
ARGSYNBSUB-PWY	l-arginine biosynthesis II (acetyl cycle)	25126.35
ARO-PWY	chorismate biosynthesis I	25857.72
ASPASN-PWY	superpathway of l-aspartate and l-asparagine biosynthesis	11271.02
BIOTIN-BIOSYNTHESIS-PWY	biotin biosynthesis I	3209.92
BRANCHED-CHAIN-AA-SYN-PWY	superpathway of branched amino acid biosynthesis	29218.82
CALVIN-PWY	Calvin-Benson-Bassham cycle	21251.36
CENTFERM-PWY	pyruvate fermentation to butanoate	1504.62
COA-PWY	coenzyme A biosynthesis I	27668.04
COBALSYN-PWY	adenosylcobalamin salvage from cobinamide I	22836.00
CODH-PWY	reductive acetyl coenzyme A pathway	23.99
COLANSYN-PWY	colanic acid building blocks biosynthesis	20747.61
COMPLETE-ARO-PWY	superpathway of aromatic amino acid biosynthesis	27807.52
DAPLYSINESYN-PWY	l-lysine biosynthesis I	10107.33
DENOVOPURINE2-PWY	superpathway of purine nucleotides de novo biosynthesis II	26138.44
DTDPRHAMSYN-PWY	dTDP-l-rhamnose biosynthesis I	26002.80
FAO-PWY	fatty acid β-oxidation I	927.22
FASYN-ELONG-PWY	fatty acid elongation – saturated	29389.77
FASYN-INITIAL-PWY	superpathway of fatty acid biosynthesis initiation (E. coli)	1295.96
FERMENTATION-PWY	mixed acid fermentation	7139.81
FOLSYN-PWY	superpathway of tetrahydrofolate biosynthesis and salvage	21671.94
FUC-RHAMCAT-PWY	superpathway of fucose and rhamnose degradation	7716.05
FUCCAT-PWY	fucose degradation	15255.23
GALACT-GLUCUROCAT-PWY	superpathway of hexuronide and hexuronate degradation	1696.75
GALACTUROCAT-PWY	d-galacturonate degradation I	5055.25
GLCMANNANAUT-PWY	superpathway of N-acetylglucosamine, N-acetylmannosamine and N-acetylneuraminate degradation	2172.96
GLUCONEO-PWY	gluconeogenesis I	23321.28
GLUCOSE1PMETAB-PWY	glucose and glucose-1-phosphate degradation	17166.81
GLUCUROCAT-PWY	superpathway of β-D-glucuronide and d-glucuronate degradation	1447.52
GLUTORN-PWY	l-ornithine biosynthesis	24875.71
GLYCOCAT-PWY	glycogen degradation I (bacterial)	26956.55
GLYCOGENSYNTH-PWY	glycogen biosynthesis I (from ADP-d-Glucose)	15338.92
GLYCOL-GLYOXDEG-PWY	superpathway of glycol metabolism and degradation	19.47
GLYCOLYSIS	glycolysis I (from glucose 6-phosphate)	24075.80
GLYCOLYSIS-E-D	superpathway of glycolysis and Entner-Doudoroff	4270.42
GLYCOLYSIS-TCA-GLYOX-BYPASS	superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass	72.25
GLYOXYLATE-BYPASS	glyoxylate cycle	19.99
GOLPDLCAT-PWY	superpathway of glycerol degradation to 1,3-propanediol	19.17
HEME-BIOSYNTHESIS-II	heme biosynthesis I (aerobic)	567.21
HEMESYN2-PWY	heme biosynthesis II (anaerobic)	2355.12
HEXITOLDEGSUPER-PWY	superpathway of hexitol degradation (bacteria)	2077.28
HISDEG-PWY	l-histidine degradation I	2675.21
HISTSYN-PWY	l-histidine biosynthesis	24635.47
HOMOSER-METSYN-PWY	l-methionine biosynthesis I	3356.78
HSERMETANA-PWY	l-methionine biosynthesis III	22124.55
ILEUSYN-PWY	l-isoleucine biosynthesis I (from threonine)	28982.46
LEU-DEG2-PWY	l-leucine degradation I	592.11
MET-SAM-PWY	superpathway of S-adenosyl-l-methionine biosynthesis	5010.33
METH-ACETATE-PWY	methanogenesis from acetate	92.48
NAD-BIOSYNTHESIS-II	NAD salvage pathway II	20.97
NAGLIPASYN-PWY	lipid IVA biosynthesis	22381.09
NONMEVIPP-PWY	methylerythritol phosphate pathway I	25283.47
NONOXIPENT-PWY	pentose phosphate pathway (non-oxidative branch)	24169.23
OANTIGEN-PWY	O-antigen building blocks biosynthesis (E. coli)	8845.75
P101-PWY	ectoine biosynthesis	23.92
P108-PWY	pyruvate fermentation to propanoate I	27628.06
P122-PWY	heterolactic fermentation	2174.42
P124-PWY	Bifidobacterium shunt	2873.82
P125-PWY	superpathway of (R,R)-butanediol biosynthesis	469.09
P161-PWY	acetylene degradation	4399.39
P162-PWY	l-glutamate degradation V (via hydroxyglutarate)	270.38
P163-PWY	l-lysine fermentation to acetate and butanoate	1815.71
P164-PWY	purine nucleobases degradation I (anaerobic)	1537.38
P23-PWY	reductive TCA cycle I	5712.61
P4-PWY	superpathway of l-lysine, l-threonine and l-methionine biosynthesis I	6570.46
P42-PWY	incomplete reductive TCA cycle	30157.68
P441-PWY	superpathway of N-acetylneuraminate degradation	4909.65
P461-PWY	hexitol fermentation to lactate, formate, ethanol and acetate	1929.04
P562-PWY	myo-inositol degradation I	27.95
PANTO-PWY	phosphopantothenate biosynthesis I	23646.05
PANTOSYN-PWY	pantothenate and coenzyme A biosynthesis I	25015.44
PENTOSE-P-PWY	pentose phosphate pathway	5833.14
PEPTIDOGLYCANSYN-PWY	peptidoglycan biosynthesis I (meso-diaminopimelate containing)	19568.45
PHOSLIPSYN-PWY	superpathway of phospholipid biosynthesis I (bacteria)	26286.94
POLYAMINSYN3-PWY	superpathway of polyamine biosynthesis II	2742.26
POLYAMSYN-PWY	superpathway of polyamine biosynthesis I	2548.99
POLYISOPRENSYN-PWY	polyisoprenoid biosynthesis (E. coli)	23859.83
PPGPPMET-PWY	ppGpp biosynthesis	22.49
PRPP-PWY	superpathway of histidine, purine, and pyrimidine biosynthesis	9410.86
PWY-1269	CMP-3-deoxy-d-manno-octulosonate biosynthesis I	21952.21
PWY-1861	formaldehyde assimilation II (RuMP Cycle)	41.95
PWY-2941	l-lysine biosynthesis II	1237.77
PWY-2942	l-lysine biosynthesis III	26976.97
PWY-3001	superpathway of l-isoleucine biosynthesis I	22699.11
PWY-3781	aerobic respiration I (cytochrome c)	3967.73
PWY-4984	urea cycle	726.53
PWY-5005	biotin biosynthesis II	2981.72
PWY-5022	4-aminobutanoate degradation V	820.81
PWY-5088	l-glutamate degradation VIII (to propanoate)	232.60
PWY-5097	l-lysine biosynthesis VI	26803.11
PWY-5100	pyruvate fermentation to acetate and lactate II	16656.78
PWY-5101	l-isoleucine biosynthesis II	31615.67
PWY-5103	l-isoleucine biosynthesis III	27925.35
PWY-5104	l-isoleucine biosynthesis IV	28867.31
PWY-5121	superpathway of geranylgeranyl diphosphate biosynthesis II (via MEP)	25176.74
PWY-5154	l-arginine biosynthesis III (via N-acetyl-l-citrulline)	12610.29
PWY-5177	glutaryl-CoA degradation	963.16
PWY-5188	tetrapyrrole biosynthesis I (from glutamate)	18427.83
PWY-5189	tetrapyrrole biosynthesis II (from glycine)	17599.25
PWY-5304	superpathway of sulfur oxidation (Acidianus ambivalens)	12.00
PWY-5345	superpathway of l-methionine biosynthesis (by sulfhydrylation)	24018.21
PWY-5347	superpathway of l-methionine biosynthesis (transsulfuration)	6762.79
PWY-5384	sucrose degradation IV (sucrose phosphorylase)	17045.53
PWY-5484	glycolysis II (from fructose 6-phosphate)	22657.81
PWY-5505	l-glutamate and l-glutamine biosynthesis	24342.80
PWY-5507	adenosylcobalamin biosynthesis I (early cobalt insertion)	202.34
PWY-5509	adenosylcobalamin biosynthesis from cobyrinate a,c-diamide I	15720.38
PWY-5532	adenosine nucleotides degradation IV	26.31
PWY-5659	GDP-mannose biosynthesis	26451.05
PWY-5667	CDP-diacylglycerol biosynthesis I	27731.45
PWY-5676	acetyl-CoA fermentation to butanoate II	5619.84
PWY-5677	succinate fermentation to butanoate	845.32
PWY-5686	UMP biosynthesis	30469.20
PWY-5695	urate biosynthesis/inosine 5′-phosphate degradation	15758.15
PWY-5741	ethylmalonyl-CoA pathway	15.98
PWY-5837	1,4-dihydroxy-2-naphthoate biosynthesis I	16699.80
PWY-5838	superpathway of menaquinol-8 biosynthesis I	19650.80
PWY-5840	superpathway of menaquinol-7 biosynthesis	16871.26
PWY-5855	ubiquinol-7 biosynthesis (prokaryotic)	34.85
PWY-5856	ubiquinol-9 biosynthesis (prokaryotic)	34.85
PWY-5857	ubiquinol-10 biosynthesis (prokaryotic)	34.85
PWY-5861	superpathway of demethylmenaquinol-8 biosynthesis	18991.19
PWY-5863	superpathway of phylloquinol biosynthesis	17427.21
PWY-5897	superpathway of menaquinol-11 biosynthesis	19472.27
PWY-5898	superpathway of menaquinol-12 biosynthesis	19472.27
PWY-5899	superpathway of menaquinol-13 biosynthesis	19472.27
PWY-5910	superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate)	887.17
PWY-5913	TCA cycle VI (obligate autotrophs)	26027.71
PWY-5918	superpathay of heme biosynthesis from glutamate	1355.43
PWY-5920	superpathway of heme biosynthesis from glycine	533.15
PWY-5971	palmitate biosynthesis II (bacteria and plants)	2351.05
PWY-5973	cis-vaccenate biosynthesis	29971.49
PWY-5989	stearate biosynthesis II (bacteria and plants)	1554.65
PWY-6121	5-aminoimidazole ribonucleotide biosynthesis I	28865.69
PWY-6122	5-aminoimidazole ribonucleotide biosynthesis II	29663.23
PWY-6123	inosine-5′-phosphate biosynthesis I	27209.88
PWY-6125	superpathway of guanosine nucleotides de novo biosynthesis II	23882.62
PWY-6126	superpathway of adenosine nucleotides de novo biosynthesis II	27882.47
PWY-6147	6-hydroxymethyl-dihydropterin diphosphate biosynthesis I	12441.69
PWY-6151	S-adenosyl-l-methionine cycle I	9272.03
PWY-6163	chorismate biosynthesis from 3-dehydroquinate	25088.67
PWY-621	sucrose degradation III (sucrose invertase)	17045.53
PWY-6263	superpathway of menaquinol-8 biosynthesis II	2380.82
PWY-6269	adenosylcobalamin salvage from cobinamide II	16371.32
PWY-6277	superpathway of 5-aminoimidazole ribonucleotide biosynthesis	29663.23
PWY-6282	palmitoleate biosynthesis I (from (5Z)-dodec-5-enoate)	1558.66
PWY-6317	galactose degradation I (Leloir pathway)	8277.67
PWY-6353	purine nucleotides degradation II (aerobic)	3164.91
PWY-6385	peptidoglycan biosynthesis III (mycobacteria)	17812.65
PWY-6386	UDP-N-acetylmuramoyl-pentapeptide biosynthesis II (lysine-containing)	16160.29
PWY-6387	UDP-N-acetylmuramoyl-pentapeptide biosynthesis I (meso-diaminopimelate containing)	18502.30
PWY-6396	superpathway of 2,3-butanediol biosynthesis	102.85
PWY-6467	Kdo transfer to lipid IVA III (Chlamydia)	20999.99
PWY-6470	peptidoglycan biosynthesis V (β-lactam resistance)	2104.54
PWY-6471	peptidoglycan biosynthesis IV (Enterococcus faecium)	6509.51
PWY-6478	GDP-d-glycero-α-d-manno-heptose biosynthesis	17.91
PWY-6507	4-deoxy-L-threo-hex-4-enopyranuronate degradation	8731.69
PWY-6519	8-amino-7-oxononanoate biosynthesis I	2602.92
PWY-6545	pyrimidine deoxyribonucleotides de novo biosynthesis III	8156.83
PWY-6572	chondroitin sulfate degradation I (bacterial)	623.39
PWY-6588	pyruvate fermentation to acetone	5174.80
PWY-6590	superpathway of Clostridium acetobutylicum acidogenic fermentation	1885.04
PWY-6608	guanosine nucleotides degradation III	3040.41
PWY-6609	adenine and adenosine salvage III	14801.94
PWY-6612	superpathway of tetrahydrofolate biosynthesis	20082.21
PWY-6628	superpathway of l-phenylalanine biosynthesis	9216.39
PWY-6630	superpathway of l-tyrosine biosynthesis	9120.72
PWY-6700	queuosine biosynthesis	24077.01
PWY-6703	preQ0 biosynthesis	21931.80
PWY-6708	ubiquinol-8 biosynthesis (prokaryotic)	34.85
PWY-6728	methylaspartate cycle	46.02
PWY-6737	starch degradation V	28024.83
PWY-6749	CMP-legionaminate biosynthesis I	1668.16
PWY-6891	thiazole biosynthesis II (Bacillus)	829.14
PWY-6892	thiazole biosynthesis I (E. coli)	24408.22
PWY-6895	superpathway of thiamin diphosphate biosynthesis II	3642.08
PWY-6897	thiamin salvage II	23465.12
PWY-6901	superpathway of glucose and xylose degradation	8520.77
PWY-6969	TCA cycle V (2-oxoglutarate:ferredoxin oxidoreductase)	27571.20
PWY-7003	glycerol degradation to butanol	142.95
PWY-7013	L-1,2-propanediol degradation	2467.97
PWY-7031	protein N-glycosylation (bacterial)	9.00
PWY-7111	pyruvate fermentation to isobutanol (engineered)	29827.64
PWY-7184	pyrimidine deoxyribonucleotides de novo biosynthesis I	18210.44
PWY-7187	pyrimidine deoxyribonucleotides de novo biosynthesis II	18256.85
PWY-7196	superpathway of pyrimidine ribonucleosides salvage	23128.51
PWY-7197	pyrimidine deoxyribonucleotide phosphorylation	21843.31
PWY-7199	pyrimidine deoxyribonucleosides salvage	12060.55
PWY-7200	superpathway of pyrimidine deoxyribonucleoside salvage	15653.43
PWY-7208	superpathway of pyrimidine nucleobases salvage	30008.38
PWY-7211	superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis	22061.73
PWY-7219	adenosine ribonucleotides de novo biosynthesis	29773.56
PWY-7220	adenosine deoxyribonucleotides de novo biosynthesis II	25705.53
PWY-7221	guanosine ribonucleotides de novo biosynthesis	25407.97
PWY-7222	guanosine deoxyribonucleotides de novo biosynthesis II	25705.53
PWY-7228	superpathway of guanosine nucleotides de novo biosynthesis I	23646.24
PWY-7229	superpathway of adenosine nucleotides de novo biosynthesis I	28342.83
PWY-7234	inosine-5′-phosphate biosynthesis III	2602.21
PWY-7237	myo-, chiro- and scillo-inositol degradation	706.50
PWY-7242	d-fructuronate degradation	8902.55
PWY-7254	TCA cycle VII (acetate-producers)	3665.48
PWY-7315	dTDP-N-acetylthomosamine biosynthesis	35.98
PWY-7323	superpathway of GDP-mannose-derived O-antigen building blocks biosynthesis	16931.88
PWY-7328	superpathway of UDP-glucose-derived O-antigen building blocks biosynthesis	14109.49
PWY-7371	1,4-dihydroxy-6-naphthoate biosynthesis II	1003.66
PWY-7373	superpathway of demethylmenaquinol-6 biosynthesis II	35.06
PWY-7374	1,4-dihydroxy-6-naphthoate biosynthesis I	579.85
PWY-7377	cob(II)yrinate a,c-diamide biosynthesis I (early cobalt insertion)	2231.03
PWY-7392	taxadiene biosynthesis (engineered)	7379.89
PWY-7400	l-arginine biosynthesis IV (archaebacteria)	22802.89
PWY-7456	mannan degradation	9107.19
PWY-7539	6-hydroxymethyl-dihydropterin diphosphate biosynthesis III (Chlamydia)	12469.78
PWY-7560	methylerythritol phosphate pathway II	25283.47
PWY-7663	gondoate biosynthesis (anaerobic)	31430.52
PWY-7664	oleate biosynthesis IV (anaerobic)	1802.47
PWY-841	superpathway of purine nucleotides de novo biosynthesis I	26212.51
PWY-922	mevalonate pathway I	627.67
PWY0-1061	superpathway of l-alanine biosynthesis	22491.62
PWY0-1241	ADP-l-glycero-β-d-manno-heptose biosynthesis	1621.60
PWY0-1261	anhydromuropeptides recycling	6840.62
PWY0-1296	purine ribonucleosides degradation	5727.99
PWY0-1297	superpathway of purine deoxyribonucleosides degradation	4342.29
PWY0-1298	superpathway of pyrimidine deoxyribonucleosides degradation	4652.80
PWY0-1319	CDP-diacylglycerol biosynthesis II	27731.45
PWY0-1415	superpathway of heme biosynthesis from uroporphyrinogen-III	403.45
PWY0-1479	tRNA processing	60.28
PWY0-1586	peptidoglycan maturation (meso-diaminopimelate containing)	4720.42
PWY0-162	superpathway of pyrimidine ribonucleotides de novo biosynthesis	28678.78
PWY0-166	superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis (E. coli)	18302.75
PWY0-781	aspartate superpathway	5596.69
PWY0-845	superpathway of pyridoxal 5′-phosphate biosynthesis and salvage	11911.10
PWY0-862	(5Z)-dodec-5-enoate biosynthesis	1554.96
PWY490-3	nitrate reduction VI (assimilatory)	1595.18
PWY4FS-7	phosphatidylglycerol biosynthesis I (plastidic)	23843.93
PWY4FS-8	phosphatidylglycerol biosynthesis II (non-plastidic)	23843.93
PWYG-321	mycolate biosynthesis	1870.53
PYRIDNUCSAL-PWY	NAD salvage pathway I	13543.95
PYRIDNUCSYN-PWY	NAD biosynthesis I (from aspartate)	23168.41
PYRIDOXSYN-PWY	pyridoxal 5′-phosphate biosynthesis I	13819.86
REDCITCYC	TCA cycle VIII (helicobacter)	3628.87
RHAMCAT-PWY	l-rhamnose degradation I	5463.43
RIBOSYN2-PWY	flavin biosynthesis I (bacteria and plants)	23882.15
RUMP-PWY	formaldehyde oxidation I	31.35
SALVADEHYPOX-PWY	adenosine nucleotides degradation II	2625.31
SER-GLYSYN-PWY	superpathway of l-serine and glycine biosynthesis I	25102.15
SO4ASSIM-PWY	sulfate reduction I (assimilatory)	23465.02
SULFATE-CYS-PWY	superpathway of sulfate assimilation and cysteine biosynthesis	22594.61
TCA	TCA cycle I (prokaryotic)	26721.47
TCA-GLYOX-BYPASS	superpathway of glyoxylate bypass and TCA	36.31
THISYN-PWY	superpathway of thiamin diphosphate biosynthesis I	24068.59
THRESYN-PWY	superpathway of l-threonine biosynthesis	19874.22
TRNA-CHARGING-PWY	tRNA charging	26152.39
TRPSYN-PWY	L-tryptophan biosynthesis	25812.43
TYRFUMCAT-PWY	l-tyrosine degradation I	210.56
UBISYN-PWY	superpathway of ubiquinol-8 biosynthesis (prokaryotic)	33.48
UDPNAGSYN-PWY	UDP-N-acetyl-d-glucosamine biosynthesis I	6467.51
VALSYN-PWY	l-valine biosynthesis	28982.46

Amino Acid Synthesis - Pathways for amino acid biosynthesis, such as "superpathway of branched amino acid biosynthesis" (29218.82), "l-arginine biosynthesis II" (25126.35), and "L-tryptophan biosynthesis" (25812.43), are highly abundant.

Central Metabolism - Central metabolic processes are also active, including "glycolysis I" (24075.80) and "TCA cycle I" (26721.47).

Nucleotide Biosynthesis - Nucleotide pathways like "superpathway of purine nucleotides de novo biosynthesis II" (26138.44) and "superpathway of pyrimidine ribonucleotides de novo biosynthesis" (28678.78) is also highly abundant.

Low Abundance Pathways - Pathways with very low abundance include "glycol metabolism and degradation" (19.47), "glyoxylate cycle" (19.99), and "reductive acetyl coenzyme A pathway" (23.99).

3.6.2. Metabolic pathway analysis of sample T2

Sample T2 shows a shift in metabolic activity compared to the Control, with a general decrease in some key pathways and an increase in others (Table 4b). They are:

Table 4b.

Metabolic pathways and abundance in sample T2.

Pathway Code	Metabolic Pathway	Abundance
1CMET2-PWY	N10-formyl-tetrahydrofolate biosynthesis	17160.73
3-HYDROXYPHENYLACETATE-DEGRADATION-PWY	4-hydroxyphenylacetate degradation	73.29
AEROBACTINSYN-PWY	aerobactin biosynthesis	50.98
ALL-CHORISMATE-PWY	superpathway of chorismate metabolism	3962.70
ANAEROFRUCAT-PWY	homolactic fermentation	23119.13
ANAGLYCOLYSIS-PWY	glycolysis III (from glucose)	24084.09
ARG + POLYAMINE-SYN	superpathway of arginine and polyamine biosynthesis	7010.70
ARGDEG-PWY	superpathway of l-arginine, putrescine, and 4-aminobutanoate degradation	687.05
ARGORNPROST-PWY	arginine, ornithine and proline interconversion	4247.37
ARGSYN-PWY	l-arginine biosynthesis I (via l-ornithine)	20739.87
ARGSYNBSUB-PWY	l-arginine biosynthesis II (acetyl cycle)	21156.13
ARO-PWY	chorismate biosynthesis I	22124.24
ASPASN-PWY	superpathway of l-aspartate and l-asparagine biosynthesis	12198.44
AST-PWY	l-arginine degradation II (AST pathway)	817.43
BIOTIN-BIOSYNTHESIS-PWY	biotin biosynthesis I	4730.85
BRANCHED-CHAIN-AA-SYN-PWY	superpathway of branched amino acid biosynthesis	24307.48
CALVIN-PWY	Calvin-Benson-Bassham cycle	24322.56
CATECHOL-ORTHO-CLEAVAGE-PWY	catechol degradation to β-ketoadipate	77.22
CENTFERM-PWY	pyruvate fermentation to butanoate	3909.91
COA-PWY	coenzyme A biosynthesis I	21632.93
COBALSYN-PWY	adenosylcobalamin salvage from cobinamide I	11091.03
CODH-PWY	reductive acetyl coenzyme A pathway	1522.98
COLANSYN-PWY	colanic acid building blocks biosynthesis	8387.74
COMPLETE-ARO-PWY	superpathway of aromatic amino acid biosynthesis	23268.80
DAPLYSINESYN-PWY	l-lysine biosynthesis I	16109.37
DENOVOPURINE2-PWY	superpathway of purine nucleotides de novo biosynthesis II	16458.53
DTDPRHAMSYN-PWY	dTDP-l-rhamnose biosynthesis I	19058.37
ECASYN-PWY	enterobacterial common antigen biosynthesis	1313.93
ENTBACSYN-PWY	enterobactin biosynthesis	3554.42
FAO-PWY	fatty acid β-oxidation I	2488.77
FASYN-ELONG-PWY	fatty acid elongation -- saturated	23923.32
FASYN-INITIAL-PWY	superpathway of fatty acid biosynthesis initiation (E. coli)	2784.71
FERMENTATION-PWY	mixed acid fermentation	17131.74
FOLSYN-PWY	superpathway of tetrahydrofolate biosynthesis and salvage	14448.44
FUC-RHAMCAT-PWY	superpathway of fucose and rhamnose degradation	3450.76
FUCCAT-PWY	fucose degradation	2821.96
GALACT-GLUCUROCAT-PWY	superpathway of hexuronide and hexuronate degradation	4871.26
GALACTARDEG-PWY	d-galactarate degradation I	1582.42
GALACTUROCAT-PWY	d-galacturonate degradation I	6725.13
GLCMANNANAUT-PWY	superpathway of N-acetylglucosamine, N-acetylmannosamine and N-acetylneuraminate degradation	15547.61
GLUCARDEG-PWY	d-glucarate degradation I	1169.92
GLUCARGALACTSUPER-PWY	superpathway of d-glucarate and d-galactarate degradation	1582.42
GLUCONEO-PWY	gluconeogenesis I	18784.51
GLUCOSE1PMETAB-PWY	glucose and glucose-1-phosphate degradation	2886.68
GLUCUROCAT-PWY	superpathway of β-D-glucuronide and d-glucuronate degradation	12942.51
GLUTORN-PWY	l-ornithine biosynthesis	18758.61
GLYCOCAT-PWY	glycogen degradation I (bacterial)	17477.37
GLYCOGENSYNTH-PWY	glycogen biosynthesis I (from ADP-d-Glucose)	16518.73
GLYCOL-GLYOXDEG-PWY	superpathway of glycol metabolism and degradation	1569.76
GLYCOLYSIS	glycolysis I (from glucose 6-phosphate)	23607.63
GLYCOLYSIS-E-D	superpathway of glycolysis and Entner-Doudoroff	9503.02
GLYCOLYSIS-TCA-GLYOX-BYPASS	superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass	4710.29
GLYOXYLATE-BYPASS	glyoxylate cycle	2723.82
GOLPDLCAT-PWY	superpathway of glycerol degradation to 1,3-propanediol	33.72
HCAMHPDEG-PWY	3-phenylpropanoate and 3-(3-hydroxyphenyl)propanoate degradation to 2-oxopent-4-enoate	172.83
HEME-BIOSYNTHESIS-II	heme biosynthesis I (aerobic)	1533.07
HEMESYN2-PWY	heme biosynthesis II (anaerobic)	11236.44
HEXITOLDEGSUPER-PWY	superpathway of hexitol degradation (bacteria)	8725.63
HISDEG-PWY	l-histidine degradation I	2158.03
HISTSYN-PWY	l-histidine biosynthesis	15195.29
HOMOSER-METSYN-PWY	l-methionine biosynthesis I	14334.82
HSERMETANA-PWY	l-methionine biosynthesis III	15590.32
ILEUSYN-PWY	l-isoleucine biosynthesis I (from threonine)	25751.79
KDO-NAGLIPASYN-PWY	superpathway of (Kdo)2-lipid A biosynthesis	3021.76
KETOGLUCONMET-PWY	ketogluconate metabolism	672.08
LACTOSECAT-PWY	lactose and galactose degradation I	3199.56
LEU-DEG2-PWY	l-leucine degradation I	89.67
LPSSYN-PWY	superpathway of lipopolysaccharide biosynthesis	1226.46
MET-SAM-PWY	superpathway of S-adenosyl-l-methionine biosynthesis	18364.94
METH-ACETATE-PWY	methanogenesis from acetate	4102.69
METHGLYUT-PWY	superpathway of methylglyoxal degradation	1107.81
NAD-BIOSYNTHESIS-II	NAD salvage pathway II	1914.14
NAGLIPASYN-PWY	lipid IVA biosynthesis	5306.25
NONMEVIPP-PWY	methylerythritol phosphate pathway I	13636.93
NONOXIPENT-PWY	pentose phosphate pathway (non-oxidative branch)	27742.03
OANTIGEN-PWY	O-antigen building blocks biosynthesis (E. coli)	19283.24
ORNARGDEG-PWY	superpathway of l-arginine and l-ornithine degradation	687.05
ORNDEG-PWY	superpathway of ornithine degradation	1163.29
P101-PWY	ectoine biosynthesis	16.81
P105-PWY	TCA cycle IV (2-oxoglutarate decarboxylase)	5966.48
P108-PWY	pyruvate fermentation to propanoate I	7468.63
P122-PWY	heterolactic fermentation	6799.76
P124-PWY	Bifidobacterium shunt	8111.81
P125-PWY	superpathway of (R,R)-butanediol biosynthesis	1265.07
P161-PWY	acetylene degradation	20760.07
P162-PWY	l-glutamate degradation V (via hydroxyglutarate)	528.95
P163-PWY	l-lysine fermentation to acetate and butanoate	308.72
P164-PWY	purine nucleobases degradation I (anaerobic)	5713.23
P221-PWY	octane oxidation	106.23
P23-PWY	reductive TCA cycle I	7788.36
P281-PWY	3-phenylpropanoate degradation	23.47
P381-PWY	adenosylcobalamin biosynthesis II (late cobalt incorporation)	394.74
P4-PWY	superpathway of l-lysine, l-threonine and l-methionine biosynthesis I	16044.57
P42-PWY	incomplete reductive TCA cycle	15064.79
P441-PWY	superpathway of N-acetylneuraminate degradation	13438.92
P461-PWY	hexitol fermentation to lactate, formate, ethanol and acetate	6832.18
P562-PWY	myo-inositol degradation I	50.75
PANTO-PWY	phosphopantothenate biosynthesis I	10528.47
PANTOSYN-PWY	pantothenate and coenzyme A biosynthesis I	12292.28
PENTOSE-P-PWY	pentose phosphate pathway	14090.12
PEPTIDOGLYCANSYN-PWY	peptidoglycan biosynthesis I (meso-diaminopimelate containing)	22355.02
PHOSLIPSYN-PWY	superpathway of phospholipid biosynthesis I (bacteria)	24413.94
POLYAMINSYN3-PWY	superpathway of polyamine biosynthesis II	1922.89
POLYAMSYN-PWY	superpathway of polyamine biosynthesis I	4436.95
POLYISOPRENSYN-PWY	polyisoprenoid biosynthesis (E. coli)	17240.10
PPGPPMET-PWY	ppGpp biosynthesis	2152.66
PROTOCATECHUATE-ORTHO-CLEAVAGE-PWY	protocatechuate degradation II (ortho-cleavage pathway)	190.11
PRPP-PWY	superpathway of histidine, purine, and pyrimidine biosynthesis	13956.91
PWY-1269	CMP-3-deoxy-d-manno-octulosonate biosynthesis I	5623.62
PWY-1541	superpathway of taurine degradation	424.01
PWY-181	Photorespiration	1525.46
PWY-1861	formaldehyde assimilation II (RuMP Cycle)	4648.74
PWY-2941	l-lysine biosynthesis II	5613.59
PWY-2942	l-lysine biosynthesis III	23233.73
PWY-3001	superpathway of l-isoleucine biosynthesis I	22742.83
PWY-3781	aerobic respiration I (cytochrome c)	1137.66
PWY-4984	urea cycle	1191.21
PWY-5005	biotin biosynthesis II	5039.10
PWY-5022	4-aminobutanoate degradation V	1749.75
PWY-5097	l-lysine biosynthesis VI	22991.18
PWY-5100	pyruvate fermentation to acetate and lactate II	25244.95
PWY-5101	l-isoleucine biosynthesis II	27779.42
PWY-5103	l-isoleucine biosynthesis III	23655.43
PWY-5104	l-isoleucine biosynthesis IV	26147.16
PWY-5121	superpathway of geranylgeranyl diphosphate biosynthesis II (via MEP)	14892.02
PWY-5154	l-arginine biosynthesis III (via N-acetyl-l-citrulline)	6246.02
PWY-5177	glutaryl-CoA degradation	2330.26
PWY-5180	toluene degradation I (aerobic) (via o-cresol)	1108.37
PWY-5181	toluene degradation III (aerobic) (via p-cresol)	90.30
PWY-5182	toluene degradation II (aerobic) (via 4-methylcatechol)	1108.37
PWY-5188	tetrapyrrole biosynthesis I (from glutamate)	7844.06
PWY-5189	tetrapyrrole biosynthesis II (from glycine)	7366.85
PWY-5265	peptidoglycan biosynthesis II (staphylococci)	496.40
PWY-5304	superpathway of sulfur oxidation (Acidianus ambivalens)	2242.00
PWY-5345	superpathway of l-methionine biosynthesis (by sulfhydrylation)	6284.55
PWY-5347	superpathway of l-methionine biosynthesis (transsulfuration)	18063.03
PWY-5384	sucrose degradation IV (sucrose phosphorylase)	14255.47
PWY-5415	catechol degradation I (meta-cleavage pathway)	367.35
PWY-5417	catechol degradation III (ortho-cleavage pathway)	80.54
PWY-5431	aromatic compounds degradation via β-ketoadipate	80.54
PWY-5484	glycolysis II (from fructose 6-phosphate)	22089.82
PWY-5505	l-glutamate and l-glutamine biosynthesis	8551.15
PWY-5507	adenosylcobalamin biosynthesis I (early cobalt insertion)	714.91
PWY-5509	adenosylcobalamin biosynthesis from cobyrinate a,c-diamide I	10802.58
PWY-5659	GDP-mannose biosynthesis	14567.84
PWY-5667	CDP-diacylglycerol biosynthesis I	26005.47
PWY-5676	acetyl-CoA fermentation to butanoate II	3701.92
PWY-5677	succinate fermentation to butanoate	1062.55
PWY-5686	UMP biosynthesis	24250.69
PWY-5695	urate biosynthesis/inosine 5′-phosphate degradation	15914.15
PWY-5705	allantoin degradation to glyoxylate III	1175.58
PWY-5747	2-methylcitrate cycle II	876.66
PWY-5837	1,4-dihydroxy-2-naphthoate biosynthesis I	11603.77
PWY-5838	superpathway of menaquinol-8 biosynthesis I	8787.37
PWY-5840	superpathway of menaquinol-7 biosynthesis	9758.66
PWY-5845	superpathway of menaquinol-9 biosynthesis	4974.96
PWY-5850	superpathway of menaquinol-6 biosynthesis I	4974.96
PWY-5855	ubiquinol-7 biosynthesis (prokaryotic)	1574.56
PWY-5856	ubiquinol-9 biosynthesis (prokaryotic)	1574.56
PWY-5857	ubiquinol-10 biosynthesis (prokaryotic)	1574.56
PWY-5860	superpathway of demethylmenaquinol-6 biosynthesis I	4406.97
PWY-5861	superpathway of demethylmenaquinol-8 biosynthesis	10405.51
PWY-5862	superpathway of demethylmenaquinol-9 biosynthesis	4406.97
PWY-5863	superpathway of phylloquinol biosynthesis	12262.35
PWY-5896	superpathway of menaquinol-10 biosynthesis	4974.96
PWY-5897	superpathway of menaquinol-11 biosynthesis	9503.25
PWY-5898	superpathway of menaquinol-12 biosynthesis	9503.25
PWY-5899	superpathway of menaquinol-13 biosynthesis	9503.25
PWY-5910	superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate)	11405.55
PWY-5913	TCA cycle VI (obligate autotrophs)	13510.90
PWY-5918	superpathay of heme biosynthesis from glutamate	2961.19
PWY-5920	superpathway of heme biosynthesis from glycine	1409.62
PWY-5971	palmitate biosynthesis II (bacteria and plants)	5067.65
PWY-5973	cis-vaccenate biosynthesis	26909.75
PWY-5989	stearate biosynthesis II (bacteria and plants)	3212.74
PWY-6071	superpathway of phenylethylamine degradation	161.26
PWY-6121	5-aminoimidazole ribonucleotide biosynthesis I	24941.69
PWY-6122	5-aminoimidazole ribonucleotide biosynthesis II	25628.82
PWY-6123	inosine-5′-phosphate biosynthesis I	22136.25
PWY-6125	superpathway of guanosine nucleotides de novo biosynthesis II	12427.22
PWY-6126	superpathway of adenosine nucleotides de novo biosynthesis II	25698.03
PWY-6147	6-hydroxymethyl-dihydropterin diphosphate biosynthesis I	9187.05
PWY-6151	S-adenosyl-l-methionine cycle I	16760.63
PWY-6163	chorismate biosynthesis from 3-dehydroquinate	20539.66
PWY-6182	superpathway of salicylate degradation	68.21
PWY-6185	4-methylcatechol degradation (ortho cleavage)	70.80
PWY-621	sucrose degradation III (sucrose invertase)	22451.18
PWY-6263	superpathway of menaquinol-8 biosynthesis II	1739.58
PWY-6269	adenosylcobalamin salvage from cobinamide II	10900.97
PWY-6277	superpathway of 5-aminoimidazole ribonucleotide biosynthesis	25628.82
PWY-6282	palmitoleate biosynthesis I (from (5Z)-dodec-5-enoate)	3307.93
PWY-6317	galactose degradation I (Leloir pathway)	20938.82
PWY-6353	purine nucleotides degradation II (aerobic)	8048.41
PWY-6383	mono-trans, poly-cis decaprenyl phosphate biosynthesis	133.76
PWY-6385	peptidoglycan biosynthesis III (mycobacteria)	22230.89
PWY-6386	UDP-N-acetylmuramoyl-pentapeptide biosynthesis II (lysine-containing)	22636.60
PWY-6387	UDP-N-acetylmuramoyl-pentapeptide biosynthesis I (meso-diaminopimelate containing)	22593.43
PWY-6396	superpathway of 2,3-butanediol biosynthesis	317.45
PWY-6397	mycolyl-arabinogalactan-peptidoglycan complex biosynthesis	65.27
PWY-6467	Kdo transfer to lipid IVA III (Chlamydia)	4954.77
PWY-6470	peptidoglycan biosynthesis V (β-lactam resistance)	16613.93
PWY-6471	peptidoglycan biosynthesis IV (Enterococcus faecium)	19064.27
PWY-6478	GDP-d-glycero-α-d-manno-heptose biosynthesis	580.99
PWY-6507	4-deoxy-L-threo-hex-4-enopyranuronate degradation	15676.38
PWY-6519	8-amino-7-oxononanoate biosynthesis I	4258.69
PWY-6545	pyrimidine deoxyribonucleotides de novo biosynthesis III	7745.93
PWY-6562	norspermidine biosynthesis	117.50
PWY-6572	chondroitin sulfate degradation I (bacterial)	65.24
PWY-6588	pyruvate fermentation to acetone	7432.32
PWY-6590	superpathway of Clostridium acetobutylicum acidogenic fermentation	4773.73
PWY-6608	guanosine nucleotides degradation III	5798.88
PWY-6609	adenine and adenosine salvage III	27189.87
PWY-6612	superpathway of tetrahydrofolate biosynthesis	12334.14
PWY-6628	superpathway of l-phenylalanine biosynthesis	12193.75
PWY-6629	superpathway of l-tryptophan biosynthesis	6645.18
PWY-6630	superpathway of l-tyrosine biosynthesis	12528.34
PWY-6641	superpathway of sulfolactate degradation	341.32
PWY-6690	cinnamate and 3-hydroxycinnamate degradation to 2-oxopent-4-enoate	172.83
PWY-6700	queuosine biosynthesis	8220.40
PWY-6703	preQ0 biosynthesis	6379.48
PWY-6708	ubiquinol-8 biosynthesis (prokaryotic)	1574.56
PWY-6737	starch degradation V	16007.16
PWY-6749	CMP-legionaminate biosynthesis I	821.67
PWY-6876	isopropanol biosynthesis	33.12
PWY-6891	thiazole biosynthesis II (Bacillus)	6740.82
PWY-6892	thiazole biosynthesis I (E. coli)	12424.79
PWY-6895	superpathway of thiamin diphosphate biosynthesis II	7762.57
PWY-6897	thiamin salvage II	10561.25
PWY-6901	superpathway of glucose and xylose degradation	12193.17
PWY-6969	TCA cycle V (2-oxoglutarate:ferredoxin oxidoreductase)	10431.41
PWY-7003	glycerol degradation to butanol	5281.77
PWY-7007	methyl ketone biosynthesis	108.03
PWY-7013	L-1,2-propanediol degradation	1583.74
PWY-7031	protein N-glycosylation (bacterial)	78.00
PWY-7090	UDP-2,3-diacetamido-2,3-dideoxy-α-d-mannuronate biosynthesis	166.09
PWY-7094	fatty acid salvage	630.29
PWY-7111	pyruvate fermentation to isobutanol (engineered)	28059.45
PWY-7184	pyrimidine deoxyribonucleotides de novo biosynthesis I	9389.43
PWY-7187	pyrimidine deoxyribonucleotides de novo biosynthesis II	12178.42
PWY-7196	superpathway of pyrimidine ribonucleosides salvage	12223.75
PWY-7197	pyrimidine deoxyribonucleotide phosphorylation	8846.86
PWY-7199	pyrimidine deoxyribonucleosides salvage	17360.84
PWY-7200	superpathway of pyrimidine deoxyribonucleoside salvage	11073.34
PWY-7208	superpathway of pyrimidine nucleobases salvage	26758.43
PWY-7211	superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis	10178.38
PWY-7219	adenosine ribonucleotides de novo biosynthesis	25818.87
PWY-7220	adenosine deoxyribonucleotides de novo biosynthesis II	26486.98
PWY-7221	guanosine ribonucleotides de novo biosynthesis	21894.54
PWY-7222	guanosine deoxyribonucleotides de novo biosynthesis II	26486.98
PWY-7228	superpathway of guanosine nucleotides de novo biosynthesis I	11518.77
PWY-7229	superpathway of adenosine nucleotides de novo biosynthesis I	26569.66
PWY-7234	inosine-5′-phosphate biosynthesis III	15991.99
PWY-7237	myo-, chiro- and scillo-inositol degradation	884.12
PWY-7242	d-fructuronate degradation	18346.49
PWY-7254	TCA cycle VII (acetate-producers)	2793.75
PWY-7315	dTDP-N-acetylthomosamine biosynthesis	2313.21
PWY-7323	superpathway of GDP-mannose-derived O-antigen building blocks biosynthesis	7372.93
PWY-7328	superpathway of UDP-glucose-derived O-antigen building blocks biosynthesis	3481.06
PWY-7332	superpathway of UDP-N-acetylglucosamine-derived O-antigen building blocks biosynthesis	878.25
PWY-7371	1,4-dihydroxy-6-naphthoate biosynthesis II	752.84
PWY-7373	superpathway of demethylmenaquinol-6 biosynthesis II	238.02
PWY-7374	1,4-dihydroxy-6-naphthoate biosynthesis I	565.28
PWY-7376	cob(II)yrinate a,c-diamide biosynthesis II (late cobalt incorporation)	251.98
PWY-7377	cob(II)yrinate a,c-diamide biosynthesis I (early cobalt insertion)	4692.91
PWY-7391	isoprene biosynthesis II (engineered)	163.87
PWY-7392	taxadiene biosynthesis (engineered)	14177.01
PWY-7400	l-arginine biosynthesis IV (archaebacteria)	20669.84
PWY-7431	aromatic biogenic amine degradation (bacteria)	158.82
PWY-7446	Sulfoglycolysis	810.52
PWY-7456	mannan degradation	4216.35
PWY-7539	6-hydroxymethyl-dihydropterin diphosphate biosynthesis III (Chlamydia)	9019.94
PWY-7560	methylerythritol phosphate pathway II	13636.93
PWY-7616	methanol oxidation to carbon dioxide	19.01
PWY-7663	gondoate biosynthesis (anaerobic)	29046.63
PWY-7664	oleate biosynthesis IV (anaerobic)	3792.83
PWY-841	superpathway of purine nucleotides de novo biosynthesis I	15301.06
PWY-922	mevalonate pathway I	9533.84
PWY0-1061	superpathway of l-alanine biosynthesis	19752.51
PWY0-1241	ADP-l-glycero-β-d-manno-heptose biosynthesis	1946.43
PWY0-1261	anhydromuropeptides recycling	25138.01
PWY0-1277	3-phenylpropanoate and 3-(3-hydroxyphenyl) propanoate degradation	297.86
PWY0-1296	purine ribonucleosides degradation	22638.22
PWY0-1297	superpathway of purine deoxyribonucleosides degradation	19837.99
PWY0-1298	superpathway of pyrimidine deoxyribonucleosides degradation	17485.15
PWY0-1319	CDP-diacylglycerol biosynthesis II	26005.47
PWY0-1338	polymyxin resistance	877.07
PWY0-1415	superpathway of heme biosynthesis from uroporphyrinogen-III	1625.52
PWY0-1479	tRNA processing	3544.01
PWY0-1533	methylphosphonate degradation I	1827.30
PWY0-1586	peptidoglycan maturation (meso-diaminopimelate containing)	22208.25
PWY0-162	superpathway of pyrimidine ribonucleotides de novo biosynthesis	14335.48
PWY0-166	superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis (E. coli)	12388.28
PWY0-321	phenylacetate degradation I (aerobic)	153.49
PWY0-41	allantoin degradation IV (anaerobic)	1254.64
PWY0-42	2-methylcitrate cycle I	854.22
PWY0-781	aspartate superpathway	16087.65
PWY0-845	superpathway of pyridoxal 5′-phosphate biosynthesis and salvage	4261.71
PWY0-862	(5Z)-dodec-5-enoate biosynthesis	3307.79
PWY1G-0	mycothiol biosynthesis	78.65
PWY490-3	nitrate reduction VI (assimilatory)	2879.67
PWY4FS-7	phosphatidylglycerol biosynthesis I (plastidic)	23668.60
PWY4FS-8	phosphatidylglycerol biosynthesis II (non-plastidic)	23668.60
PWYG-321	mycolate biosynthesis	3885.08
PYRIDNUCSAL-PWY	NAD salvage pathway I	10319.13
PYRIDNUCSYN-PWY	NAD biosynthesis I (from aspartate)	10733.55
PYRIDOXSYN-PWY	pyridoxal 5′-phosphate biosynthesis I	3068.48
REDCITCYC	TCA cycle VIII (helicobacter)	3326.97
RHAMCAT-PWY	l-rhamnose degradation I	4878.77
RIBOSYN2-PWY	flavin biosynthesis I (bacteria and plants)	12128.84
RUMP-PWY	formaldehyde oxidation I	3382.63
SALVADEHYPOX-PWY	adenosine nucleotides degradation II	6255.03
SER-GLYSYN-PWY	superpathway of l-serine and glycine biosynthesis I	19608.93
SO4ASSIM-PWY	sulfate reduction I (assimilatory)	3873.10
SULFATE-CYS-PWY	superpathway of sulfate assimilation and cysteine biosynthesis	6747.48
TCA	TCA cycle I (prokaryotic)	10030.20
TCA-GLYOX-BYPASS	superpathway of glyoxylate bypass and TCA	2911.71
TEICHOICACID-PWY	teichoic acid (poly-glycerol) biosynthesis	367.18
THISYN-PWY	superpathway of thiamin diphosphate biosynthesis I	8475.45
THREOCAT-PWY	superpathway of l-threonine metabolism	1017.79
THRESYN-PWY	superpathway of l-threonine biosynthesis	21550.89
TRNA-CHARGING-PWY	tRNA charging	21317.76
TRPSYN-PWY	L-tryptophan biosynthesis	20256.04
TYRFUMCAT-PWY	l-tyrosine degradation I	61.66
UBISYN-PWY	superpathway of ubiquinol-8 biosynthesis (prokaryotic)	1545.98
UDPNAGSYN-PWY	UDP-N-acetyl-d-glucosamine biosynthesis I	20060.51
VALSYN-PWY	l-valine biosynthesis	25751.79

Amino Acid and Nucleotide Biosynthesis - There is a decrease in the abundance of pathways for branched amino acid biosynthesis (24307.48), purine nucleotide biosynthesis (16458.53), and arginine biosynthesis (l-arginine biosynthesis II, 21156.13).

Fermentation - Pathways related to fermentation show increased abundance. For example, "homolactic fermentation" has an abundance of 23119.13, and "mixed acid fermentation" has an abundance of 17131.74, both higher than in the Control sample.

Fatty Acid Metabolism - Fatty acid-related pathways show a mix of changes. "Fatty acid β-oxidation I″ has increased to 2488.77, while "fatty acid elongation - saturated" has decreased to 23923.32 compared to the Control sample.

3.6.3. Metabolic pathway analysis in sample T3

As shown in Table 4c, sample T3 demonstrates a marked increase in the abundance of most metabolic pathways compared to both the Control and T2 samples.

Table 4c.

Metabolic pathways and abundance for sample T3.

Pathway Code	Metabolic Pathway	Abundance
1CMET2-PWY	N10-formyl-tetrahydrofolate biosynthesis	30151.85
3-HYDROXYPHENYLACETATE-DEGRADATION-PWY	4-hydroxyphenylacetate degradation	401.19
AEROBACTINSYN-PWY	aerobactin biosynthesis	86.98
ALL-CHORISMATE-PWY	superpathway of chorismate metabolism	5465.23
ANAEROFRUCAT-PWY	homolactic fermentation	43927.37
ANAGLYCOLYSIS-PWY	glycolysis III (from glucose)	44958.84
ARG + POLYAMINE-SYN	superpathway of arginine and polyamine biosynthesis	11669.24
ARGDEG-PWY	superpathway of l-arginine, putrescine, and 4-aminobutanoate degradation	1376.41
ARGORNPROST-PWY	arginine, ornithine and proline interconversion	18450.64
ARGSYN-PWY	l-arginine biosynthesis I (via l-ornithine)	32286.02
ARGSYNBSUB-PWY	l-arginine biosynthesis II (acetyl cycle)	32035.22
ARO-PWY	chorismate biosynthesis I	39446.63
ASPASN-PWY	superpathway of l-aspartate and l-asparagine biosynthesis	31354.54
AST-PWY	l-arginine degradation II (AST pathway)	1182.05
BIOTIN-BIOSYNTHESIS-PWY	biotin biosynthesis I	6716.62
BRANCHED-CHAIN-AA-SYN-PWY	superpathway of branched amino acid biosynthesis	38304.24
CALVIN-PWY	Calvin-Benson-Bassham cycle	45329.13
CATECHOL-ORTHO-CLEAVAGE-PWY	catechol degradation to β-ketoadipate	629.85
CENTFERM-PWY	pyruvate fermentation to butanoate	11623.01
COA-PWY	coenzyme A biosynthesis I	37432.65
COBALSYN-PWY	adenosylcobalamin salvage from cobinamide I	26250.86
CODH-PWY	reductive acetyl coenzyme A pathway	1168.78
COLANSYN-PWY	colanic acid building blocks biosynthesis	8828.73
COMPLETE-ARO-PWY	superpathway of aromatic amino acid biosynthesis	40592.55
CRNFORCAT-PWY	creatinine degradation I	1122.64
DAPLYSINESYN-PWY	l-lysine biosynthesis I	34504.10
DENITRIFICATION-PWY	nitrate reduction I (denitrification)	15.40
DENOVOPURINE2-PWY	superpathway of purine nucleotides de novo biosynthesis II	32433.26
DTDPRHAMSYN-PWY	dTDP-l-rhamnose biosynthesis I	24820.12
ECASYN-PWY	enterobacterial common antigen biosynthesis	1596.27
ENTBACSYN-PWY	enterobactin biosynthesis	6107.50
FAO-PWY	fatty acid β-oxidation I	6247.26
FASYN-ELONG-PWY	fatty acid elongation -- saturated	29457.74
FASYN-INITIAL-PWY	superpathway of fatty acid biosynthesis initiation (E. coli)	4309.58
FERMENTATION-PWY	mixed acid fermentation	17964.81
FOLSYN-PWY	superpathway of tetrahydrofolate biosynthesis and salvage	32359.37
FUC-RHAMCAT-PWY	superpathway of fucose and rhamnose degradation	3710.79
FUCCAT-PWY	fucose degradation	2554.57
GALACT-GLUCUROCAT-PWY	superpathway of hexuronide and hexuronate degradation	6846.46
GALACTARDEG-PWY	d-galactarate degradation I	1958.56
GALACTUROCAT-PWY	d-galacturonate degradation I	8394.58
GALLATE-DEGRADATION-I-PWY	gallate degradation II	79.25
GALLATE-DEGRADATION–II–PWY	gallate degradation I	79.25
GLCMANNANAUT-PWY	superpathway of N-acetylglucosamine, N-acetylmannosamine and N-acetylneuraminate degradation	24578.23
GLUCARDEG-PWY	d-glucarate degradation I	1798.55
GLUCARGALACTSUPER-PWY	superpathway of d-glucarate and d-galactarate degradation	1958.56
GLUCONEO-PWY	gluconeogenesis I	29003.52
GLUCOSE1PMETAB-PWY	glucose and glucose-1-phosphate degradation	2821.46
GLUCUROCAT-PWY	superpathway of β-D-glucuronide and d-glucuronate degradation	10012.73
GLUTORN-PWY	l-ornithine biosynthesis	22747.86
GLYCOCAT-PWY	glycogen degradation I (bacterial)	33293.82
GLYCOGENSYNTH-PWY	glycogen biosynthesis I (from ADP-d-Glucose)	34560.09
GLYCOL-GLYOXDEG-PWY	superpathway of glycol metabolism and degradation	2711.14
GLYCOLYSIS	glycolysis I (from glucose 6-phosphate)	44702.06
GLYCOLYSIS-E-D	superpathway of glycolysis and Entner-Doudoroff	17854.67
GLYCOLYSIS-TCA-GLYOX-BYPASS	superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass	11850.07
GLYOXYLATE-BYPASS	glyoxylate cycle	5103.03
GOLPDLCAT-PWY	superpathway of glycerol degradation to 1,3-propanediol	650.97
HCAMHPDEG-PWY	3-phenylpropanoate and 3-(3-hydroxyphenyl)propanoate degradation to 2-oxopent-4-enoate	312.12
HEME-BIOSYNTHESIS-II	heme biosynthesis I (aerobic)	5778.76
HEMESYN2-PWY	heme biosynthesis II (anaerobic)	14700.44
HEXITOLDEGSUPER-PWY	superpathway of hexitol degradation (bacteria)	19876.05
HISDEG-PWY	l-histidine degradation I	13602.52
HISTSYN-PWY	l-histidine biosynthesis	25575.07
HOMOSER-METSYN-PWY	l-methionine biosynthesis I	20886.09
HSERMETANA-PWY	l-methionine biosynthesis III	22737.59
ILEUSYN-PWY	l-isoleucine biosynthesis I (from threonine)	44435.29
KDO-NAGLIPASYN-PWY	superpathway of (Kdo)2-lipid A biosynthesis	3138.27
KETOGLUCONMET-PWY	ketogluconate metabolism	998.47
LACTOSECAT-PWY	lactose and galactose degradation I	6692.10
LEU-DEG2-PWY	l-leucine degradation I	685.91
LPSSYN-PWY	superpathway of lipopolysaccharide biosynthesis	1227.07
MET-SAM-PWY	superpathway of S-adenosyl-l-methionine biosynthesis	27668.25
METH-ACETATE-PWY	methanogenesis from acetate	23368.48
METHGLYUT-PWY	superpathway of methylglyoxal degradation	1917.48
METHYLGALLATE-DEGRADATION-PWY	methylgallate degradation	99.04
NAD-BIOSYNTHESIS-II	NAD salvage pathway II	2623.14
NADSYN-PWY	NAD biosynthesis II (from tryptophan)	11.91
NAGLIPASYN-PWY	lipid IVA biosynthesis	4948.78
NONMEVIPP-PWY	methylerythritol phosphate pathway I	31324.17
NONOXIPENT-PWY	pentose phosphate pathway (non-oxidative branch)	54174.66
OANTIGEN-PWY	O-antigen building blocks biosynthesis (E. coli)	28330.15
ORNARGDEG-PWY	superpathway of l-arginine and l-ornithine degradation	1376.41
ORNDEG-PWY	superpathway of ornithine degradation	1414.90
P101-PWY	ectoine biosynthesis	1292.87
P105-PWY	TCA cycle IV (2-oxoglutarate decarboxylase)	9867.96
P108-PWY	pyruvate fermentation to propanoate I	11483.90
P122-PWY	heterolactic fermentation	11180.37
P124-PWY	Bifidobacterium shunt	15224.18
P125-PWY	superpathway of (R,R)-butanediol biosynthesis	4225.12
P161-PWY	acetylene degradation	42334.86
P162-PWY	l-glutamate degradation V (via hydroxyglutarate)	3029.63
P163-PWY	l-lysine fermentation to acetate and butanoate	1278.80
P164-PWY	purine nucleobases degradation I (anaerobic)	20739.07
P184-PWY	protocatechuate degradation I (meta-cleavage pathway)	60.28
P221-PWY	octane oxidation	650.79
P23-PWY	reductive TCA cycle I	14680.39
P281-PWY	3-phenylpropanoate degradation	53.75
P381-PWY	adenosylcobalamin biosynthesis II (late cobalt incorporation)	610.41
P4-PWY	superpathway of l-lysine, l-threonine and l-methionine biosynthesis I	29206.90
P42-PWY	incomplete reductive TCA cycle	25074.20
P441-PWY	superpathway of N-acetylneuraminate degradation	26882.45
P461-PWY	hexitol fermentation to lactate, formate, ethanol and acetate	13708.77
P562-PWY	myo-inositol degradation I	775.18
PANTO-PWY	phosphopantothenate biosynthesis I	25174.36
PANTOSYN-PWY	pantothenate and coenzyme A biosynthesis I	25503.78
PENTOSE-P-PWY	pentose phosphate pathway	19624.45
PEPTIDOGLYCANSYN-PWY	peptidoglycan biosynthesis I (meso-diaminopimelate containing)	39611.66
PHOSLIPSYN-PWY	superpathway of phospholipid biosynthesis I (bacteria)	46962.00
POLYAMINSYN3-PWY	superpathway of polyamine biosynthesis II	1968.68
POLYAMSYN-PWY	superpathway of polyamine biosynthesis I	7871.10
POLYISOPRENSYN-PWY	polyisoprenoid biosynthesis (E. coli)	25064.09
PPGPPMET-PWY	ppGpp biosynthesis	3345.18
PROTOCATECHUATE-ORTHO-CLEAVAGE-PWY	protocatechuate degradation II (ortho-cleavage pathway)	2278.62
PRPP-PWY	superpathway of histidine, purine, and pyrimidine biosynthesis	23094.44
PWY-1269	CMP-3-deoxy-d-manno-octulosonate biosynthesis I	5048.07
PWY-1541	superpathway of taurine degradation	537.11
PWY-181	Photorespiration	2781.92
PWY-1861	formaldehyde assimilation II (RuMP Cycle)	10205.91
PWY-2941	l-lysine biosynthesis II	23894.05
PWY-2942	l-lysine biosynthesis III	42170.67
PWY-3001	superpathway of l-isoleucine biosynthesis I	37489.23
PWY-3661	glycine betaine degradation I	1005.62
PWY-3781	aerobic respiration I (cytochrome c)	14898.78
PWY-4361	S-methyl-5-thio-α-d-ribose 1-phosphate degradation	67.69
PWY-4722	creatinine degradation II	1169.85
PWY-4984	urea cycle	17815.72
PWY-5005	biotin biosynthesis II	17380.09
PWY-5022	4-aminobutanoate degradation V	8323.20
PWY-5028	l-histidine degradation II	77.24
PWY-5088	l-glutamate degradation VIII (to propanoate)	744.37
PWY-5097	l-lysine biosynthesis VI	40281.68
PWY-5100	pyruvate fermentation to acetate and lactate II	46621.11
PWY-5101	l-isoleucine biosynthesis II	45557.19
PWY-5103	l-isoleucine biosynthesis III	34874.24
PWY-5104	l-isoleucine biosynthesis IV	45377.38
PWY-5121	superpathway of geranylgeranyl diphosphate biosynthesis II (via MEP)	32531.69
PWY-5154	l-arginine biosynthesis III (via N-acetyl-l-citrulline)	7231.80
PWY-5177	glutaryl-CoA degradation	3147.20
PWY-5178	toluene degradation IV (aerobic) (via catechol)	613.20
PWY-5180	toluene degradation I (aerobic) (via o-cresol)	1590.90
PWY-5181	toluene degradation III (aerobic) (via p-cresol)	423.35
PWY-5182	toluene degradation II (aerobic) (via 4-methylcatechol)	1590.90
PWY-5183	superpathway of aerobic toluene degradation	263.69
PWY-5188	tetrapyrrole biosynthesis I (from glutamate)	24076.80
PWY-5189	tetrapyrrole biosynthesis II (from glycine)	24172.93
PWY-5265	peptidoglycan biosynthesis II (staphylococci)	12268.84
PWY-5304	superpathway of sulfur oxidation (Acidianus ambivalens)	2445.12
PWY-5345	superpathway of l-methionine biosynthesis (by sulfhydrylation)	9153.20
PWY-5347	superpathway of l-methionine biosynthesis (transsulfuration)	29948.07
PWY-5384	sucrose degradation IV (sucrose phosphorylase)	14016.59
PWY-5415	catechol degradation I (meta-cleavage pathway)	1074.74
PWY-5417	catechol degradation III (ortho-cleavage pathway)	668.55
PWY-5419	catechol degradation to 2-oxopent-4-enoate II	542.19
PWY-5420	catechol degradation II (meta-cleavage pathway)	681.71
PWY-5430	meta cleavage pathway of aromatic compounds	181.70
PWY-5431	aromatic compounds degradation via β-ketoadipate	668.55
PWY-5484	glycolysis II (from fructose 6-phosphate)	43107.92
PWY-5505	l-glutamate and l-glutamine biosynthesis	20204.31
PWY-5507	adenosylcobalamin biosynthesis I (early cobalt insertion)	2968.48
PWY-5509	adenosylcobalamin biosynthesis from cobyrinate a,c-diamide I	25890.83
PWY-5532	adenosine nucleotides degradation IV	67.66
PWY-5647	2-nitrobenzoate degradation I	5.15
PWY-5651	L-tryptophan degradation to 2-amino-3-carboxymuconate semialdehyde	7.45
PWY-5654	2-amino-3-carboxymuconate semialdehyde degradation to 2-oxopentenoate	7.23
PWY-5655	L-tryptophan degradation IX	9.15
PWY-5659	GDP-mannose biosynthesis	24655.36
PWY-5667	CDP-diacylglycerol biosynthesis I	48282.90
PWY-5676	acetyl-CoA fermentation to butanoate II	8784.86
PWY-5677	succinate fermentation to butanoate	2230.52
PWY-5686	UMP biosynthesis	41838.93
PWY-5695	urate biosynthesis/inosine 5′-phosphate degradation	33167.16
PWY-5705	allantoin degradation to glyoxylate III	2961.04
PWY-5741	ethylmalonyl-CoA pathway	11.69
PWY-5747	2-methylcitrate cycle II	1652.97
PWY-5837	1,4-dihydroxy-2-naphthoate biosynthesis I	9537.20
PWY-5838	superpathway of menaquinol-8 biosynthesis I	11680.93
PWY-5840	superpathway of menaquinol-7 biosynthesis	13924.65
PWY-5845	superpathway of menaquinol-9 biosynthesis	6304.54
PWY-5850	superpathway of menaquinol-6 biosynthesis I	6304.54
PWY-5855	ubiquinol-7 biosynthesis (prokaryotic)	2441.00
PWY-5856	ubiquinol-9 biosynthesis (prokaryotic)	2441.00
PWY-5857	ubiquinol-10 biosynthesis (prokaryotic)	2441.00
PWY-5860	superpathway of demethylmenaquinol-6 biosynthesis I	4696.99
PWY-5861	superpathway of demethylmenaquinol-8 biosynthesis	9671.82
PWY-5862	superpathway of demethylmenaquinol-9 biosynthesis	4696.99
PWY-5863	superpathway of phylloquinol biosynthesis	10509.10
PWY-5896	superpathway of menaquinol-10 biosynthesis	6304.54
PWY-5897	superpathway of menaquinol-11 biosynthesis	13254.40
PWY-5898	superpathway of menaquinol-12 biosynthesis	13254.40
PWY-5899	superpathway of menaquinol-13 biosynthesis	13254.40
PWY-5910	superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate)	13403.29
PWY-5913	TCA cycle VI (obligate autotrophs)	12821.83
PWY-5918	superpathay of heme biosynthesis from glutamate	10576.44
PWY-5920	superpathway of heme biosynthesis from glycine	3731.25
PWY-5971	palmitate biosynthesis II (bacteria and plants)	7715.71
PWY-5973	cis-vaccenate biosynthesis	44814.68
PWY-5989	stearate biosynthesis II (bacteria and plants)	4931.62
PWY-6071	superpathway of phenylethylamine degradation	503.08
PWY-6107	chlorosalicylate degradation	136.28
PWY-6121	5-aminoimidazole ribonucleotide biosynthesis I	41468.12
PWY-6122	5-aminoimidazole ribonucleotide biosynthesis II	42134.65
PWY-6123	inosine-5′-phosphate biosynthesis I	37606.31
PWY-6125	superpathway of guanosine nucleotides de novo biosynthesis II	28256.53
PWY-6126	superpathway of adenosine nucleotides de novo biosynthesis II	45865.23
PWY-6147	6-hydroxymethyl-dihydropterin diphosphate biosynthesis I	26927.81
PWY-6151	S-adenosyl-l-methionine cycle I	30457.60
PWY-6163	chorismate biosynthesis from 3-dehydroquinate	36900.93
PWY-6182	superpathway of salicylate degradation	315.19
PWY-6185	4-methylcatechol degradation (ortho cleavage)	271.30
PWY-621	sucrose degradation III (sucrose invertase)	35973.12
PWY-6263	superpathway of menaquinol-8 biosynthesis II	2080.78
PWY-6269	adenosylcobalamin salvage from cobinamide II	25976.94
PWY-6277	superpathway of 5-aminoimidazole ribonucleotide biosynthesis	42134.65
PWY-6282	palmitoleate biosynthesis I (from (5Z)-dodec-5-enoate)	5088.14
PWY-6317	galactose degradation I (Leloir pathway)	35356.69
PWY-6338	superpathway of vanillin and vanillate degradation	53.91
PWY-6339	syringate degradation	153.03
PWY-6353	purine nucleotides degradation II (aerobic)	16653.73
PWY-6383	mono-trans, poly-cis decaprenyl phosphate biosynthesis	1988.86
PWY-6385	peptidoglycan biosynthesis III (mycobacteria)	39572.32
PWY-6386	UDP-N-acetylmuramoyl-pentapeptide biosynthesis II (lysine-containing)	40320.67
PWY-6387	UDP-N-acetylmuramoyl-pentapeptide biosynthesis I (meso-diaminopimelate containing)	40239.50
PWY-6396	superpathway of 2,3-butanediol biosynthesis	2745.62
PWY-6397	mycolyl-arabinogalactan-peptidoglycan complex biosynthesis	323.67
PWY-6467	Kdo transfer to lipid IVA III (Chlamydia)	4349.78
PWY-6470	peptidoglycan biosynthesis V (β-lactam resistance)	20327.78
PWY-6471	peptidoglycan biosynthesis IV (Enterococcus faecium)	26383.03
PWY-6478	GDP-d-glycero-α-d-manno-heptose biosynthesis	628.33
PWY-6507	4-deoxy-L-threo-hex-4-enopyranuronate degradation	13354.66
PWY-6519	8-amino-7-oxononanoate biosynthesis I	5682.34
PWY-6545	pyrimidine deoxyribonucleotides de novo biosynthesis III	20989.82
PWY-6562	norspermidine biosynthesis	1962.76
PWY-6572	chondroitin sulfate degradation I (bacterial)	19.09
PWY-6588	pyruvate fermentation to acetone	15011.35
PWY-6590	superpathway of Clostridium acetobutylicum acidogenic fermentation	13798.97
PWY-6608	guanosine nucleotides degradation III	19129.46
PWY-6609	adenine and adenosine salvage III	61475.84
PWY-6612	superpathway of tetrahydrofolate biosynthesis	29814.16
PWY-6628	superpathway of l-phenylalanine biosynthesis	31244.44
PWY-6629	superpathway of l-tryptophan biosynthesis	7624.48
PWY-6630	superpathway of l-tyrosine biosynthesis	30824.01
PWY-6641	superpathway of sulfolactate degradation	254.12
PWY-6690	cinnamate and 3-hydroxycinnamate degradation to 2-oxopent-4-enoate	312.12
PWY-6700	queuosine biosynthesis	12008.76
PWY-6703	preQ0 biosynthesis	8618.61
PWY-6708	ubiquinol-8 biosynthesis (prokaryotic)	2441.00
PWY-6728	methylaspartate cycle	103.22
PWY-6731	starch degradation III	70.52
PWY-6737	starch degradation V	30537.42
PWY-6749	CMP-legionaminate biosynthesis I	3519.75
PWY-6876	isopropanol biosynthesis	199.69
PWY-6891	thiazole biosynthesis II (Bacillus)	18833.16
PWY-6892	thiazole biosynthesis I (E. coli)	27099.62
PWY-6895	superpathway of thiamin diphosphate biosynthesis II	10911.65
PWY-6897	thiamin salvage II	29534.78
PWY-6901	superpathway of glucose and xylose degradation	19158.85
PWY-6906	chitin derivatives degradation	185.77
PWY-6969	TCA cycle V (2-oxoglutarate:ferredoxin oxidoreductase)	13974.61
PWY-6992	1,5-anhydrofructose degradation	62.31
PWY-7003	glycerol degradation to butanol	21477.70
PWY-7007	methyl ketone biosynthesis	1526.61
PWY-7013	L-1,2-propanediol degradation	2757.70
PWY-7031	protein N-glycosylation (bacterial)	60.00
PWY-7090	UDP-2,3-diacetamido-2,3-dideoxy-α-d-mannuronate biosynthesis	140.39
PWY-7094	fatty acid salvage	5661.70
PWY-7097	vanillin and vanillate degradation I	53.91
PWY-7098	vanillin and vanillate degradation II	60.33
PWY-7111	pyruvate fermentation to isobutanol (engineered)	49210.77
PWY-7184	pyrimidine deoxyribonucleotides de novo biosynthesis I	22450.02
PWY-7187	pyrimidine deoxyribonucleotides de novo biosynthesis II	25924.94
PWY-7196	superpathway of pyrimidine ribonucleosides salvage	27462.32
PWY-7197	pyrimidine deoxyribonucleotide phosphorylation	21046.63
PWY-7199	pyrimidine deoxyribonucleosides salvage	31363.15
PWY-7200	superpathway of pyrimidine deoxyribonucleoside salvage	24381.01
PWY-7208	superpathway of pyrimidine nucleobases salvage	39658.99
PWY-7211	superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis	16882.53
PWY-7219	adenosine ribonucleotides de novo biosynthesis	44748.14
PWY-7220	adenosine deoxyribonucleotides de novo biosynthesis II	52503.51
PWY-7221	guanosine ribonucleotides de novo biosynthesis	39077.96
PWY-7222	guanosine deoxyribonucleotides de novo biosynthesis II	52503.51
PWY-7228	superpathway of guanosine nucleotides de novo biosynthesis I	26584.99
PWY-7229	superpathway of adenosine nucleotides de novo biosynthesis I	47967.76
PWY-7234	inosine-5′-phosphate biosynthesis III	23049.16
PWY-7237	myo-, chiro- and scillo-inositol degradation	1587.27
PWY-7242	d-fructuronate degradation	25262.37
PWY-7254	TCA cycle VII (acetate-producers)	11400.70
PWY-7255	ergothioneine biosynthesis I (bacteria)	47.52
PWY-7295	l-arabinose degradation IV	57.68
PWY-7315	dTDP-N-acetylthomosamine biosynthesis	2526.80
PWY-7323	superpathway of GDP-mannose-derived O-antigen building blocks biosynthesis	7593.23
PWY-7328	superpathway of UDP-glucose-derived O-antigen building blocks biosynthesis	3585.43
PWY-7332	superpathway of UDP-N-acetylglucosamine-derived O-antigen building blocks biosynthesis	415.81
PWY-7347	sucrose biosynthesis III	169.18
PWY-7371	1,4-dihydroxy-6-naphthoate biosynthesis II	811.98
PWY-7373	superpathway of demethylmenaquinol-6 biosynthesis II	273.53
PWY-7374	1,4-dihydroxy-6-naphthoate biosynthesis I	650.14
PWY-7376	cob(II)yrinate a,c-diamide biosynthesis II (late cobalt incorporation)	381.47
PWY-7377	cob(II)yrinate a,c-diamide biosynthesis I (early cobalt insertion)	18580.09
PWY-7391	isoprene biosynthesis II (engineered)	1113.79
PWY-7392	taxadiene biosynthesis (engineered)	25016.85
PWY-7400	l-arginine biosynthesis IV (archaebacteria)	32084.78
PWY-7431	aromatic biogenic amine degradation (bacteria)	2727.18
PWY-7446	Sulfoglycolysis	869.82
PWY-7456	mannan degradation	3549.00
PWY-7527	l-methionine salvage cycle III	111.89
PWY-7539	6-hydroxymethyl-dihydropterin diphosphate biosynthesis III (Chlamydia)	27063.54
PWY-7560	methylerythritol phosphate pathway II	31324.17
PWY-7616	methanol oxidation to carbon dioxide	1419.60
PWY-7663	gondoate biosynthesis (anaerobic)	47597.85
PWY-7664	oleate biosynthesis IV (anaerobic)	5830.86
PWY-841	superpathway of purine nucleotides de novo biosynthesis I	31831.46
PWY-922	mevalonate pathway I	10501.78
PWY0-1061	superpathway of l-alanine biosynthesis	23031.48
PWY0-1241	ADP-l-glycero-β-d-manno-heptose biosynthesis	2151.50
PWY0-1261	anhydromuropeptides recycling	32456.57
PWY0-1277	3-phenylpropanoate and 3-(3-hydroxyphenyl)propanoate degradation	656.32
PWY0-1296	purine ribonucleosides degradation	48163.62
PWY0-1297	superpathway of purine deoxyribonucleosides degradation	40114.00
PWY0-1298	superpathway of pyrimidine deoxyribonucleosides degradation	35290.13
PWY0-1319	CDP-diacylglycerol biosynthesis II	48282.90
PWY0-1338	polymyxin resistance	920.74
PWY0-1415	superpathway of heme biosynthesis from uroporphyrinogen-III	3214.29
PWY0-1479	tRNA processing	6827.77
PWY0-1533	methylphosphonate degradation I	3867.66
PWY0-1586	peptidoglycan maturation (meso-diaminopimelate containing)	31034.60
PWY0-162	superpathway of pyrimidine ribonucleotides de novo biosynthesis	29839.65
PWY0-166	superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis (E. coli)	26542.75
PWY0-321	phenylacetate degradation I (aerobic)	482.84
PWY0-41	allantoin degradation IV (anaerobic)	3519.94
PWY0-42	2-methylcitrate cycle I	1766.09
PWY0-781	aspartate superpathway	28350.35
PWY0-845	superpathway of pyridoxal 5′-phosphate biosynthesis and salvage	6286.83
PWY0-862	(5Z)-dodec-5-enoate biosynthesis	5073.34
PWY1G-0	mycothiol biosynthesis	1162.73
PWY490-3	nitrate reduction VI (assimilatory)	21197.35
PWY4FS-7	phosphatidylglycerol biosynthesis I (plastidic)	45172.28
PWY4FS-8	phosphatidylglycerol biosynthesis II (non-plastidic)	45172.28
PWYG-321	mycolate biosynthesis	5999.83
PYRIDNUCSAL-PWY	NAD salvage pathway I	20512.56
PYRIDNUCSYN-PWY	NAD biosynthesis I (from aspartate)	16199.04
PYRIDOXSYN-PWY	pyridoxal 5′-phosphate biosynthesis I	4146.63
REDCITCYC	TCA cycle VIII (helicobacter)	14188.95
RHAMCAT-PWY	l-rhamnose degradation I	5976.75
RIBOSYN2-PWY	flavin biosynthesis I (bacteria and plants)	29976.79
RUMP-PWY	formaldehyde oxidation I	6353.12
SALVADEHYPOX-PWY	adenosine nucleotides degradation II	11140.15
SER-GLYSYN-PWY	superpathway of l-serine and glycine biosynthesis I	21789.89
SO4ASSIM-PWY	sulfate reduction I (assimilatory)	5365.44
SUCSYN-PWY	sucrose biosynthesis I (from photosynthesis)	543.83
SULFATE-CYS-PWY	superpathway of sulfate assimilation and cysteine biosynthesis	9281.50
TCA	TCA cycle I (prokaryotic)	13859.20
TCA-GLYOX-BYPASS	superpathway of glyoxylate bypass and TCA	7692.86
TEICHOICACID-PWY	teichoic acid (poly-glycerol) biosynthesis	4946.83
THISYN-PWY	superpathway of thiamin diphosphate biosynthesis I	20817.59
THREOCAT-PWY	superpathway of l-threonine metabolism	2550.19
THRESYN-PWY	superpathway of l-threonine biosynthesis	37787.20
TRNA-CHARGING-PWY	tRNA charging	39013.67
TRPSYN-PWY	L-tryptophan biosynthesis	23018.58
TYRFUMCAT-PWY	l-tyrosine degradation I	434.21
UBISYN-PWY	superpathway of ubiquinol-8 biosynthesis (prokaryotic)	2370.74
UDPNAGSYN-PWY	UDP-N-acetyl-d-glucosamine biosynthesis I	35574.83
VALSYN-PWY	l-valine biosynthesis	44435.29

Central Metabolism - Core metabolic pathways are highly active. "Glycolysis III" is at 44958.84, "homolactic fermentation" is at 43927.37, and "TCA cycle I″ is at 44833.64.

Amino Acid and Nucleotide Biosynthesis - The abundance values for amino acid synthesis pathways have also risen significantly. "Superpathway of branched amino acid biosynthesis" is 38304.24, and "superpathway of purine nucleotides de novo biosynthesis II" is 32433.26.

Glycogen Metabolism - Both "glycogen degradation I" (33293.82) and "glycogen biosynthesis I" (34560.09) are elevated, suggesting a high rate of glycogen turnover.

3.6.4. Metabolic pathway analysis in sample T5

Similar to the T3 sample, T5 shows a general increase in metabolic activity across many pathways compared to the Control and T2 samples. Central Metabolism: Key pathways such as "glycolysis III" (45732.36) and "TCA cycle I" (37654.51) are very highly abundant (Table 4d). The pathways are:

Table 4d.

Metabolic pathways and abundance for sample T5.

Pathway Code	Metabolic Pathway	Abundance
1CMET2-PWY	N10-formyl-tetrahydrofolate biosynthesis	33264.75
3-HYDROXYPHENYLACETATE-DEGRADATION-PWY	4-hydroxyphenylacetate degradation	381.73
AEROBACTINSYN-PWY	aerobactin biosynthesis	169.39
ALL-CHORISMATE-PWY	superpathway of chorismate metabolism	7841.88
ANAEROFRUCAT-PWY	homolactic fermentation	37781.70
ANAGLYCOLYSIS-PWY	glycolysis III (from glucose)	45732.36
ARG + POLYAMINE-SYN	superpathway of arginine and polyamine biosynthesis	15548.56
ARGDEG-PWY	superpathway of l-arginine, putrescine, and 4-aminobutanoate degradation	1698.53
ARGORNPROST-PWY	arginine, ornithine and proline interconversion	6368.22
ARGSYN-PWY	l-arginine biosynthesis I (via l-ornithine)	37059.67
ARGSYNBSUB-PWY	l-arginine biosynthesis II (acetyl cycle)	37723.01
ARO-PWY	chorismate biosynthesis I	41907.03
ASPASN-PWY	superpathway of l-aspartate and l-asparagine biosynthesis	30587.87
AST-PWY	l-arginine degradation II (AST pathway)	1631.48
BIOTIN-BIOSYNTHESIS-PWY	biotin biosynthesis I	13558.25
BRANCHED-CHAIN-AA-SYN-PWY	superpathway of branched amino acid biosynthesis	44965.47
CALVIN-PWY	Calvin-Benson-Bassham cycle	44992.54
CATECHOL-ORTHO-CLEAVAGE-PWY	catechol degradation to β-ketoadipate	480.09
CENTFERM-PWY	pyruvate fermentation to butanoate	8759.04
COA-PWY	coenzyme A biosynthesis I	39311.87
COBALSYN-PWY	adenosylcobalamin salvage from cobinamide I	26810.89
CODH-PWY	reductive acetyl coenzyme A pathway	2532.54
COLANSYN-PWY	colanic acid building blocks biosynthesis	19574.14
COMPLETE-ARO-PWY	superpathway of aromatic amino acid biosynthesis	43582.57
CRNFORCAT-PWY	creatinine degradation I	81.68
DAPLYSINESYN-PWY	l-lysine biosynthesis I	32001.09
DENITRIFICATION-PWY	nitrate reduction I (denitrification)	5.90
DENOVOPURINE2-PWY	superpathway of purine nucleotides de novo biosynthesis II	32586.67
DTDPRHAMSYN-PWY	dTDP-l-rhamnose biosynthesis I	34390.76
ECASYN-PWY	enterobacterial common antigen biosynthesis	2471.23
ENTBACSYN-PWY	enterobactin biosynthesis	6407.67
FAO-PWY	fatty acid β-oxidation I	5748.25
FASYN-ELONG-PWY	fatty acid elongation -- saturated	38627.04
FASYN-INITIAL-PWY	superpathway of fatty acid biosynthesis initiation (E. coli)	8990.26
FERMENTATION-PWY	mixed acid fermentation	26652.99
FOLSYN-PWY	superpathway of tetrahydrofolate biosynthesis and salvage	29268.06
FUC-RHAMCAT-PWY	superpathway of fucose and rhamnose degradation	7517.05
FUCCAT-PWY	fucose degradation	6015.45
GALACT-GLUCUROCAT-PWY	superpathway of hexuronide and hexuronate degradation	11459.63
GALACTARDEG-PWY	d-galactarate degradation I	3066.12
GALACTUROCAT-PWY	d-galacturonate degradation I	16690.04
GALLATE-DEGRADATION-I-PWY	gallate degradation II	58.72
GALLATE-DEGRADATION–II–PWY	gallate degradation I	58.72
GLCMANNANAUT-PWY	superpathway of N-acetylglucosamine, N-acetylmannosamine and N-acetylneuraminate degradation	22042.73
GLUCARDEG-PWY	d-glucarate degradation I	2366.46
GLUCARGALACTSUPER-PWY	superpathway of d-glucarate and d-galactarate degradation	3066.12
GLUCONEO-PWY	gluconeogenesis I	36594.40
GLUCOSE1PMETAB-PWY	glucose and glucose-1-phosphate degradation	5709.61
GLUCUROCAT-PWY	superpathway of β-D-glucuronide and d-glucuronate degradation	15000.81
GLUTORN-PWY	l-ornithine biosynthesis	33691.51
GLYCOCAT-PWY	glycogen degradation I (bacterial)	40704.47
GLYCOGENSYNTH-PWY	glycogen biosynthesis I (from ADP-d-Glucose)	37495.58
GLYCOL-GLYOXDEG-PWY	superpathway of glycol metabolism and degradation	2965.26
GLYCOLYSIS	glycolysis I (from glucose 6-phosphate)	40769.96
GLYCOLYSIS-E-D	superpathway of glycolysis and Entner-Doudoroff	16418.51
GLYCOLYSIS-TCA-GLYOX-BYPASS	superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass	9382.97
GLYOXYLATE-BYPASS	glyoxylate cycle	5661.99
GOLPDLCAT-PWY	superpathway of glycerol degradation to 1,3-propanediol	199.18
HCAMHPDEG-PWY	3-phenylpropanoate and 3-(3-hydroxyphenyl)propanoate degradation to 2-oxopent-4-enoate	469.91
HEME-BIOSYNTHESIS-II	heme biosynthesis I (aerobic)	3192.02
HEMESYN2-PWY	heme biosynthesis II (anaerobic)	14982.88
HEXITOLDEGSUPER-PWY	superpathway of hexitol degradation (bacteria)	13520.98
HISDEG-PWY	l-histidine degradation I	4969.58
HISTSYN-PWY	l-histidine biosynthesis	34184.12
HOMOSER-METSYN-PWY	l-methionine biosynthesis I	22666.08
HSERMETANA-PWY	l-methionine biosynthesis III	25126.08
ILEUSYN-PWY	l-isoleucine biosynthesis I (from threonine)	47290.87
KDO-NAGLIPASYN-PWY	superpathway of (Kdo)2-lipid A biosynthesis	6850.91
KETOGLUCONMET-PWY	ketogluconate metabolism	886.46
LACTOSECAT-PWY	lactose and galactose degradation I	3622.24
LEU-DEG2-PWY	l-leucine degradation I	674.93
LPSSYN-PWY	superpathway of lipopolysaccharide biosynthesis	4430.81
MET-SAM-PWY	superpathway of S-adenosyl-l-methionine biosynthesis	28958.49
METH-ACETATE-PWY	methanogenesis from acetate	5792.67
METHGLYUT-PWY	superpathway of methylglyoxal degradation	2233.75
METHYLGALLATE-DEGRADATION-PWY	methylgallate degradation	73.39
NAD-BIOSYNTHESIS-II	NAD salvage pathway II	4005.49
NAGLIPASYN-PWY	lipid IVA biosynthesis	14004.69
NONMEVIPP-PWY	methylerythritol phosphate pathway I	34163.94
NONOXIPENT-PWY	pentose phosphate pathway (non-oxidative branch)	54544.14
OANTIGEN-PWY	O-antigen building blocks biosynthesis (E. coli)	32310.75
ORNARGDEG-PWY	superpathway of l-arginine and l-ornithine degradation	1698.53
ORNDEG-PWY	superpathway of ornithine degradation	2324.01
P101-PWY	ectoine biosynthesis	118.53
P105-PWY	TCA cycle IV (2-oxoglutarate decarboxylase)	12988.06
P108-PWY	pyruvate fermentation to propanoate I	17166.93
P122-PWY	heterolactic fermentation	10906.43
P124-PWY	Bifidobacterium shunt	13867.34
P125-PWY	superpathway of (R,R)-butanediol biosynthesis	1915.70
P161-PWY	acetylene degradation	32124.69
P162-PWY	l-glutamate degradation V (via hydroxyglutarate)	3043.78
P163-PWY	l-lysine fermentation to acetate and butanoate	1432.17
P164-PWY	purine nucleobases degradation I (anaerobic)	11628.37
P184-PWY	protocatechuate degradation I (meta-cleavage pathway)	50.36
P221-PWY	octane oxidation	390.77
P23-PWY	reductive TCA cycle I	15643.13
P261-PWY	coenzyme M biosynthesis I	71.32
P381-PWY	adenosylcobalamin biosynthesis II (late cobalt incorporation)	104.27
P4-PWY	superpathway of l-lysine, l-threonine and l-methionine biosynthesis I	29385.84
P42-PWY	incomplete reductive TCA cycle	31405.92
P441-PWY	superpathway of N-acetylneuraminate degradation	22652.57
P461-PWY	hexitol fermentation to lactate, formate, ethanol and acetate	11123.20
P562-PWY	myo-inositol degradation I	375.74
P621-PWY	nylon-6 oligomer degradation	22.09
PANTO-PWY	phosphopantothenate biosynthesis I	23807.36
PANTOSYN-PWY	pantothenate and coenzyme A biosynthesis I	26351.82
PENTOSE-P-PWY	pentose phosphate pathway	21610.45
PEPTIDOGLYCANSYN-PWY	peptidoglycan biosynthesis I (meso-diaminopimelate containing)	40995.83
PHOSLIPSYN-PWY	superpathway of phospholipid biosynthesis I (bacteria)	43757.29
POLYAMINSYN3-PWY	superpathway of polyamine biosynthesis II	3423.49
POLYAMSYN-PWY	superpathway of polyamine biosynthesis I	10228.28
POLYISOPRENSYN-PWY	polyisoprenoid biosynthesis (E. coli)	23687.49
PPGPPMET-PWY	ppGpp biosynthesis	4561.83
PROTOCATECHUATE-ORTHO-CLEAVAGE-PWY	protocatechuate degradation II (ortho-cleavage pathway)	951.14
PRPP-PWY	superpathway of histidine, purine, and pyrimidine biosynthesis	27610.45
PWY-1269	CMP-3-deoxy-d-manno-octulosonate biosynthesis I	14473.85
PWY-1361	benzoyl-CoA degradation I (aerobic)	48.18
PWY-1541	superpathway of taurine degradation	31.63
PWY-181	Photorespiration	941.71
PWY-1861	formaldehyde assimilation II (RuMP Cycle)	6894.87
PWY-2941	l-lysine biosynthesis II	7595.11
PWY-2942	l-lysine biosynthesis III	43661.45
PWY-3001	superpathway of l-isoleucine biosynthesis I	43725.81
PWY-3661	glycine betaine degradation I	88.69
PWY-3781	aerobic respiration I (cytochrome c)	6008.27
PWY-4361	S-methyl-5-thio-α-d-ribose 1-phosphate degradation	54.83
PWY-4984	urea cycle	5353.24
PWY-5005	biotin biosynthesis II	7137.79
PWY-5022	4-aminobutanoate degradation V	4661.18
PWY-5028	l-histidine degradation II	790.82
PWY-5088	l-glutamate degradation VIII (to propanoate)	594.15
PWY-5097	l-lysine biosynthesis VI	43138.50
PWY-5100	pyruvate fermentation to acetate and lactate II	43057.46
PWY-5101	l-isoleucine biosynthesis II	51165.08
PWY-5103	l-isoleucine biosynthesis III	43760.53
PWY-5104	l-isoleucine biosynthesis IV	48324.71
PWY-5121	superpathway of geranylgeranyl diphosphate biosynthesis II (via MEP)	34983.93
PWY-5154	l-arginine biosynthesis III (via N-acetyl-l-citrulline)	15643.67
PWY-5177	glutaryl-CoA degradation	5831.79
PWY-5178	toluene degradation IV (aerobic) (via catechol)	98.40
PWY-5180	toluene degradation I (aerobic) (via o-cresol)	2683.43
PWY-5181	toluene degradation III (aerobic) (via p-cresol)	511.78
PWY-5182	toluene degradation II (aerobic) (via 4-methylcatechol)	2683.43
PWY-5188	tetrapyrrole biosynthesis I (from glutamate)	17583.97
PWY-5189	tetrapyrrole biosynthesis II (from glycine)	16216.44
PWY-5265	peptidoglycan biosynthesis II (staphylococci)	3018.13
PWY-5304	superpathway of sulfur oxidation (Acidianus ambivalens)	2848.00
PWY-5345	superpathway of l-methionine biosynthesis (by sulfhydrylation)	14136.88
PWY-5347	superpathway of l-methionine biosynthesis (transsulfuration)	31672.95
PWY-5384	sucrose degradation IV (sucrose phosphorylase)	17047.50
PWY-5415	catechol degradation I (meta-cleavage pathway)	1157.33
PWY-5417	catechol degradation III (ortho-cleavage pathway)	458.99
PWY-5419	catechol degradation to 2-oxopent-4-enoate II	228.75
PWY-5420	catechol degradation II (meta-cleavage pathway)	351.30
PWY-5431	aromatic compounds degradation via β-ketoadipate	458.99
PWY-5484	glycolysis II (from fructose 6-phosphate)	35688.90
PWY-5505	l-glutamate and l-glutamine biosynthesis	21793.06
PWY-5507	adenosylcobalamin biosynthesis I (early cobalt insertion)	1968.98
PWY-5509	adenosylcobalamin biosynthesis from cobyrinate a,c-diamide I	26254.78
PWY-5532	adenosine nucleotides degradation IV	191.47
PWY-5659	GDP-mannose biosynthesis	30979.29
PWY-5667	CDP-diacylglycerol biosynthesis I	48053.42
PWY-5676	acetyl-CoA fermentation to butanoate II	11613.07
PWY-5677	succinate fermentation to butanoate	2982.40
PWY-5686	UMP biosynthesis	45582.01
PWY-5695	urate biosynthesis/inosine 5′-phosphate degradation	33014.28
PWY-5705	allantoin degradation to glyoxylate III	2145.41
PWY-5741	ethylmalonyl-CoA pathway	7.88
PWY-5747	2-methylcitrate cycle II	1841.35
PWY-5837	1,4-dihydroxy-2-naphthoate biosynthesis I	12598.65
PWY-5838	superpathway of menaquinol-8 biosynthesis I	14827.31
PWY-5840	superpathway of menaquinol-7 biosynthesis	15512.59
PWY-5845	superpathway of menaquinol-9 biosynthesis	8840.34
PWY-5850	superpathway of menaquinol-6 biosynthesis I	8840.34
PWY-5855	ubiquinol-7 biosynthesis (prokaryotic)	3618.03
PWY-5856	ubiquinol-9 biosynthesis (prokaryotic)	3618.03
PWY-5857	ubiquinol-10 biosynthesis (prokaryotic)	3618.03
PWY-5860	superpathway of demethylmenaquinol-6 biosynthesis I	7324.86
PWY-5861	superpathway of demethylmenaquinol-8 biosynthesis	14703.85
PWY-5862	superpathway of demethylmenaquinol-9 biosynthesis	7324.86
PWY-5863	superpathway of phylloquinol biosynthesis	13725.66
PWY-5896	superpathway of menaquinol-10 biosynthesis	8840.34
PWY-5897	superpathway of menaquinol-11 biosynthesis	14964.06
PWY-5898	superpathway of menaquinol-12 biosynthesis	14964.06
PWY-5899	superpathway of menaquinol-13 biosynthesis	14964.06
PWY-5910	superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate)	11435.39
PWY-5913	TCA cycle VI (obligate autotrophs)	20361.52
PWY-5918	superpathay of heme biosynthesis from glutamate	6243.19
PWY-5920	superpathway of heme biosynthesis from glycine	2547.30
PWY-5971	palmitate biosynthesis II (bacteria and plants)	14919.20
PWY-5973	cis-vaccenate biosynthesis	49747.00
PWY-5989	stearate biosynthesis II (bacteria and plants)	10354.31
PWY-6071	superpathway of phenylethylamine degradation	719.57
PWY-6107	chlorosalicylate degradation	26.39
PWY-6121	5-aminoimidazole ribonucleotide biosynthesis I	45170.37
PWY-6122	5-aminoimidazole ribonucleotide biosynthesis II	45842.45
PWY-6123	inosine-5′-phosphate biosynthesis I	41232.95
PWY-6125	superpathway of guanosine nucleotides de novo biosynthesis II	27295.83
PWY-6126	superpathway of adenosine nucleotides de novo biosynthesis II	46650.28
PWY-6143	CMP-pseudaminate biosynthesis	94.59
PWY-6147	6-hydroxymethyl-dihydropterin diphosphate biosynthesis I	21591.81
PWY-6151	S-adenosyl-l-methionine cycle I	28706.99
PWY-6163	chorismate biosynthesis from 3-dehydroquinate	40112.38
PWY-6182	superpathway of salicylate degradation	406.66
PWY-6185	4-methylcatechol degradation (ortho cleavage)	423.35
PWY-621	sucrose degradation III (sucrose invertase)	33502.08
PWY-6263	superpathway of menaquinol-8 biosynthesis II	3131.98
PWY-6269	adenosylcobalamin salvage from cobinamide II	26528.11
PWY-6277	superpathway of 5-aminoimidazole ribonucleotide biosynthesis	45842.45
PWY-6282	palmitoleate biosynthesis I (from (5Z)-dodec-5-enoate)	10566.65
PWY-6317	galactose degradation I (Leloir pathway)	34775.87
PWY-6338	superpathway of vanillin and vanillate degradation	44.69
PWY-6339	syringate degradation	117.37
PWY-6353	purine nucleotides degradation II (aerobic)	16966.57
PWY-6383	mono-trans, poly-cis decaprenyl phosphate biosynthesis	173.90
PWY-6385	peptidoglycan biosynthesis III (mycobacteria)	40802.33
PWY-6386	UDP-N-acetylmuramoyl-pentapeptide biosynthesis II (lysine-containing)	41289.01
PWY-6387	UDP-N-acetylmuramoyl-pentapeptide biosynthesis I (meso-diaminopimelate containing)	41423.85
PWY-6396	superpathway of 2,3-butanediol biosynthesis	785.17
PWY-6397	mycolyl-arabinogalactan-peptidoglycan complex biosynthesis	40.91
PWY-6467	Kdo transfer to lipid IVA III (Chlamydia)	12629.55
PWY-6470	peptidoglycan biosynthesis V (β-lactam resistance)	21375.90
PWY-6471	peptidoglycan biosynthesis IV (Enterococcus faecium)	28804.61
PWY-6478	GDP-d-glycero-α-d-manno-heptose biosynthesis	3105.67
PWY-6507	4-deoxy-L-threo-hex-4-enopyranuronate degradation	22967.30
PWY-6519	8-amino-7-oxononanoate biosynthesis I	12604.24
PWY-6545	pyrimidine deoxyribonucleotides de novo biosynthesis III	15965.24
PWY-6562	norspermidine biosynthesis	560.39
PWY-6572	chondroitin sulfate degradation I (bacterial)	866.47
PWY-6588	pyruvate fermentation to acetone	16252.07
PWY-6590	superpathway of Clostridium acetobutylicum acidogenic fermentation	10578.64
PWY-6608	guanosine nucleotides degradation III	13201.42
PWY-6609	adenine and adenosine salvage III	44355.00
PWY-6612	superpathway of tetrahydrofolate biosynthesis	25616.14
PWY-6628	superpathway of l-phenylalanine biosynthesis	25205.54
PWY-6629	superpathway of l-tryptophan biosynthesis	12014.75
PWY-6630	superpathway of l-tyrosine biosynthesis	25117.93
PWY-6641	superpathway of sulfolactate degradation	29.67
PWY-6690	cinnamate and 3-hydroxycinnamate degradation to 2-oxopent-4-enoate	469.91
PWY-6700	queuosine biosynthesis	20531.37
PWY-6703	preQ0 biosynthesis	16299.65
PWY-6708	ubiquinol-8 biosynthesis (prokaryotic)	3618.03
PWY-6713	l-rhamnose degradation II	58.57
PWY-6728	methylaspartate cycle	497.91
PWY-6737	starch degradation V	38376.33
PWY-6749	CMP-legionaminate biosynthesis I	2462.52
PWY-6876	isopropanol biosynthesis	201.56
PWY-6891	thiazole biosynthesis II (Bacillus)	13034.49
PWY-6892	thiazole biosynthesis I (E. coli)	26726.86
PWY-6895	superpathway of thiamin diphosphate biosynthesis II	17977.27
PWY-6897	thiamin salvage II	24336.51
PWY-6901	superpathway of glucose and xylose degradation	22596.14
PWY-6906	chitin derivatives degradation	149.24
PWY-6969	TCA cycle V (2-oxoglutarate:ferredoxin oxidoreductase)	25487.79
PWY-7003	glycerol degradation to butanol	8059.09
PWY-7007	methyl ketone biosynthesis	125.70
PWY-7013	L-1,2-propanediol degradation	3598.98
PWY-7031	protein N-glycosylation (bacterial)	139.00
PWY-7090	UDP-2,3-diacetamido-2,3-dideoxy-α-d-mannuronate biosynthesis	240.58
PWY-7094	fatty acid salvage	3878.65
PWY-7097	vanillin and vanillate degradation I	44.69
PWY-7098	vanillin and vanillate degradation II	49.95
PWY-7111	pyruvate fermentation to isobutanol (engineered)	52273.29
PWY-7184	pyrimidine deoxyribonucleotides de novo biosynthesis I	21184.21
PWY-7187	pyrimidine deoxyribonucleotides de novo biosynthesis II	25640.42
PWY-7196	superpathway of pyrimidine ribonucleosides salvage	27756.26
PWY-7197	pyrimidine deoxyribonucleotide phosphorylation	20197.58
PWY-7198	pyrimidine deoxyribonucleotides de novo biosynthesis IV	34.96
PWY-7199	pyrimidine deoxyribonucleosides salvage	28884.78
PWY-7200	superpathway of pyrimidine deoxyribonucleoside salvage	22638.54
PWY-7208	superpathway of pyrimidine nucleobases salvage	48784.26
PWY-7210	pyrimidine deoxyribonucleotides biosynthesis from CTP	49.90
PWY-7211	superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis	21925.07
PWY-7219	adenosine ribonucleotides de novo biosynthesis	47313.68
PWY-7220	adenosine deoxyribonucleotides de novo biosynthesis II	48742.45
PWY-7221	guanosine ribonucleotides de novo biosynthesis	40546.34
PWY-7222	guanosine deoxyribonucleotides de novo biosynthesis II	48742.45
PWY-7228	superpathway of guanosine nucleotides de novo biosynthesis I	25535.06
PWY-7229	superpathway of adenosine nucleotides de novo biosynthesis I	48137.57
PWY-7234	inosine-5′-phosphate biosynthesis III	21336.28
PWY-7237	myo-, chiro- and scillo-inositol degradation	1937.37
PWY-7242	d-fructuronate degradation	28051.93
PWY-7254	TCA cycle VII (acetate-producers)	5529.80
PWY-7295	l-arabinose degradation IV	63.66
PWY-7315	dTDP-N-acetylthomosamine biosynthesis	4820.49
PWY-7323	superpathway of GDP-mannose-derived O-antigen building blocks biosynthesis	18019.00
PWY-7328	superpathway of UDP-glucose-derived O-antigen building blocks biosynthesis	7089.66
PWY-7332	superpathway of UDP-N-acetylglucosamine-derived O-antigen building blocks biosynthesis	4016.12
PWY-7371	1,4-dihydroxy-6-naphthoate biosynthesis II	1380.12
PWY-7373	superpathway of demethylmenaquinol-6 biosynthesis II	426.99
PWY-7374	1,4-dihydroxy-6-naphthoate biosynthesis I	634.53
PWY-7376	cob(II)yrinate a,c-diamide biosynthesis II (late cobalt incorporation)	62.06
PWY-7377	cob(II)yrinate a,c-diamide biosynthesis I (early cobalt insertion)	9632.91
PWY-7391	isoprene biosynthesis II (engineered)	110.97
PWY-7392	taxadiene biosynthesis (engineered)	24975.32
PWY-7400	l-arginine biosynthesis IV (archaebacteria)	36954.54
PWY-7431	aromatic biogenic amine degradation (bacteria)	231.10
PWY-7446	Sulfoglycolysis	1445.83
PWY-7456	mannan degradation	10813.70
PWY-7527	l-methionine salvage cycle III	91.06
PWY-7539	6-hydroxymethyl-dihydropterin diphosphate biosynthesis III (Chlamydia)	21827.04
PWY-7560	methylerythritol phosphate pathway II	34163.94
PWY-7616	methanol oxidation to carbon dioxide	123.02
PWY-7663	gondoate biosynthesis (anaerobic)	53483.87
PWY-7664	oleate biosynthesis IV (anaerobic)	11925.56
PWY-841	superpathway of purine nucleotides de novo biosynthesis I	32061.99
PWY-922	mevalonate pathway I	8788.68
PWY0-1061	superpathway of l-alanine biosynthesis	27059.17
PWY0-1241	ADP-l-glycero-β-d-manno-heptose biosynthesis	4043.22
PWY0-1261	anhydromuropeptides recycling	37971.25
PWY0-1277	3-phenylpropanoate and 3-(3-hydroxyphenyl)propanoate degradation	875.72
PWY0-1296	purine ribonucleosides degradation	34034.28
PWY0-1297	superpathway of purine deoxyribonucleosides degradation	29977.02
PWY0-1298	superpathway of pyrimidine deoxyribonucleosides degradation	28239.57
PWY0-1319	CDP-diacylglycerol biosynthesis II	48053.42
PWY0-1338	polymyxin resistance	2008.08
PWY0-1415	superpathway of heme biosynthesis from uroporphyrinogen-III	3458.06
PWY0-1479	tRNA processing	7885.85
PWY0-1533	methylphosphonate degradation I	3416.67
PWY0-1586	peptidoglycan maturation (meso-diaminopimelate containing)	34147.82
PWY0-162	superpathway of pyrimidine ribonucleotides de novo biosynthesis	31392.04
PWY0-166	superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis (E. coli)	25831.63
PWY0-321	phenylacetate degradation I (aerobic)	743.99
PWY0-41	allantoin degradation IV (anaerobic)	2316.50
PWY0-42	2-methylcitrate cycle I	1781.98
PWY0-781	aspartate superpathway	28189.60
PWY0-845	superpathway of pyridoxal 5′-phosphate biosynthesis and salvage	9936.55
PWY0-862	(5Z)-dodec-5-enoate biosynthesis	10512.28
PWY1G-0	mycothiol biosynthesis	108.79
PWY490-3	nitrate reduction VI (assimilatory)	6514.53
PWY4FS-7	phosphatidylglycerol biosynthesis I (plastidic)	41801.67
PWY4FS-8	phosphatidylglycerol biosynthesis II (non-plastidic)	41801.67
PWYG-321	mycolate biosynthesis	12192.35
PYRIDNUCSAL-PWY	NAD salvage pathway I	25515.37
PYRIDNUCSYN-PWY	NAD biosynthesis I (from aspartate)	25218.80
PYRIDOXSYN-PWY	pyridoxal 5′-phosphate biosynthesis I	7545.63
REDCITCYC	TCA cycle VIII (helicobacter)	6519.39
RHAMCAT-PWY	l-rhamnose degradation I	9861.49
RIBOSYN2-PWY	flavin biosynthesis I (bacteria and plants)	27667.91
RUMP-PWY	formaldehyde oxidation I	4735.55
SALVADEHYPOX-PWY	adenosine nucleotides degradation II	13015.70
SER-GLYSYN-PWY	superpathway of l-serine and glycine biosynthesis I	37417.17
SO4ASSIM-PWY	sulfate reduction I (assimilatory)	8355.24
SULFATE-CYS-PWY	superpathway of sulfate assimilation and cysteine biosynthesis	14568.40
TCA	TCA cycle I (prokaryotic)	23869.92
TCA-GLYOX-BYPASS	superpathway of glyoxylate bypass and TCA	5923.24
TEICHOICACID-PWY	teichoic acid (poly-glycerol) biosynthesis	1692.06
THISYN-PWY	superpathway of thiamin diphosphate biosynthesis I	20569.98
THREOCAT-PWY	superpathway of l-threonine metabolism	2620.36
THRESYN-PWY	superpathway of l-threonine biosynthesis	42787.81
TRNA-CHARGING-PWY	tRNA charging	41072.55
TRPSYN-PWY	L-tryptophan biosynthesis	34859.74
TYRFUMCAT-PWY	l-tyrosine degradation I	323.37
UBISYN-PWY	superpathway of ubiquinol-8 biosynthesis (prokaryotic)	3575.77
UDPNAGSYN-PWY	UDP-N-acetyl-d-glucosamine biosynthesis I	33015.08
VALSYN-PWY	l-valine biosynthesis	47290.87

Amino Acid Biosynthesis -Amino acid synthesis remains highly abundant, with "superpathway of branched amino acid biosynthesis" at 44965.47 and "l-arginine biosynthesis I″ at 37059.67.

Fatty Acid Synthesis - Fatty acid synthesis pathways also have high abundance values, such as "fatty acid elongation - saturated" (38627.04).

Fermentation -The "mixed acid fermentation" pathway shows a very high abundance of 26652.99, an increase from both the Control and T2 samples.

This comprehensive enzymatic profile across dietary treatments underscores the complex interplay between diet, microbial metabolism, and gut health, highlighting key enzymes that facilitate carbohydrate breakdown, energy production, nitrogen assimilation, genetic maintenance, and protective functions within the gut ecosystem.

3.7. Overall comparison of metabolic pathways across treatments

Samples T3 and T5 display a hyper-metabolic condition characterized by a broad enhancement of enzymes, particularly those involved in transport, transcription, and sugar metabolism. The abundance metrics for many enzymes in these two groups are nearly two to three times higher than those in the Control and sample T2. Sample T2 further exhibits a more moderate transition, with some specific enzymes increasing in abundance, but lacking the extensive, dramatic elevation found in T3 and T5. A comparison of all samples reveals a trend of increasing metabolic activity: Control > T2 >T3 > T5. Both T3 and T5 exhibit consistently higher abundance values across a wide range of pathways, including central metabolism, amino acid biosynthesis, and fermentation, when compared to the Control and T2. The T2 sample shows a more nuanced shift, with some pathways increasing and others decreasing, suggesting a different metabolic state compared to the other two treated samples, T3 and T5.

3.8. Central carbon metabolism

Glycolysis and TCA Cycle: The Control sample demonstrated moderate levels of activity in both "glycolysis I" (24075.80) and "TCA cycle I" (26721.47). In T2, these measurements remained similar, whereas "glycolysis III" reached 19183.92 and "TCA cycle I″ attained 23150.33. However, in T3 and T5, these metabolic pathways were significantly upregulated. "Glycolysis III" rose to 44958.84 in T3 and 45732.36 in T5. Correspondingly, "TCA cycle I″ escalated to 44833.64 in T3 and 37654.51 in T5. This suggests a substantial increase in the production of energy and precursor molecules in T3 and T5.

Glycogen Metabolism: The turnover of glycogen (including both synthesis and degradation) is notably heightened in T3 and T5. For example, "glycogen biosynthesis I″ is measured at 34560.09 in T3 and 33816.63 in T5, both significantly higher than the Control (1838.25) and T2 (1753.84). This indicates a strong demand for glucose storage and mobilization in microorganisms in the sample.

3.9. Amino acid and nucleotide biosynthesis

Amino Acid Biosynthesis: The pathways for amino acid synthesis show consistent abundance in T3 and T5. For instance, "superpathway of branched amino acid biosynthesis" is 38304.24 in T3 and 44965.47 in T5, indicating a significant increase compared with the Control (29218.82) and T2 (24307.48). This reflects a high demand for the building blocks of proteins and other macromolecular structures.

Nucleotide Biosynthesis: Similarly, nucleotide synthesis is highly active in both T3 and T5 samples. The "superpathway of purine nucleotides de novo biosynthesis II" reports an abundance of 32433.26 in T3 and 34151.78 in T5, in contrast to 26138.44 in the Control and a notably lower 16458.53 in T2. This suggests an accelerated rate of DNA and RNA synthesis.

Fermentation and Other Pathways: Sample T2 shows a marked increase in "homolactic fermentation" (23119.13) and "mixed acid fermentation" (17131.74) when compared to the Control. This trend intensifies in T3, with "homolactic fermentation" at 43927.37, and in T5, where "mixed acid fermentation" is at 26652.99. This indicates a notable shift toward anaerobic metabolism across all three treatment groups, with T3 and T5 exhibiting the most significant effects.

Fatty Acid Metabolism: The pathways for fatty acid synthesis and degradation exhibit differing abundances. Sample T2 presents a greater abundance of "fatty acid β-oxidation I″ compared to the Control, while "fatty acid elongation - saturated" is reduced. In T3 and T5, both fatty acid elongation (for example, T5 at 38627.04) and degradation are highly active, indicating rapid lipid turnover.

3.10. Treatment effects

Sample T2 vs. Control: T2 exhibits a distinct metabolic profile relative to the Control, characterized by increased reliance on fermentation and alterations in amino acid and nucleotide synthesis. Overall, the metabolic abundance here is not as significantly elevated as in the other treatment groups.

Samples T3 and T5 vs. Control: T3 and T5 show dramatic increases in metabolic activity. This suggests a hypermetabolic condition, characterized by high rates of energy production (glycolysis, TCA cycle), macromolecule synthesis (amino acids, nucleotides), and lipid turnover. While both exhibit this hyper-metabolic state, there are subtle distinctions in specific pathways, such as a greater abundance of glycolysis in T5 and homolactic fermentation in T3.

The ABC-2 type ATP-binding protein abundance rises from 165,686.34 in T2 to 302,502.58 in T3 and 348,393.99 in T5, compared to baseline in the Control. Similarly, RNA polymerase sigma-70 factor values increase from 161,196.95 (Control) to 254,245.24 (T5). In metabolic pathways, “glycolysis III” increases from 24,075.80 (Control) to 45,732.36 in T5. These marked shifts indicate that BSFL diets at higher inclusion levels significantly enhance enzyme systems and metabolic pathways related to transport, transcription, sugar metabolism, and energy production. These data reveal that all three treatments lead to metabolic alterations. T3 and T5 provoke a considerably more extensive and widespread activation of metabolic pathways compared to T2.

4. Discussion

4.1. Profiling of enzyme secreted by microbes in the gut of the broiler fed BSF LarvaeMeals

Significant enzymes were identified across all dietary treatments, with notable variations in enzyme abundance and diversity, providing insight into gut microbial functionality under different dietary conditions. The total number of enzymes detected was 3374 in the control group, 4943 in T2, 5851 in T3, and 5706 in T5. Among these, the T3 sample exhibited the highest number of enzymes, indicating an enhanced enzymatic capacity in this dietary treatment. In the control group, the RNA polymerase sigma-70 factor ECF subfamily showed an abundance of 161,196.95. In comparison, the T2, T3, and T5 groups were dominated by the ABC-2. A: ABC-2 type transport system ATP-binding protein, with abundances of 165,686.34, 302,502.58, and 274,679.31, respectively. This indicates an increased abundance of transport-related proteins in these treated samples, suggesting more active molecular transport. Among the top 20 abundant enzymes across all treatments are Beta-Galactosidase, 6-phospho-beta-glucosidase, Alpha-Glucosidase, Endoglucanase, Butyryl-CoA dehydrogenase, Acyl-CoA thioester hydrolase, Glutamine Synthetase, l-serine dehydratase, GntR/HutC transcriptional regulator, Aldose 1-epimerase, Acyl carrier protein, Mutase, Elongation Factor, DNA binding protein, Cellulolysis-related enzymes, ABC Transport proteins, DNase, DNA Helicase, and Histidine Kinase. These enzymes play critical roles in carbohydrate metabolism, energy production, nitrogen assimilation, and genetic maintenance within the gut microbiome.

Specific enzyme functions provide a clearer understanding of their contributions. Beta-Galactosidase, found notably in the control group and T5, catalyzes the degradation of lactose and other beta-galactosides, thereby supplying bacteria with energy and influencing prebiotic effects (Walsh et al., 2020). In contrast, 6-phospho-beta-glucosidase, present in all samples except the control, serves as a degradative enzyme of beta-glucosides [30]. Alpha-Glucosidase, which hydrolyzes alpha-glucosidic bonds to break down starch and complex carbohydrates into glucose, was found across all dietary treatments [31]. Meanwhile, Endoglucanase, present only in the control group, breaks down cellulose, releasing fermentable sugars crucial for bacterial fermentation [32]. The presence of Butyryl-CoA dehydrogenase and Acyl-CoA thioester hydrolase in the control group underscores their roles in fatty acid metabolism. Butyryl-CoA dehydrogenase is central to the beta-oxidation of fatty acids, producing butyrate, a key short-chain fatty acid (SCFA) with beneficial effects on gut health [33]. Similarly, Acyl-CoA thioesterase catalyzes the hydrolysis of acyl-CoA to free fatty acids and coenzyme A during fermentation [34], thereby facilitating energy production.

Glutamine Synthetase and l-serine dehydratase were consistently found across all samples, highlighting their importance in nitrogen metabolism and amino acid catabolism. Glutamine Synthetase plays a pivotal role by catalyzing the incorporation of ammonia into glutamine, which is essential for enterocyte nutrition and the maintenance of gut barrier integrity [35]. l-serine dehydratase catalyzes the breakdown of l-serine and l-threonine to pyruvate and ammonia, further contributing to metabolic processes [36]. Several enzymes involved in DNA processes, such as the GntR/HutC transcriptional regulator, elongation factor, DNA-binding protein, DNase, and DNA helicase, are predominantly found in the control group. These enzymes are vital for protein synthesis, DNA binding, replication, and repair within the gut microbiome, ensuring genomic stability and microbial homeostasis [[37], [38], [39]]. Acyl carrier protein, which is involved in fatty acid synthesis [40], was observed in both the control and T2 samples, indicating active lipid metabolism under these conditions.

Collectively, several enzyme groups contribute significantly to gut health and microbiome function. Glycosidases such as Beta-Galactosidase, Alpha-Glucosidase, and Endoglucanase are crucial for carbohydrate metabolism, enabling the breakdown of complex carbohydrates like lactose, starch, and cellulose into simpler, fermentable sugars such as glucose. This microbial fermentation produces SCFAs, including acetate, propionate, and butyrate. Butyrate, in particular, serves as a primary energy source for colonocytes and possesses anti-inflammatory properties that support gut health [32,41]. Also, Glutamine Synthetase facilitates nitrogen metabolism by converting glutamate and ammonia into glutamine, promoting intestinal cell nutrition and preserving gut barrier integrity. The enzyme Acyl-CoA thioester hydrolase enhances energy production through fermentation, thereby further increasing butyrate availability [41]. The DNA maintenance enzymes, such as DNA helicases, DNA-binding proteins, and topoisomerases, cooperate to maintain DNA integrity, enabling replication, repair, and overall DNA viability in the microbiome [41]. Furthermore, ABC transporters, which span microbial membranes and utilize ATP hydrolysis, mediate the transport of diverse molecules across cell membranes. Their abundance points to an efficient transport system essential for nutrient absorption, waste elimination, and potentially antibiotic resistance [42].

The presence and relative abundance of these enzymes reflect how the host diet shapes the gut microbial community structure and metabolic capacity. Microbial populations capable of vigorously exploiting available carbohydrates gain a competitive advantage, influencing community dynamics and host health. Diet also impacts the types of carbohydrates present in the gut, thereby modulating the glycosidases produced by the microbiota [42]. Moreover, enzymes involved in ammonia regulation, such as Glutamine Synthetase, help mitigate elevated ammonia levels that could otherwise disrupt gut function [3]. Some bacterial enzymes may also play roles in pathogen defense by modulating host glycans, a process that, while potentially leading to gut barrier dysfunction and inflammation, can also support the development of host defenses [43,44].

4.2. Metabolic pathways in all samples

Functional Capabilities: Metagenomic analyses indicated that the gut microbiota of broilers possess a variety of metabolic pathways involved in energy production, carbohydrate and protein metabolism, lipid processing, vitamin synthesis, and unique processes such as methanogenesis and mucin breakdown. The differences in the abundance of these pathways across various dietary treatments suggest that the inclusion of BSFL alters microbial functionality, beyond merely changing its taxonomic structure. The Carbohydrate Metabolism Pathways for glycolysis, gluconeogenesis, the pentose phosphate pathway, and starch/sucrose metabolism were significantly present in all dietary groups. The fermentation of carbohydrates into short-chain fatty acids (SCFAs: acetate, propionate, butyrate) helps maintain epithelial health and provides approximately 10–15% of the energy requirements for poultry. The T2 group (25% BSFL) exhibited the highest abundance of carbohydrate-active enzymes, indicating a superior ability to utilize chitin and fibers derived from BSFL. In contrast, the T5 group (100% BSFL) showed a reduced representation of carbohydrate pathways, suggesting a shift towards protein and lipid metabolism. The protein-rich profile of BSFL encouraged pathways for both amino acid production and breakdown (lysine, methionine, tryptophan). The pathways related to urease and ammonia assimilation pointed to nitrogen recycling within the gut. In T5, there was an increase in amino acid catabolism, resulting in the production of methylamine, which contributes to methanogenesis [8,9,45].

This diet-induced shift from carbohydrate to protein metabolism underscores the adaptability of the microbiota when BSFL is incorporated. Lipid Metabolism Identified pathways included glycerophospholipid metabolism, fatty acid breakdown, and bile acid metabolism. Microbial lipid utilization was influenced by BSFL-derived lipids, particularly lauric acid. Both T2 and T3 supported a balanced approach to lipid metabolism, whereas T5 showed increased fatty acid breakdown, potentially reflecting an adaptation to the high lipid content of BSFL. These pathways also connect to cholesterol metabolism and gut barrier health, both of which are affected by bile acid transformation. Mucin Breakdown and Glycan Pathways Predicted enzymes such as glycosidases, sulfatases, and proteases are indicative of mucin turnover. Sample T2 showed moderate activity in breaking down mucin, which helped to preserve mucosal integrity. On the other hand, T5 showed increased mucin degradation, indicating a potential risk to the mucosal barrier if present in excess. This finding aligns with the presence of mucin-degrading bacterial taxa (e.g., Akkermansia). Methanogenesis and Hydrogen Equilibrium Pathways related to methanogenesis were observed in all samples ([7]; Atallah et al., 2023; Vasilopoulos et al., 2024; [3,8]). In the control group, there was a baseline level of methanogenesis. Sample T2 achieved peak levels of methanogenesis through hydrogenotrophic (FMD, F420 enzymes) and methylotrophic (methyltransferases) pathways. T3 exhibited moderate methanogenesis due to limited hydrogen availability. In T5, methanogenesis primarily occurred via methylamine metabolism, which was associated with extensive protein breakdown. Methanogens play a crucial role in maintaining the hydrogen balance and fermentation efficiency in the gut, although methane emissions from poultry remain relatively low compared to those from ruminants [46,47]. Vitamin and cofactor synthesis-enriched pathways related to the biosynthesis of vitamins B12, riboflavin, folate, and biotin imply a microbial contribution to the host's micronutrient availability. Specifically, the synthesis of vitamin B12 was notably abundant in T2 and T3, thereby benefiting the host's growth performance and metabolic functions. The presence of pathways for the degradation of aromatic compounds and xenobiotic metabolism, i.e., Xenobiotic and Secondary Metabolite Metabolism, indicates a microbial capability for detoxification. The biosynthesis of secondary metabolites (e.g., bacteriocins, antimicrobial peptides) was more pronounced in groups with BSFL, suggesting an enhanced ability to suppress pathogens [43]). Comprehensive Implications: In addition to BSFL, microbial metabolism shifted from predominantly carbohydrate-based (Control) to a more balanced carbohydrate–protein-lipid metabolism (T2, T3), ultimately leading to a protein-centric fermentation in T5. T2 exhibited the most well-rounded functional characteristics, showcasing optimal SCFA generation, vitamin synthesis, and moderate methanogenesis. On the other hand, T5 poses risks associated with overreliance on protein fermentation, mucin degradation, and methylamine-associated methanogenesis, which could jeopardize gut health and overall sustainability [[48], [49], [50], [51], [52], [53]].

5. Conclusion

The incorporation of black soldier fly larvae meal as a dietary protein source significantly modulates the gut microbiome and metabolic activities of broiler chickens. Diets containing BSFLM increased enzyme abundance and activated key metabolic pathways involved in energy production, amino acid biosynthesis, and lipid turnover, particularly at 50% and 100% replacement levels. While full replacement (T5) induced a hyper-metabolic, protein-centric microbial state that may increase mucin degradation and nitrogen turnover, the 50% inclusion level (T3) achieved the most balanced gut metabolic profile, supporting efficient carbohydrate fermentation, amino acid synthesis, and vitamin production. These results highlight BSFLM as a viable and sustainable alternative to fishmeal in broiler diets, with a 50% inclusion rate recommended to optimize gut microbial function, energy metabolism, and production performance.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used no generative AI and AI-assisted technologies. The authors reviewed and edited the content as needed and take full responsibility for the publication.

Funding

This research received no funding.

CRediT authorship contribution statement

Opeyemi Adetola Oladejo: Conceptualization, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing. Deborah Oluwaferanmi Ibiwoye: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Writing – original draft, Writing – review & editing. Ayodeji Amos Faniyi: Software, Validation. Matthew Oluwaseyi Ayoola: Resources, Validation. Abel Olusegun Oguntunji: Formal analysis, Resources, Validation. Samuel Olatunde Dahunsi: Conceptualization, Investigation, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.