Integrated transcriptomic and metabolomic analyses uncover novel genetic targets for enhanced l-tryptophan production in Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum is a promising microbial chassis for the industrial production of l-tryptophan, an essential amino acid with diverse applications and high market value. In our previous work, we constructed an l-tryptophan-overproducing C. glutamicum strain, TR26, through multiple rounds of rational metabolic engineering. Here, comparative transcriptomic and metabolomic analyses were conducted between TR26 and its progenitor MB001 to elucidate the underlying molecular mechanisms and identify potential bottlenecks for l-tryptophan production in TR26. Based on the differentially expressed genes identified, systematic gene overexpression and knockdown experiments led to the identification of two novel genetic targets, glnK and sugR. Specifically, the repression of glnK and overexpression of sugR in TR26 increased the l-tryptophan titer by 10.3 % and 16.5 % in fed-batch fermentation, and the yield by 7.2 % and 20.2 %, respectively. Further transcription profiling and intracellular metabolite analysis indicated that these improvements were associated with altered nitrogen metabolism, more efficient allocation of cellular resources, and enhanced supply of phosphoenolpyruvate (PEP), a key precursor in aromatic amino acid biosynthesis. This study expands our understanding of the regulation mechanisms governing l-tryptophan synthesis in C. glutamicum and provides valuable insights for further optimization of industrial cell factories.

1. Introduction

L-tryptophan is an essential amino acid for humans and animals, playing critical roles in protein synthesis and the regulation of various physiological functions. It has been widely applied in the pharmaceutical, nutraceutical, food and feed industries, underscoring its high economic and biotechnological value [1,2]. Over recent decades, microbial fermentation has become the predominant method for l-tryptophan production due to its high efficiency, cost-effectiveness, and environmental sustainability. Mechanism study and protein engineering of the key enzymes for l-tryptophan synthesis have been extensively conducted in industrial hosts, including Escherichia coli [[3], [4], [5], [6], [7], [8], [9], [10], [11]] and Corynebacterium glutamicum [12,13]. Based on these research, the two hosts have been successfully engineered for l-tryptophan production through various strategies, such as strengthening the l-tryptophan synthesis pathway [[14], [15], [16], [17], [18], [19]], optimizing precursor supply [[19], [20], [21], [22], [23], [24], [25]], improving transporter systems [24,[26], [27], [28], [29], [30], [31]], and suppressing competing metabolic routes [[31], [32], [33], [34]]. With well-established genetic tools, E. coli strains have been engineered to achieve l-tryptophan titers over 50 g/L, with yields ranging from 0.15 to 0.24 g/g glucose (Table 1). In comparison, C. glutamicum offers advantages in terms of biosafety, industrial robustness, and substrate versatility, making it a preferred host for the production of various amino acids [[35], [36], [37], [38], [39], [40], [41], [42], [43]]. Moreover, based on intensive investigation in the genome characteristics and metabolic regulation of C. glutamicum [[44], [45], [46], [47]], high-performing industrial chassis were developed [48,49], promoting the application of C. glutamicum for l-tryptophan synthesis. Through a combination of random mutagenesis and rational design, a C. glutamicum strain capable of producing up to 58 g/L l-tryptophan has been constructed, demonstrating its strong potential for industrial applications [23]. Despite these advances, the achieved l-tryptophan yields remain significantly below the theoretical maximum, which may be attributed to insufficient understanding of the regulatory mechanisms governing l-tryptophan biosynthesis. Therefore, gaining deeper insights into the regulatory networks involved in l-tryptophan synthesis and identifying novel genetic targets are crucial steps toward developing next-generation industrial strains with improved production capabilities.

Table 1.

l-tryptophan production by engineered industrial hosts.

Strain	Titer (g/L)	Yield (g/g)	Productivity (g/L/h)	Reference
E. coli S028	40.3	0.150	0.60	[25]
E. coli JB102	42.3	0.176	–	[65]
E. coli KW023	39.7	0.167	1.60	[19]
E. coli TRTH0709/pMEL03	48.7	0.219	–	[31]
E. coli TRTHBPAB	47.2	0.157	–	[34]
E. coli SX11	41.7	0.227	1.04	[20]
E. coli TRP12	52.1	0.171	1.45	[29]
E. coli T13	53.7	0.238	–	[18]
E. coli Trp30	42.5	0.178	0.89	[24]
C. glutamicum KY9218/pIK9960	58.0	–	–	[23]
C. glutamicum TR26	50.5	0.170	1.05	[30]

Multi-omics technologies have emerged as powerful tools for elucidating the global gene expression patterns and metabolic characteristics of industrial microbial strains. Advances in next-generation sequencing have significantly expanded transcriptomic resources, enabling comprehensive analyses of key industrial traits such as hyperproduction phenotypes and stress tolerance. These analyses provide valuable insights into cellular metabolism and regulatory networks [[51], [52], [53], [54], [55], [56], [57]]. Transcriptomic profiling, in particular, has proven effective in identifying novel genetic targets, thereby facilitating the rational engineering of improved production strains. For example, comparative transcriptome analysis of an l-tryptophan-overproducing E. coli led to the identification of the transcriptional repressor YihL, which negatively regulates tryptophan biosynthesis [24]. Deletion of yihL resulted in a 16.3 % increase in l-tryptophan titer, demonstrating the utility of this approach in enhancing production capabilities [24]. Recently, transcriptome analysis was also used to discover a link between l-tryptophan synthesis and iron sulfur cluster formation in C. glutamicum, providing a novel target for strain optimization [50]. Similarly, transcriptome analysis has been employed to investigate the mechanisms underlying l-phenylalanine overproduction in E. coli, leading to the discovery of MarA as a key regulator that enhances product tolerance and improves biosynthetic efficiency [58]. In parallel, metabolomic analysis provides complementary insights by revealing global carbon flux distributions and intracellular metabolite profiles. This approach is particularly effective in identifying intermediate metabolite accumulations and metabolic bottlenecks that limit product yield [30,59,60]. Integrating transcriptomic and metabolomic data thus offers a systems-level understanding of cellular behavior and enables more precise metabolic engineering strategies for industrial strain development [59,[61], [62], [63], [64]].

In our previous work, we developed an l-tryptophan overproducing C. glutamicum strain TR26 through rational metabolic engineering strategies [30], including: 1) enhancement of the shikimate and tryptophan pathway (overexpression of aroG^D146N-aroDC and trpE^S40F-trpD-trpCBA genes); 2) improvement of precursor supply (Δpyk, overexpression of serA^Δ197, glnA^E304A, tkt, pkt^{T2A/I6T/H260Y}); 3) deletion of competing pathways (ΔldhΔpat); 4) integration of heterogeneous tryptophan exporter gene yddG. The strain TR26 achieved an l-tryptophan titer of 50.5 g/L and a yield of 0.17 g/g glucose in 48 h in fed-batch fermentation, which was comparable to other reported strains (Table 1). In the present study, we perform comparative transcriptomic and metabolomic analyses of TR26 and its wild-type progenitor strain MB001 to uncover the molecular and metabolic determinants underlying the enhanced l-tryptophan production phenotype. By systematically examining intracellular metabolite profiles and global gene expression patterns, we identify key regulatory and metabolic features associated with tryptophan overproduction. Furthermore, through targeted downregulation and overexpression of the selected differentially expressed genes (DEGs), we discover novel genetic targets that significantly influence l-tryptophan biosynthesis in C. glutamicum. Follow-up transcriptional profiling and metabolite analysis provide mechanistic insights into the roles of these newly identified regulators in modulating metabolic fluxes and pathway activity. This work improves our understanding of the regulatory mechanisms governing l-tryptophan synthesis in C. glutamicum, offering valuable insights for strain engineering towards industrial application.

2. Materials and methods

2.1. Bacterial strains and plasmids

C. glutamicum MB001 is a prophage-free strain derived from ATCC 13032 [66]. C. glutamicum TR26, an l-tryptophan-overproducing strain, was obtained from MB001 through multiple rounds of rational genetic engineering [30]. E. coli DH5α was used as the host for plasmid construction. The pD9SG plasmid [67], an E. coli-C. glutamicum shuttle vector containing a dCas9 protein expression cassette and sgRNA sequences, was employed for gene repression in C. glutamicum. For gene overexpression studies, the pEC-K18mob2 plasmid [68] was used.

2.2. Plasmids and strains construction

For the construction of gene repression plasmids, gene-specific sgRNA fragments flanked by BsaI overhangs were generated by annealing complementary oligonucleotides and subsequently ligated into the BsaI sites of the plasmid pD9SG using T4 DNA ligase. One sgRNA was designed for each candidate gene following the established procedure [69]. The sgRNA sequences targeting each candidate gene are listed in Table S1.

For gene overexpression, functional genes were amplified with a conserved ribosome binding site (RBS: 5ʹ-aaaggaggttgtc-3ʹ) and the rrnB terminator sequence. These expression cassettes were then cloned into the EcoRI/XbaI sites of the plasmid pEC-K18mob2 via Gibson assembly following standard protocols [70]. Gene expression levels were controlled by the native lac promoter on the plasmid backbone. All primers used for amplification of target genes are listed in Table S2. The primers PEC_F (5ʹ-gtggctgttttggcggatgag-3ʹ) and rrnB_R (5ʹ-aagcttgcatgcctgcaggtcgactagagtttgtagaaacgcaaaaaggcc-3ʹ) were used to amplify the rrnB terminator sequence.

2.3. Media and culture conditions

For shake-flask fermentations, C. glutamicum strains were first cultivated in 20 mL of LSS1 medium in 250 mL baffled flasks at 30 °C and 200 rpm for 14–16 h [71]. The resulting seed cultures were then inoculated into 30 mL of LPG2 medium in 500 mL baffled flasks supplemented with 30 g/L CaCO₃ for pH maintenance [72]. The LSS1 medium contains: sucrose 50 g/L, corn steep liquor 10 g/L, (NH₄)₂SO₄ 8.3 g/L, KH₂PO₄ 2 g/L, urea 1 g/L, MgSO₄⋅7H₂O 0.83 g/L, FeSO₄⋅7H₂O 10 mg/L, ZnSO₄⋅7H₂O 10 mg/L, CuSO₄⋅5H₂O 1 mg/L, β-alanine 10 mg/L, thiamine-HCl 1.5 mg/L, biotin 0.5 mg/L, nicotinic acid 5 mg/L. The LPG2 medium contains: glucose 100 g/L, corn steep liquor 10 g/L, (NH₄)₂SO₄ 45 g/L, KH₂PO₄ 0.5 g/L, urea 4.5 g/L, MgSO₄⋅7H₂O 0.5 g/L, FeSO₄⋅7H₂O 10 mg/L, MnSO₄⋅4H₂O 1 mg/L, β-alanine 10 mg/L, thiamine-HCl 5 mg/L, biotin 0.3 mg/L, nicotinic acid 5 mg/L. All shake-flask fermentations were conducted in duplicate at 30 °C, 200 rpm, and an initial pH of 7.2.

Microtiter-scale fermentations were carried out using a Biolector® I system in 48-well microtiter plates. Briefly, C. glutamicum strains were pre-cultured in 1 mL of modified LSS1 medium containing 20 g/L 3-(N-Morpholino)propanesulfonic acid (MOPS) instead of CaCO₃ for pH control, at 30 °C and 990 rpm for 12–14 h. The seed cultures were then inoculated into 1 mL of modified LPG2 medium containing 50 g/L glucose and 20 g/L MOPS (without CaCO₃), and incubated under the same conditions for an additional 36 h. All microtiter fermentations were conducted in duplicate at 30 °C, 990 rpm, and 75 % relative humidity. When necessary, 25 μg/L kanamycin was added. Additionally, 1 mmol/L IPTG was included to induce dCas9 expression for targeted gene repression upon inoculation of the seed culture into the fermentation medium.

Fed-batch fermentations were conducted in 5 L bioreactors with 2 L of LPG2 at 30 °C. The pH of the fermentation was maintained at 7.0 through the automatic addition of a 25 % (v/v) ammonia solution. Dissolved oxygen levels were kept above 30 % of air saturation by adjusting the agitation speed at an aeration rate of 2 L/min. A feeding solution containing 600 g/L glucose was administered to ensure the glucose concentration remained below 10 g/L.

2.4. Analysis of cell growth and extracellular metabolites

Cell growth was monitored by measuring the optical density at 600 nm (OD₆₀₀) of the culture broth. Glucose and acetic acid concentrations were quantified using high-performance liquid chromatography (HPLC) equipped with an Aminex HPX-87H Column (300 × 7.8 mm). The mobile phase consisted of 5 mM H₂SO₄, with a flow rate of 0.8 mL/min at 65 °C. The injection volume was 20 μL.

L-tryptophan concentrations were determined by HPLC with a Dikma Diamonsil AAA Column (5 μm, 250 × 4.6 mm), following derivatization with phenyl isothiocyanate according to standard protocols [73]. l-tryptophan was separated using mobile phase A (50 mM CH₃COONa, pH 6.5) and mobile phase B (methanol:acetonitrile:water = 1:3:1) with gradient elution. The total flow rate was maintained at 1 mL/min at 45 °C. The detective wavelength was set at 254 nm and the injection volume was 10 μL.

2.5. Metabolome analysis

Cells were harvested during the exponential growth phase, and 2 mL of each culture sample was collected. The cell suspension was centrifuged at 4 °C, 12000 rpm for 1 min to remove the culture medium. The cell pellets were gently washed three times with PBS buffer (pH 7.2–7.4), and the supernatant was removed. Immediately, 1.6 mL of pre-cooled (−80 °C) 80 % methanol was added to resuspend the cell pellets by vortexing. The samples were then incubated at −20 °C for 30 min, followed by ultrasonication for 2 min. Afterwards, the samples were incubated at −80 °C for 2 h for cell lysis. The suspensions were subsequently centrifuged at 4 °C, 14000 rpm for 15 min to remove the cellular debris and protein. The supernatants were transferred to fresh 2 mL microcentrifuge tubes, dried by vacuum, and stored at −80 °C until further analysis.

Intracellular metabolites were analyzed using SCIEXTriple Quad 6500+ liquid chromatography-tandem mass spectrometry (LC-MS/MS) system (AB Sciex, Singapore), equipped with an IonDrive detector and Qtrap-6500 mass spectrometer. Metabolites with significant concentration variations were identified based on the following criteria: average fold change ≥1.5, t-test p ≤ 0.05.

2.6. Transcriptome analysis

Cells were harvested at both the exponential and stationary growth phases, and 2 mL of each sample was collected. The cell suspensions were centrifuged at 4 °C, 12000 rpm for 1 min to remove the culture medium. The resulting cell pellets were washed three times with PBS buffer (pH 7.2–7.4) and immediately frozen in liquid nitrogen for subsequent analysis. RNA extraction and transcriptome sequencing were performed by AZENTA life science. Sequencing was carried out on the Illumina platform. Raw sequencing data were quality-controlled and preprocessed using FastQC (v0.10.1) and Cutadapt (v1.9.1) to remove low-quality reads and adapter sequences. Clean reads were aligned to the reference genome of C. glutamicum MB001 (CP005959.1) using Bowtie2 (v2.2.6). Differential gene expression analysis was performed using the DESeq2 Bioconductor package. Genes showing statistically significant differential expression were identified based on the following criteria: average fold change ≥2.0, t-test p ≤ 0.05.

3. Results and discussion

3.1. Metabolomic analysis of C. glutamicum TR26 and MB001

C. glutamicum TR26 is an l-tryptophan-overproducing strain derived from the wild-type strain MB001 through multiple genome modifications based on rational metabolic engineering strategies. To investigate the underlying mechanisms and identify potential metabolic bottlenecks associated with l-tryptophan biosynthesis in TR26, a comparative metabolomic analysis was first performed between TR26 and MB001, focusing on variations in intracellular concentrations of central metabolic intermediates and amino acids.

As illustrated in Fig. 1A, a total of 75 metabolites involved in central metabolic pathways were detected. Among these, four exhibited significantly reduced concentrations, while fourteen showed significantly increased concentrations in TR26 compared to MB001. Notably, the concentration of the byproduct l-lactate was markedly reduced in TR26 due to the deletion of the lactate dehydrogenase gene ldh. On the other hand, the intracellular level of shikimate, a key intermediate in the biosynthesis of aromatic amino acids, increased by 117-fold in TR26 (Fig. S1). This suggests that although flux from shikimate to l-tryptophan was enhanced via overexpression of aroC and aroD, further optimization of the shikimate pathway may still be required to maximize l-tryptophan production [30]. In addition, the intracellular concentrations of phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), which are essential precursors for l-tryptophan synthesis, showed slight increases in TR26 (Fig. S1), indicating that precursor availability does not appear to be a limiting factor under the current genetic background. However, a general decline in the levels of pyruvate and tricarboxylic acid (TCA) cycle intermediates was observed in TR26 (Fig. S1), suggesting a redirection of carbon flux away from biomass formation toward l-tryptophan biosynthesis.

Fig. 1.

Comparative metabolomic analysis of TR26 and MB001. (A) Concentration changes of intracellular metabolites within central metabolic pathways. (B) Concentration changes of intracellular amino acids. Metabolites with average fold change ≥1.5, t-test p ≤ 0.05 were considered significantly different.

Regarding the intracellular amino acid profiles, TR26 exhibited significant increases in the concentrations of four amino acids and decreases in another four compared to MB001 (Fig. 1B). Most notably, the intracellular concentration of l-tryptophan in TR26 was 65-fold higher than that in MB001 (Fig. S1), although it was significantly lower than that previously reported for strain TR13 without heterogeneous l-tryptophan exporter [30]. This observation supports the hypothesis that the introduction of the l-tryptophan exporter protein has effectively enhanced product efflux. Moreover, the 4.1-fold increase in l-lysine concentration indicates the need for targeted modulation of its biosynthetic pathway to avoid unnecessary carbon allocation. In contrast, no significant accumulation of l-tyrosine or l-phenylalanine was observed in TR26 relative to MB001, which may be attributed to the deletion of the pat gene and the consequent alleviation of carbon flux competition among aromatic amino acid biosynthetic pathways. Nevertheless, significant reductions in the concentrations of l-serine and l-glutamine—both of which serve as direct precursors for l-tryptophan biosynthesis—were observed in TR26, pointing to the emergence of new metabolic constraints that may limit further improvements in l-tryptophan production.

3.2. Transcriptomic analysis of C. glutamicum TR26 and MB001

To deeper understand the mechanisms underlying l-tryptophan overproduction in the engineered C. glutamicum TR26 and to provide potential strategies for further strain optimization, a comparative transcriptomic analysis was performed between TR26 and MB001. As shown in Fig. 2A, 455 genes were significantly down-regulated and 524 genes were significantly up-regulated in the exponential phase in TR26 compared to MB001. In the stationary phase, 392 genes were significantly down-regulated and 343 genes were up-regulated in TR26 (Fig. 2B). KEGG pathway enrichment analysis revealed that the DEGs in the exponential phase were predominantly associated with microbial metabolism in diverse environments, amino acids biosynthesis, and ABC transporters (Fig. S2), indicating substantial alterations in amino acid metabolism and widespread metabolic impacts resulting from the genetic engineering strategies. In the stationary phase, DEGs were primarily enriched in pathways related to amino acids biosynthesis, ribosome biogenesis, and two-component systems (Fig. S2), suggesting complex metabolic regulation regulatory changes and phase-dependent transcriptional differences in TR26, in addition to the overall reprogramming of amino acid metabolism.

Fig. 2.

Transcriptomic analysis between strains TR26 and MB001 (A) Global gene expression characteristics of strains TR26 and MB001 in the exponential phase. (B) Global gene expression characteristics of strains TR26 and MB001 in the stationary phase. (C) Gene expression profiles in strain TR26 relative to wild-type MB001 within central metabolic pathways and aromatic amino acid synthesis pathway. The right and left boxes represent variations in gene expression levels during the exponential phase and stationary phase, respectively. “X” colored in red indicates genes deleted in TR26. Genes with average fold changes ≥2, t-test p ≤ 0.05 were considered significantly different.

As shown in Fig. 2C, analysis of gene expression in central metabolic pathways revealed that most genes involved in glycolysis and the TCA cycle were down-regulated during the exponential phase in TR26. This suggests a shift in carbon flux away from biomass formation toward product synthesis, which is further supported by the significant up-regulation of the trp operon genes (trpEGDCFBA) at both growth stages. However, in the stationary phase, the expression differences of these central metabolic genes between TR26 and MB001 were largely diminished (Fig. 2C), implying a possible increase in carbon loss through enhanced TCA cycle activity. Therefore, dynamic regulation of key genes in central metabolic pathways may represent an effective strategy to enhance l-tryptophan accumulation during the later stages of fermentation. Notably, the transcription level of tkt, which was overexpressed in TR26, increased by 14.6-fold and 23.7-fold during the exponential and stationary phases, respectively (Fig. 2C), potentially contributing significantly to E4P supply for efficient l-tryptophan overproduction.

Further analysis of genes within the shikimate and aromatic amino acid synthesis pathways revealed that aroC and aroD were significantly up-regulated, confirming their critical roles in optimizing the shikimate pathway for l-tryptophan production in C. glutamicum [30]. In contrast, genes involved in the biosynthesis of competing aromatic amino acids—specifically csm, pheA, and tyrA, which are responsible for l-tyrosine and l-phenylalanine synthesis—were significantly down-regulated in TR26. This indicates effective suppression of competing pathways, a finding consistent with the metabolomic data and representing one of the key mechanisms contributing to l-tryptophan overproduction in strain TR26. Notably, although an almost consistent trend could be found between the metabolomic and transcriptomic data, deviation still exist especially in the glycolysis pathway, which could result from post-transcriptional regulation, enzyme activity modulation, and metabolite feedback mechanism.

3.3. Screening of novel genetic targets for l-tryptophan production

The DEGs identified between TR26 and MB001 may include novel target genes that can be engineered to further enhance l-tryptophan production in TR26. Notably, 214 genes were commonly down-regulated and 183 genes were up-regulated in both the exponential and stationary growth phases in TR26 relative to MB001. Functional analysis of these consistently differentially expressed genes led to the identification of 43 candidate genes potentially involved in l-tryptophan biosynthesis, none of which have been previously characterized in this context. These candidate genes are involved in diverse biological processes, including transcriptional regulation, redox reactions, energy metabolism, stress response, amino acids and aromatic compounds metabolism, as well as cell membrane functions (Table 2). These findings provide a valuable foundation for guiding targeted genetic modifications and uncovering potential regulatory mechanisms to further optimize l-tryptophan biosynthesis in strain TR26.

Table 2.

Candidate genetic targets related to l-tryptophan synthesis.

Gene	Description	Fold change (exponential phase/stationary phase)	GenBank ID
cgp_1486	transcriptional regulator	2.07/5.15	AGT05300.1
qorR	transcriptional repressor of quinone oxidoreductase qor2	5.46/6.35	AGT05339.1
cgp_1687	putative transcriptional regulatory protein	2.63/2.11	AGT05458.1
cgp_2910	putative transcriptional regulator	2.46/2.11	AGT06344.1
ssuR	transcriptional activator of sulfonate utilization	0.33/0.10	AGT04021.1
cgp_0027	putative transcriptional regulator	0.31/0.18	AGT04031.1
cysR	transcriptional activator of assimilatory sulfate reduction	0.18/0.12	AGT04132.1
iolR	transcriptional regulator	0.29/0.21	AGT04167.1
lrp	leucine-responsive transcriptional activator	0.16/0.16	AGT04268.1
whiB3	transcriptional regulator protein	0.03/0.01	AGT04595.1
cgp_0800	transcriptional activator of propionate catabolism	0.23/0.18	AGT04691.1
whiB2	putative transcriptional regulator	0.37/0.18	AGT04734.1
cgp_1053	putative transcriptional regulator	0.33/0.13	AGT04919.1
cgp_1143	putative transcriptional regulator	0.44/0.29	AGT05002.1
rbsR	transcriptional repressor of the ribose importer RbsACBD	0.10/0.30	AGT05240.1
sugR	transcriptional regulator	0.20/0.26	AGT05671.1
glnK	nitrogen regulatory protein PII	0.23/0.34	AGT05797.1
narG	respiratory nitrate reductase	4.54/3.90	AGT05179.1
cgp_0253	putative 2Fe–2S ferredoxin	6.72/4.78	AGT04215.1
cydA	cytochrome d ubiquinol oxidase subunit I	2.36/4.77	AGT05141.1
cgp_3100	chaperone DnaK heat shock protein	8.54/4.38	AGT06511.1
fdhD	putative formate dehydrogenase	13.03/5.81	AGT04526.1
cgp_2865	phosphoribosylformylglycinamidine synthase subunit	0.29/0.48	AGT06306.1
glmS	glutamine-fructose-6-phosphate transaminase	0.22/0.22	AGT06001.1
gltB	glutamate synthase large chain	0.13/0.04	AGT04192.1
pcaH	protocatechuate 3,4-dioxygenase, beta subunit	3.50/3.69	AGT06126.1
cgp_2953	putative 4-hydroxybenzaldehyde dehydrogenase	2.73/6.24	AGT06385.1
cgp_2966	putative phenol 2-monooxygenase	0.35/0.28	AGT06397.1
ald	acetaldehyde dehydrogenase	0.21/0.41	AGT06507.1
adhA	alcohol dehydrogenase	0.14/0.38	AGT06518.1
tagA2	DNA-3-methyladenine glycosylase I	3.71/10.76	AGT04155.1
papA	prolyl aminopeptidase A	2.17/3.76	AGT04587.1
cspB	cold-shock protein B	0.30/0.34	AGT04318.1
cysI	ferredoxin-sulfite reductase	0.12/0.11	AGT06527.1
ssuD1	FMNH2-dependent aliphatic sulfonate monooxygenase	0.43/0.09	AGT05210.1
ctaC	cytochrome c oxidase subunit II	0.24/0.16	AGT05932.1
cgp_2402	secreted protein NLP/P60 family	4.89/5.34	AGT05926.1
wzy	putative membrane protein involved in polysaccharide polymerization	0.35/0.32	AGT04370.1
mscL	large-conductance mechanosensitive ion channel	0.21/0.32	AGT04875.1
recA	recombinase A	10.65/24.33	AGT05695.1
recO	DNA repair protein	3.95/3.30	AGT06014.1
rpf1	RPF-protein precursor	4.07/3.71	AGT04815.1
rpf2	RPF2 precursor	2.75/4.91	AGT04905.1

To investigate the impact of down-regulating the identified candidate genes on l-tryptophan production, we employed the CRISPR interference (CRISPRi) system. Specific sgRNA targeting each candidate gene was designed and cloned into the pD9SG plasmid, which expresses both dCas9 and the sgRNA cassette. These plasmids were then introduced into strain TR26 to generate an arrayed library with individual gene repression. Strain TR26 carrying the pD9SG plasmid with a non-targeting sgRNA was used as the negative control (NC). The performance of these strains were evaluated in 48-well microtiter plates. As shown in Fig. 3A and 3B, repression of glnK, cydA, papA, ssuD1, and cgp_2402 led to increased l-tryptophan titers (>8 %) compared to the control group. Conversely, down-regulation of qorR, cgp_1687, cysR, lrp, whiB3, rbsR, sugR, narG, cgp_0253, cgp_2865, and cysI resulted in reduced l-tryptophan accumulation (>12 %). Moreover, repression of cysR, whiB2, cgp_2865, and cysI significantly impaired biomass accumulation (Fig. S3), suggesting potential negative effects on cellular physiology and growth performance.

Fig. 3.

Screening of novel genetic targets for l-tryptophan overproduction. (A) (B) l-tryptophan titers of strains with down-regulated target genes in 48-well microtiter plates. (C) l-tryptophan titers of strains with up-regulated target genes in 48-well microtiter plates. NC, TR26 containing pD9SG plasmid with non-targeting sgRNA. Ctrl, TR26 containing empty pEC-K18mob2 plasmid. Two tailed t-tests indicate statistical significance compared to the control group. ∗P < 0.1, ∗∗P < 0.05.

To further validate the functional roles of these genes, we overexpressed those whose down-regulation had negatively impacted l-tryptophan production using the pEC-K18mob2 expression plasmid. As shown in Fig. 3C, overexpression of sugR resulted in a marked increase in both l-tryptophan titer and biomass accumulation (Fig. S3). Collectively, these results indicate that repression of glnK, cydA, papA, ssuD1, and cgp_2402, as well as activation of sugR, could serve as effective genetic strategies to enhance l-tryptophan biosynthesis in C. glutamicum.

3.4. Verification of novel genetic targets by fermentation

To further verify the effects of down- or up-regulating the novel genetic targets in C. glutamicum, shake-flask fermentations were conducted. As shown in Fig. 4A, the production of l-tryptophan in the glnK-repressed strain was consistently higher than that of the negative control (NC) throughout the fermentation process, ultimately showing the highest improvement in product titer by 6.7 % with significant increase in product secretion rate. Additionally, the OD₆₀₀ value of the glnK-repressed strain was comparable to the control group (Fig. 4B), suggesting that repression of this gene did not cause significant impairment to cell growth or physiology. Furthermore, the repression of glnK led to a reduction of acetate accumulation by 26.0 % (Fig. S4), indicating improved metabolic balance and a shift in carbon flux away from byproduct formation toward l-tryptophan synthesis. The production of l-tryptophan by other strains with gene repression was not statistically higher than that of the control strain within the first 48 h, and therefore, they were not taken for further analysis. On the other hand, overexpression of sugR significantly enhanced l-tryptophan production in strain TR26 under shake-flask conditions (Fig. 4C). The sugR-overexpressing strain achieved a 20.9 % increase in l-tryptophan titer (Fig. 4C), accompanied by improved biomass accumulation (Fig. 4D) and complete suppression of acetate secretion (Fig. S4).

Fig. 4.

Fermentations of the engineered strains. (A) (C) l-tryptophan production in shake-flask fermentation. (B) (D) Cell growth in shake flask fermentation. (E) l-tryptophan production in fed-batch fermentation. (F) Cell growth in fed-batch fermentation. NC, TR26 containing pD9SG plasmid with non-targeting sgRNA. Control, TR26 containing empty pEC-K18mob2 plasmid.

Fed-batch fermentation in 5 L fermenters was subsequently carried out to verify the effects of glnK-repression and sugR-overexpression on l-tryptophan production. As depicted in Fig. 4E, the glnK-repressed strain produced l-tryptophan at a concentration of 55.6 g/L with a yield of 0.182 g/g, which is 10.3 % and 7.2 % higher than that of strain TR26. The sugR-overexpressed strain produced l-tryptophan at a concentration of 58.7 g/L with a yield of 0.204 g/g, which is 16.5 % and 20.2 % higher than that of strain TR26. These results highlight the important roles of glnK and sugR for modulating cellular metabolism and tryptophan biosynthesis.

3.5. Regulation mechanism of glnK on l-tryptophan production

The above results demonstrate that down-regulation of the glnK gene enhances l-tryptophan production in strain TR26. According to the current knowledge, GlnK is a key regulatory protein in the nitrogen metabolism system of C. glutamicum, functioning through its interaction with the global transcription factor AmtR. Under nitrogen-limiting conditions, GlnK is adenylylated by GlnD, which enables it to bind AmtR and derepress the AmtR regulons. A total of 35 genes are regulated by AmtR, including genes involved in ammonium uptake and assimilation (e.g., amt, amtB, glnA, and gltB), urea transport and metabolism (e.g., ureABCDEFG), and signal transduction (e.g., glnK and glnD) (Fig. 5A) [74,75].

Fig. 5.

Regulation mechanism of GlnK. (A) The nitrogen regulation network mediated by GlnK in C. glutamicum. Arrowheads indicate signal transduction; solid lines with blunt ends indicate regulation of enzyme activity; dashed lines indicate expression regulation. (B) (C) Transcription levels of genes related to nitrogen utilization. NC, TR26 containing pD9SG plasmid with non-targeting sgRNA; glnK, TR26 with down-regulated glnK gene mediated by CRISPRi.

Comparative transcriptomic analysis between the TR26 strain containing non-targeting sgRNA plasmid (NC) and the strain with glnK down-regulation revealed that repression of glnK led to significant down-regulation of the AmtR regulons (Fig. 5B and C), consistent with previous studies [74,75]. Notably, the transcription level of the glutamine synthase gene glnA was reduced by 51.1 % (Fig. 5C). These findings suggested a lower capacity for nitrogen uptake and assimilation, as well as l-glutamine synthesis in the glnK-repressed strain. Given the central role of nitrogen metabolism in cellular resource reallocation, this shift may indirectly favor flux redistribution toward l-tryptophan biosynthesis, highlighting a potential link between nitrogen metabolism and aromatic amino acid production.

To further investigate metabolic consequences of glnK down-regulation, intracellular amino acid concentrations were analyzed. As shown in Tables 2 and in strain TR26 with repressed glnK, the concentrations of l-glutamate and l-glutamine were reduced to 87 % and 34 %, respectively, compared to the control group. In addition, levels of other nitrogen-rich amino acids—such as l-arginine, l-proline, and l-tyrosine—were also decreased (Table 3). These results indicate that glnK repression reduces fluxes toward nitrogen assimilation and the biosynthesis of nitrogen-rich amino acids. Given that the fermentation medium contains an abundant nitrogen supply (i.e., ammonium, urea, and corn steep liquor), which can be passively transported into the cell, the observed down-regulation of nitrogen uptake-related genes (Fig. 5B) is unlikely to limit intracellular nitrogen availability. Instead, the transcriptional and metabolic changes suggest a reallocation of cellular resources away from nitrogen acquisition and the synthesis of competing amino acids, potentially favoring flux toward l-tryptophan biosynthesis. This could effectively enhance carbon and energy utilization efficiency for target product formation, offering a potential mechanistic explanation for the improved l-tryptophan yield in the glnK-repressed strain. While further studies are required to confirm this mechanism, our findings provide a basis for connecting the nitrogen metabolism and l-tryptophan production in C. glutamicum.

Table 3.

The effect of glnK repression on intracellular amino acid concentrations.

Amino acid	Fold change	t-test P
l-glutamate	0.87	0.54
l-glutamine	0.34	0.01
l-arginine	0.70	0.001
l-proline	0.50	0.001
l-tyrosine	0.41	0.06

3.6. Regulation mechanism of sugR on l-tryptophan production

The novel genetic target identified in this study for enhancing l-tryptophan production, sugR, encodes a global transcriptional regulator involved in sugar uptake and metabolism in C. glutamicum. SugR functions as a transcriptional repressor of genes encoding the components of phosphotransferase system (PTS), including ptsI, ptsH, ptsF, ptsG, and ptsS [76], as well as key genes in glycolysis pathway such as pfk, fba, and eno [77] (Fig. 6A). Comparative transcriptomic analysis confirmed that sugR overexpression led to a marked reduction in the transcription levels of these PTS and glycolysis genes (Fig. 6B and C). Conversely, the non-PTS sugar uptake-related genes iolT1 and glk were up-regulated by 3.7- and 1.6-fold, respectively, suggesting a compensatory activation of the non-PTS system for sugar transport.

Fig. 6.

Regulation mechanism of SugR. (A) Genes regulated by SugR in C. glutamicum. (B) (C) Transcription level variations of genes related to sugR regulation. Ctrl, TR26 containing empty pEC-K18mob2 plasmid; sugR, TR26 with up-regulated sugR gene.

In C. glutamicum, the PTS is the primary route for sugar uptake and relies on PEP as a phosphoryl donor, thereby competing with l-tryptophan biosynthesis for this essential precursor. In contrast, the non-PTS system utilizes ATP rather than PEP for phosphorylation and is typically inactive under normal conditions. Therefore, the repression of PTS genes and concomitant activation of non-PTS pathways upon sugR overexpression may effectively reduce PEP consumption during sugar uptake, thereby increasing PEP availability for the shikimate and aromatic amino acid biosynthetic pathways. This regulatory shift provides a mechanistic basis for the observed improvement in l-tryptophan production in the sugR-overexpressing strain, establishing sugR as a promising metabolic engineering target for aromatic amino acid production.

4. Conclusion

In conclusion, this study systematically investigated the mechanisms underlying l-tryptophan overproduction in C. glutamicum TR26 and identified novel genetic targets through an integrated comparative metabolomic and transcriptomic analyses. Comparative multi-omics analysis between TR26 and MB001 revealed several key metabolic features contributing to enhanced l-tryptophan synthesis, including the redirection of metabolic flux from the TCA cycle toward l-tryptophan synthesis, enhanced export of l-tryptophan, optimized supply of key precursors PEP and E4P, relief of bottlenecks in the shikimate pathway, and reduced competition from l-tyrosine and l-phenylalanine biosynthesis. Importantly, transcriptomic data guided the identification of two novel regulatory targets, glnK and sugR, while metabolomic profiling validated their functional relevance by linking gene expression changes to shifts in intracellular metabolite levels. Repression of glnK, a regulator involved in nitrogen metabolism, resulted in a 10.3 % increase in l-tryptophan titer and a 7.2 % increase in yield in fed-batch fermentation. This beneficial effect may be attributed to altered nitrogen uptake and assimilation, as well as improved cellular resource allocation favoring aromatic amino acid biosynthesis. Meanwhile, overexpression of sugR, a global regulator of sugar metabolism, led to a 16.5 % increase in l-tryptophan titer and a 20.2 % increase in yield in fed-batch fermentation. By repressing PTS-dependent sugar uptake and activating non-PTS pathways, sugR overexpression effectively reduced PEP consumption during sugar transport, thereby enhancing precursor availability for l-tryptophan biosynthesis. These findings provide multi-layered insights into the regulatory networks governing l-tryptophan overproduction in C. glutamicum, demonstrating the power of combining transcriptomic and metabolomic data for strain improvement in industrial biotechnology.

CRediT authorship contribution statement

Yufei Dong: Writing – original draft, Validation, Methodology, Investigation, Data curation. Rongsheng Gao: Formal analysis, Data curation. Nan Qin: Methodology, Investigation. Kunyu Liu: Methodology, Investigation. Youmeng Liu: Methodology, Investigation. Zhen Chen: Writing – review & editing, Supervision, Resources, Project administration, Investigation, Funding acquisition, Conceptualization.

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.

Appendix A. Supplementary data

The following is the Supplementary data to this article: