Prospecting Microbial Genomes for Biomolecules and Their Applications

Abstract

Bioactive molecules of microbial origin are finding increasing biotechnological applications. Their sources range from the terrestrial, marine, and endophytic to the human microbiome. These biomolecules have unique chemical structures and related groups, which enable them to improve the efficiency of the bioprocesses. This review focuses on the applications of biomolecules in bioremediation, agriculture, food, pharmaceutical industries, and human health.

Introduction

Naturally produced products have been exploited mainly for their therapeutic potential as they possess essential pharmacokinetic characteristics necessary for clinical works [1]. Most bioactive compounds are known for their growth-promoting, antimicrobial, antiviral, and antitumor activities [2, 3]. In addition to these major targets, other molecules are being searched with activities to counter parasites, nematodes, inflammation, and neurological issues [4]. Chemically, these bioactive molecules are small peptides, polyketides, terpenoids, and alkaloids [5]. Diverse environmental niches are actively explored through metagenomic approaches for bioactive compounds [6–8]. The prospecting for bioactive molecules through regular procedures is quite a tedious process, which has been proving to be inefficient and uneconomical. Molecular biology tools, especially metagenomics accompanied by bioinformatics techniques has revolutionized this search for novel biomolecules. A combined approach involving culture-independent analysis, whole genome sequencing, high-throughput data analysis is expected to help search for biomolecules of diverse origins for applications in agriculture, aquaculture, and human health. Most review articles have dealt with one aspect or field at a time. Here, we have comprehensively assessed their applications in diverse fields.

Biomolecules of Diverse Origins

Terrestrial Microbes

Microbes from terrestrial habitats are among the most frequently reported to produce bioactive molecules (Table 1; Fig. 1). Bacterially produced polyhydroxyalkanoate was used to produce 18 compounds through (R)-3-hydroxyoctanoic acid. The ten compounds had antimicrobial and antiproliferative effects on mammalian cell lines. Hyphae formation by Candida ablicans was inhibited by 3-halogenated octanoic acids, whereas quorum sensing (QS)-mediated pyocyanin production by Pseudomonas aeruginosa was inhibited by (E)-oct-2-enoic acids and (R)-3-hydroxyoctanoic [9]. Compounds derived from gut microbes can generate anti-carcinogenic compounds such as pyrogallol and gallic acid [10]. The need to develop novel enzymes, processes and products for biotechnological applications seems to be achievable through metagenomic approaches [11]. Functional metagenomic assays were used to search microbes from soil samples for inhibiting the growth of phytopathogen—Bacillus subtilis DSM10. It helped to identify a putative β-lactamase-like gene, abgT, which had esterase-like hydrolytic activity [12]. Rhizospheric soil microbial DNA-based metagenomic library proved effective in identifying genes encoding for 1-aminocyclopropane-1-carboxylate deaminase [13]. Soil microbiome was reported to be a reservoir containing plasmids, phages, and transposable elements. Antibiotic resistance was among the most prevalent traits in these mobilomes [14]. Using a metagenomic analysis strategy, gene coding for endoglucanase could be cloned from agricultural residues of rice crop. The enzyme was effective against carboxymethylcellulose, showing that it has an endoactive lytic property. The enzyme was used for metabolizing cellulose into sugars for further metabolism to ethanol by Saccharomyces cervisiae with a yield of 8 g/L over a period of 7 days [15]. Metagenomic sources were used to isolate clones carrying genes encoding cellulase with properties that enabled them to function in non-natural and extreme conditions such as high pH, high temperature, and in the presence of solvents [16]. Efforts to obtain new genes through metagenomic approach have been quite fruitful. The enzyme—L-lysine decarboxylase necessary for the decarboxylation of L-lysine to 1,5-pentanediamine was identified from an uncultured organism [17]. Green technologies are being developed to produce it commercially [18]. Bioremediation of soils contaminated with petroleum products is one of the most studied ecosystems. Microbes for hydrocarbon degradation have direct biotechnological applications. Microbes primarily α- and γ- proteobacteria with the ability to degrade xenobiotic aromatic compounds such as bisphenol, xylene, methylnaphthalene, aminobenzoate, chlorobenzoate, DDT, toluene were reported through metagenomic analysis [19]. Compared to aerobic degradation of crude oil, where Actinobacteria, Firmicutes, and Proteobacteria are critical [19], Alcanivorax, Lactococcus, and Bacteroidetes are crucial under anoxic conditions [20, 21]. An in silico analysis of the genome of Corynebacteriums glutamicum, a soil-dwelling microbe, revealed its ability to degrade nitrile-based herbicides [22].

Table 1.

Biomolecules of diverse origins obtained through metagenomic approaches from terrestrial sources

Genomic source	Biomolecule	Applications	References
Bacteria	Polyhydroxyalkanoate to (R)-3-hydroxyoctanoic acid	Antimicrobial and antiproliferative effects on mammalian cell lines	[9]
Gutmicrobes	Pyrogallol and gallic acid	Anti-carcinogenic compounds	[10]
Sediments	Enzymes	Bioprocesses and products	[11]
Microbes from soil samples	β-lactamase-like gene, abgT	Inhibiting the growth of phytopathogen—Bacillus subtilis DSM10	[12]
Rhizospheric soil microbial DNA	Identifying genes encoding for 1-aminocyclopropane-1-carboxylate deaminase	Agricultural productivity	[13]
Soil microbiome	Plasmids, phages, and transposable elements	Prevalent traits (antibiotic resistance) in these mobilomes	[14]
Metagenomic analysis strategy	Gene coding for endoglucanase	Effective against carboxymethylcellulose, showing that it has an endoactive lytic property. Used for metabolizing cellulose (from agricultural residues of rice crop) into sugars to ethanol by Saccharomyces cervisiae	[15]
Metagenomic libraries	Isolated clones carrying genes encoding cellulase	Function in non-natural and extreme conditions such as high pH, high temperature, and in the presence of solvents	[16]
Metagenomic of subtropical soil microbes	New genes from an uncultured organism	The enzyme—L-lysine decarboxylase (for decarboxylation of L-lysine to 1,5-pentanediamine)	[17, 18]
Metagenomics of petroleum-contaminated soil microbiome	Microbes (α- and γ- proteobacteria)	Bioremediation of soils contaminated with petroleum products: aerobic and anoxic conditions	[19]
Whole genome analysis	Genome of Corynebacteriums glutamicum	Bioremediation: ability to degrade nitrile-based herbicides	[22]

Figure 1 Biomolecules of diverse origins obtained through metagenomic approaches from terrestrial and marine sources

Marine Microbes

Among the diverse environmental niches, oceans have the highest diversity of microbes [23]. Marine organisms have been reported to produce compounds anti-inflammatory molecules and anti-pathogenic molecules against fungi, bacteria, parasites, nematodes, and tumors [24] (Table 2; Fig. 1). Microbial genome analysis of Flaviramulus ichthyoenteri isolated from fish intestine showed that it contains genes encoding for AHL lactonase FiaL. This enzyme had little similarity to known lactonases. In addition, other digestive enzymes such as lipases and alginate lyases were also observed [25]. High microbial diversity was found to inhabit the surficial sediments of the Arabian Sea. The presence of secondary metabolites such as PKS biosynthetic genes was reported from 40% of the isolates belonging to Bacillales, γ-proteobacteria and Micrococcaceae. Organic extracts proved bactericidal to human pathogens and cytotoxic to human breast cancer cells (MCF-7) [26]. Marine fungus Oceanitis cincinnatula was found to protect shrimp against acute hepatopancreatic necrosis disease (AHPND) caused by biofilm-forming Vibrio parahaemolyticus [27]. The interest in marine sources for using bioactive compounds for biotechnological applications through metagenomic approaches has been increasing rapidly [28, 29]. Metagenomic library prepared from mangrove soil enable the isolation of β-agarase gene. Polypeptide sequenced deduced from the gene had a high identity with glycoside hydrolase. The purified recombinant protein was active at pH 7.0 and 50 °C. The biochemical activities of the enzyme showed that it could be used in cosmetic, food, and pharmaceutical industries [30].

Table 2.

Biomolecules of diverse origins obtained through metagenomic approaches from marine sources

Genomic source	Biomolecule	Applications	References
Marine organisms	Anti-inflammatory molecules and anti-pathogenic molecules	Against fungi, bacteria, parasites, nematodes, and tumors	[24]
Bacterial whole genome Flaviramulus ichthyoenteri (isolated from fish intestine)	Acylhomoserine lactone lactonase (AHLase) FiaL along with digestive enzymes such as lipases and alginate lyases	Quorum sensing inhibitors as antipathogen in aquaculture	[25]
Microbes from surficial sediments of the Arabian Sea	Secondary metabolites such as PKS biosynthetic genes	Bactericidal to human pathogens and cytotoxic to human breast cancer cells (MCF-7)	[26]
Marine fungus Oceanitis cincinnatula	Antibiofilm (of Vibrio parahaemolyticus)	Aquaculture: Protect shrimp against acute hepatopancreatic necrosis disease (AHPND)	[27]
Metagenomic library from mangrove soil	Isolation of β-agarase gene for glycoside hydrolase	Recombinant protein was active at pH 7.0 and 50 °C. Useful in cosmetic, food, and pharmaceutical industries	[30]
Marine library	QS inhibitors—AHLase	Inhibit biofilm formation by Pseudomonas aeruginosa	[31]
Screening Metagenomic libraries	Secondary metabolite gene clusters for phosphopantetheinyl transferase (PPTase).	Necessary for activation of polyketide synthase (PKS) and Non-ribosomal peptide synthetase	[33, 34]
Metagenomic library from marine water sample	Identification of a novel peptide with antifungal properties	Peptide had a chitin-binding characteristic	[35]
metagenomic approach using ‘Candidatus Endoecteinascidia frumentensis’	Ecteinascidin 743 (ET-743)	Anti-cancer compound	[36]
Metagenomic fosmid library from marine sediments	alkaline esterase, Est12	Active at low temperatures	[37]
Metagenomic fosmid library from marine sediments	Class 3 lipase	Specific towards short and medium-length p-nitrophenyl esters especially p-nitrophenyl acetate	[38]
Ocean floor Petroleum reservoir	Cellulase	Source of thermostable enzymes	[39]

Using marine library, QS inhibitors—acylhomoserine lactonases (AHLase) with the ability to inhibit QS mediated biofilm formation by P. aeruginosa were identified using a lacZ reporter fused to AHL synthase promoter [31]. A wide range of natural products of microbial origin have been reportedly discovered through genomic libraries from marine environments [28, 32]. Gene clusters responsible for producing natural products were recovered from metagenomic libraries prepared for screening phosphopantetheinyl transferase (PPTase). These enzymes are necessary for activation of enzymes—polyketide synthase (PKS) and Non-ribosomal peptide synthetase, thus are important for activities of secondary metabolite gene clusters [33, 34]. Screening of a metagenomic library from marine water sample helped in the identification of a novel peptide with antifungal properties. The peptide had a chitin-binding characteristic [35]. Efforts to extract natural products from marine microbes have been based on metagenomics, genomics, and synthetic biology [29]. Another anti-cancer compound -Ecteinascidin 743 (ET-743), was detected using metagenomic approach using ‘Candidatus Endoecteinascidia frumentensis’ [36]. Marine sediment samples used for preparing metagenomic fosmid library was productive in identifying an alkaline esterase, Est12, active at low temperatures [37], class 3 lipase was reported to be more specific towards short and medium length p-nitrophenyl esters especially p-nitrophenyl acetate. The enzyme had an optimum pH of 8.0 and a temperature optimum of 35 °C [38]. A petroleum reservoir below the ocean floor near Norway was reported to be a good source of thermostable cellulase enzymes [39].

The initial gains achieved through metagenomic studies have been appearing to slow down. The investments made so far have not proven to be effective, especially when compared to those derived from terrestrial plants and animals [40]. Metagenomics as a tool has been instrumental in elucidating natural secondary metabolite producers from terrestrial samples, however, ecological niches in marine sources are yet to be explored further for proving beneficial to the pharmaceutical industry [41, 42].

Plant Endophytes

Endophytes existing within plant tissues are most abundant in rainforests. Endophytes have been a source of novel bioactive metabolites [43, 44]. Endophytes as sources of bioactive molecules such as emodin, hypericin, deoxypodophyllotoxin, podophylloyoxin, camptothecin and its analogs, and paclitaxel have been widely reported [45]. However, the production of these compounds on commercial scale is awaited. To overcome the difficulties encountered in reproducing endophytes under laboratory conditions, genome mining has been proposed. It is expected to elucidate the genes responsible for biosynthesis of already known compounds [46]. Multiple genes encoding for the enzymes anthranilase synthases, responsible for the synthesis of acridine, quinoline, and quinazoline alkaloids were reported to be present in the genome of Aspergillus nidulans [47]. Metagenomic DNA extract of endophytic fungi, endophytic to Rhododendron tomentosum was found to code for genes responsible for producing antioxidant and antibacterial compounds [48]. Metagenomic analysis of fungal endophytic communities associated with Paulownia spp. revealed that the most abundant was Olpidiomycota (82.66%) [49]. A similar study has shown that the fungal community was dominated by Dysphania ambrosioides (Zygomycota) and Ascomycota (100%) [50]. Recent work has revealed culturable endophytic bacterial taxa generally belong to Enterobacter, Pontea, Pseudomonas, Stenotrophomonas, Bacillus, Paenibacillus, Staphylococcus, Microbacterium, and Curtobacterium [51].

Human Microbiome

A diversity of bioactive microbial products (BAMPs), primarily metabolites and proteins, play key functional roles in the metabolisms of living organisms [52, 53]. Around 80% of the anti-cancer drugs and nearly 50% of FDA-approved drugs released between 1981 and 2010 have originated from natural products [54]. Bioactives within the human gut include molecules such as short-chain fatty acids, secreted and surface peptides and sugars and processes like antimicrobial activity, microbial metabolisms, microbial signaling, immune activation and inflammation, and host-microbe cross-talk [55, 56]. In fact, natural products have been instrumental in the advancement of basic research [57]. Human health has been shown to be greatly influenced by the microbiome it carries within the body and on skin surface [58]. The symbiotic relationships between humans and bacteria harbored by them extend to numerous biosynthetic genes and important biological functions. Bacterial populations in a niche are regulated by post-translationally modified peptides as natural products [59]. These compounds include thiopeptides, thiazole/oxazole-modified microcins, microcins, bacteriocins, and lantibiotics [60]. Biosynthetic gene clusters from microbial genomes associated with a human were identified through 752 metagenomic samples. Antibiotics like thiopeptides were reported to be widely distributed in metagenomes and genomes of the human microbiota. Lactocilin—a thiopeptide antibiotic, was produced by microbes associated with the vagina. It was effective in inhibiting a wide range of Gram-positive pathogens. The study demonstrated that bacteria with the ability to produce drug-like molecules are produced by human microbiota [61]. The role of gut microbiota on human health has been gaining importance. Microbial interaction with human host metabolism affects a diversity of functions such as development, immunity, behavior, and dysbiosis leading to disease conditions [53]. Among the diverse community of microbiota present in a human host, the search for specific effectors that influence the bacteria-host interaction was analyzed by examining clones of 3000 Mb of metagenomic DNA. The search was targeted toward those which activate NF-kB transcription factor, which is instrumental in mediating the response to environmental factors [62]. One of the effector gen family (Cbeg 12) was reported to encode for producing commendamide, which resembled a mammalian signaling molecule. It was found to activate G-protein-coupled receptor G2A/GPR132, which was implicated in diseases such as autoimmunity and atherosclerosis [62].

The Prospects

The progress in producing bioactive compounds in large quantities is blocked at the clinical stage since the amount of drug required for clinical trials is large [63]. Extensive efforts in high throughput sequencing have still not been able to facilitate the recovery of bioactive molecules. It is primarily due to the tremendous diversity and the complexity of soil microbiomes. To overcome these challenges, in situ enrichment techniques and metagenomic tools need to be combined to recover and reconstruct microbial genomes [64]. Primarily, in the case of marine organisms, we cannot rely on bioprospecting alone, and we may need to focus on metagenomics [65]. Predictive bioinformatic tools and high-throughput fluorescence methods were used to mine novel constitutive promoter sequences, which belonged to diverse phylogenetic groups [66]. In addition, metabolic engineering and heterologous expression in different hosts with high growth rates can prove helpful [67]. Although gene expression in E. coli has been widely reported, however, other organisms such B. subtilis, Pseudomonas spp., Streptomyces spp., or some eukaryotic expression systems could be employed as alternatives [68].

The human microbiome is being studied quite vigorously. However, there are still wide gaps in our comprehension of the functions of natural products which have been reported to be made by the human microbiome [69]. The need is to prioritize the targets. Secondary metabolites produced by skin, lungs and various other organs need to be analysed and co-related with microbes associated with human systems [70, 71]. The need is to integrate the abilities of researchers from diverse fields and use diverse approaches to resolve the actual potential of natural bioproducts.

Conclusion

Despite persistent efforts, a majority of the microbial diversity is still unknown. It, however, enthuses the necessary confidence in researchers that much is still untapped. Another feature that enhances this enthusiasm among researchers is the discovery of novel biotechnological tools which unravel the non-culturable microbial diversity. High throughput sequencing facilities integrated with predictive bioinformatic tools to analyze an enormous amount of data are proving instrumental in meeting the challenges posed during the prospecting of diverse sources for isolating novel bioactive compounds. We can envisage that the multi-disciplinary approach will help us realize the potential of natural bioproducts in various biotechnological applications.

Declarations

Conflict of interest

All authors declare that there is no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within five years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work.