Biosurfactants for a Sustainable Future. Группа авторов
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2.7 Functional Metagenomics: Challenge and Opportunities
Sequence/homology‐based screening is routinely used to screen metagenomic DNA using designed PCR primers or through NGS. A sequence‐based approach is primarily based on shotgun sequencing of target genes from a library of clones to look at the important metabolic pathways [72–74]. The main advantage of shotgun sequencing is that the entire genome can be reconstructed from identified fragments of library clones to determine the biosynthetic pathway [57, 73, 75]. However, shotgun sequencing may not be a viable option for determining the functional aspects of a complex microbial population or those present in low abundance [58]. Functional metagenomics‐based screening has several advantages over sequencing‐based screening. The main advantage is that novel genes or their functional products can be traced without prior knowledge of gene sequences [15]. Heterologous gene expression is one of the challenges faced by functional metagenomics. Studies suggest that sizeable fractions of the target genes are insufficiently expressed in the expression host [15]. This may be due to a lack of optimal codon usages by host transcriptive machinery, discrepancy during protein synthesis and processing, lack of a suitable substrate required for a biosynthetic pathway, the toxic nature of the gene products, or other unknown associated factors [56]. In order to evaluate a complete biosynthetic pathway, a single metagenomic clone containing all the genes for the pathway is needed. Moreover, in order to represent the entire metagenome, the library should have a sufficient number of clones, taking into account the diversity of the community.
2.7.1 Single vs Multiple Host Expression System
The expression of genes in a metagenomics library by a single host expression system is a widely used strategy in recent times. The potentiality of an expression host is determined by its ability to replicate the vectors containing inserted DNA fragments, impeding recombination, and conferring resistance to background gene products and lytic phages. Escherichia coli is the most favored host system for the expression of foreign genes. However, only 40% of the genes with functional activity have been reported to express in an E. coli host system [15]. The metabolic potential of most functional genes of remotely related microbes may not be sufficiently expressed [56]. This may also be attributed to the lack of an appropriate biosynthetic pathway substrate in a single host or to the fact that the host transcription machines may not recognize the sequence of promoters or favor the expression of foreign genes by limiting the essential factors [19]. However, some of the shortcomings of the E. coli system are rectified by augmenting it with plasmids equipped with an additional tRNA gene, simultaneous expression of chaperonin genes, etc. [76, 77].
In order to mitigate the limitations of a single host, multiple hosts are used to increase the likelihood of expression of targeted genes. Bacillus, Burkholderia, Sphingomonas, Streptomyces, and Pseudomonas [78] are the alternate host systems used for the functional screening of metagenomic libraries. Parallel screening using multiple hosts allows the successful expression of gene products from the Metagenomic Clone Library by providing a diverse host cell environment. Furthermore, a multiple host system permits the screening of metagenomic libraries for biosynthetic pathways that may remain undiscovered through a single host expression system. Despite all these limitations and prospects, functional screening using multiple hosts is one of the viable options in discovery of novel biosurfactants from oil contaminated environments.
2.7.2 Metagenomic Clone Libraries
In addition to the transcriptive machinery of the host, the expression of genes in a metagenomic library host also depends on the DNA insert size [56]. The selection of an appropriate vector based on the nature of the host expression is another hurdle in functional metagenomics. Small insert sizes may limit the detection of important biosynthetic pathways required for novel biomolecule synthesis. Fosmid and Cosmid libraries could accommodate insert sizes of ~15–40 kb, which may be limiting for cloning large size DNA fragments. Large insert libraries like BACs are preferred as they permit the cloning of genes for entire biosynthetic pathways of targeted biomolecules [79]. BACs (Bacterial Artificial Chromosome) can accommodate 100–200 kb of DNA fragments, rendering them suitable for metagenomic libraries. However, maintaining large size metagenomic DNA with a high molecular weight is the other challenge in functional screening. Owing to difficulties such as this in screening large clones, newer technologies like fluorescence‐based assay are gaining importance for the rapid detection of an enzymatic activity. Furthermore, use of a robotic assay simplifies the screening process in high‐throughput screening within a short time period [19].
2.8 Conclusion
Although the metagenomic approach for detecting microbes that produce novel biosurfactants is still at a growing stage, there is an ample opportunity to use these molecular tools in the future. As of now, these techniques have been used in the collection of environmental DNA samples for research purposes. In the above sections, we discussed metagenomic approaches to microorganism screening, which produce biosurfactants with add‐on techniques to overcome barriers faced by conventional metagenomics. Emphasis has been placed on the synergy of techniques and methodologies with a functional or sequence‐based approach for more favorable outcomes. We present how DNA‐SIP coupled with metagenomic strategies can help identify specific microbes of interest. The technique could be exploited in screening microbes or genes producing novel biosurfactants from oil‐contaminated soil. In addition to the above, we emphasize the characteristics and limitations of biosurfactant screening methods. The challenges and opportunities for functional metagenomics have been presented in detail.
Acknowledgements
The opinions, ideas, conceptions, and design presented in this chapter are of the authors themselves. The authors are grateful to NNS College for providing research and logistics facilities. The authors are grateful to Professor M.N.V. Prasad for his critical suggestions and inputs during the preparation of this chapter.
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