Metagenome Mining: A Sequence Directed Strategy for the Retrieval of Enzymes for Biocatalysis

Abstract

Biocatalytic reactions are increasingly being used as a sustainable strategy in organic synthesis and it is recognised that there is need for new enzyme discovery. To establish the utility and versatility of a metagenomics approach, metagenomic DNA extracted from the oral cavity was sequenced and used to create an in silico contig library. This enables individual open reading frames, operons or all the enzymes of a particular family to be identified and then retrieved from the original DNA by PCR. As proof of principle a lactate dehydrogenase, a malate dehydrogenase and transketolases were identified in silico, successfully cloned and assayed. This new enzyme retrieval sequence directed method gives constructive access to metagenomic diversity and importantly improves on the low hit rate experienced when using conventional metagenomic screens.Over the last 30 years enzymes have increasingly been used in commercial chemical processes. [1] Enzyme catalysts have distinct advantages over chemical catalysts such as cost, sustainability, low toxicities and the use of moderate reaction conditions. The greatest advantage however, is the wide range of catalytic activities displayed by naturally occurring enzymes and the stereoselectivities that can be achieved. Drawbacks when using enzymes can include a low organic solvent tolerance, narrow pH working ranges, and low thermostability. However these properties along with enhanced stereoselectivities can be engineered using random or directed evolution techniques. [2] One of the key requirements for this type of engineering is multiple amino acid sequences. Having many enzymes that cat-alyze the same reaction, with variation in their primary sequence is a much more effective starting point for the engineering process than any single example of an enzyme family. One successful method for identifying large numbers of novel enzymes has been to mine fully sequenced and annotated genomes of laboratory cultivable strains. Such sequence directed genome mining lends itself to the retrieval of multiple targets, however a limitation to this method is the number of annotated available species. [3] With only 0.1-5 % of bacteria cultivable in the laboratory, much of the existing diversity of bacteria are inaccessible in this way. [4] At the same time, with the advances in high throughput sequencing, whole or partial genomes of uncultivable bacteria are being made available for study. The nascent field of metagenomics gives insight into the genomes of previously unstudied bacteria and by extension a potential wealth of new biocatalysts for the generation of small molecules and large bioactive molecules such as polyketides and glycopeptides. [5] The use of metagenomics to obtain new enzymes and biocatalysts for industrial applications is steadily growing but still in its infancy. [6] Efforts to capture enzymes from metagenomic samples have relied upon the creation of physical metagenomics libraries in more tractable bacteria. [7] Poor transcription and translation, enzymes with activ...

Metagenomic Mining for Amine Dehydrogenase Discovery

“…Introduced for the first time in 1998, metagenomics is the method of studying the genetic content of an entire community from an environmental sample, circumventing the need to isolate and grow potentially unculturable micro‐organisms . Analyzing the genetic material of a community allows for the identification of potential protein coding regions, which may house novel biocatalysts . Thus far, enzymes used in biocatalytic synthesis discovered through the metagenomics approach are mainly limited to hydrolases, with some examples of transaminases, transketolases, nitrilases and dehydrogenases …”

“…Analyzing the genetic material of a community allows for the identification of potential protein coding regions, which may house novel biocatalysts . Thus far, enzymes used in biocatalytic synthesis discovered through the metagenomics approach are mainly limited to hydrolases, with some examples of transaminases, transketolases, nitrilases and dehydrogenases …”

Metagenomic ene-reductases for the bioreduction of sterically challenging enones

“…18 In a previous study, we have reported a sequence-based functional metagenomics approach to identify novel biocatalysts. 19,20 Microbial communities from the human oral cavity and more recently from a domestic drainpipe were investigated. [19][20][21] The drain proved to be an interesting niche providing a highly valuable dataset for bioprospecting and led to the identication of native enzymes with notable characteristics, such as organic solvent stability oen only observed in highly engineered enzymes.…”

“…19,20 Microbial communities from the human oral cavity and more recently from a domestic drainpipe were investigated. [19][20][21] The drain proved to be an interesting niche providing a highly valuable dataset for bioprospecting and led to the identication of native enzymes with notable characteristics, such as organic solvent stability oen only observed in highly engineered enzymes. 21 In the present study, in silico searches of the drain metagenome led to the identication of eight novel and active ERs from the OYE family ( Fig.…”

Computational modelling of enzymes: predicting and understanding the selectivity of an epoxide hydrolase

“…Through the use of (meta)genomic screening methods, this largely untapped resource will likely yield many industrially useful enzymes [314,[336][337][338][339][340][341][342]. Furthermore, the protein sequences that are discovered during this "genome mining" will improve the accuracy of computational models in predicting protein structures [343], allowing a better determination of the relationship between structure and function.…”