Discovery of New Carbonyl Reductases Using Functional Metagenomics and Applications in Biocatalysis

Enzyme discovery for use in the manufacture of chemicals, requiring high stereoselectivities, continues to be an important avenue of research. Here, a sequence directed metagenomics approach is described to identify short chain carbonyl reductases. PCR from a metagenomic template generated 37 enzymes, with an average 25% sequence identity, twelve of which showed interesting activities in initial screens. Six of the most productive enzymes were then tested against a panel of 21 substrates, including bulkier substrates that have been noted as challenging in biocatalytic reductions. Two enzymes were selected for further studies with the Wieland Miescher ketone. Notably, enzyme SDR-17, when co-expressed with a co-factor recycling system produced the anti-(4aR,5S) isomer in excellent isolated yields of 89% and 99% e.e. These results demonstrate the viability of a sequence directed metagenomics approach for the identification of multiple homologous sequences with low similarity, that can yield highly stereoselective enzymes with applicability in industrial biocatalysis. (Figure presented.)

A metagenomics approach for new biocatalyst discovery: application to transaminases and the synthesis of allylic amines

Transaminase enzymes have significant potential for the sustainable synthesis of amines using mild aqueous reaction conditions. Here a metagenomics mining strategy has been used for new transaminase enzyme discovery. Starting from oral cavity microbiome samples, DNA sequencing and bioinformatics analyses were performed. Subsequent in silico mining of a library of contiguous reads built from the sequencing data identified 11 putative Class III transaminases which were cloned and overexpressed. Several screening protocols were used and three enzymes selected of interest due to activities towards substrates covering a wide structural diversity. Transamination of functionalized cinnamaldehydes was then investigated for the production of valuable amine building blocks

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

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

Functional metagenomics: metagenome mining for industrial biocatalysis

Biocatalysts are increasingly being used for organic synthesis in industry and novel enzymes are needed to meet this growing demand. Metagenomics is an emerging field of study that has the potential to be a source for the diversity and number of enzymes needed by industry. To explore the potential of a sequence directed metagenomics approach, microbial DNA extracted from the human oral cavity was sequenced and used to create an in silico metagenomic library. This in silico library was used as the template from which primers could be designed to retrieve DNA sequences coding for enzymes from the oral metagenomic DNA. Subsequently a second metagenomic sample from a drain was sequenced and processed into an in silico library. A range of enzyme classes were targeted for retrieval, Lactate/Malate dehydrogenase, Transketolase, Transaminases and Carbonyl reductases. A variety of cloning methodologies were tested, to streamline the process between identification, amplification and expression of the genes, in order to maximise the number of active enzymes that could be retrieved from the metagenome. In total 100 genes were identified for retrieval from the two metagenomes, 69 of which were successfully cloned. From the 69 gene sequences retrieved, 29 translated into active enzymes. This compares favourably with contemporary methodologies for metagenomic enzyme discovery based on the screening of clone libraries. An average value for the hit rate of clone based screening is 1 active enzyme for every 6000 clones screened. Using the number of contigs in the in silico library as a proxy for the number of clones screened, the hit rate of the sequence directed methodology is 1 active enzyme per 1999 “clones” screened. When compared with other sequence directed methodologies that rely on degenerate primers for retrieval of genes, the numbers of genes amplified are comparable. However the sequence specific primers used in the research presented here retrieve a wider variety of sequence. Degenerate primers retrieve genes with high sequence similarity, 90-99% homology. Sequence specific primers used in this research have retrieved genes from the same enzyme class with sequence identity as low as 15-20%. Retrieval of targeted genes from the two metagenomes and the assay of active enzymes stand as proof of principle, that a sequence directed method gives constructive access to metagenomes and importantly improves on the numbers, types and diversity of enzymes produced when compared with other metagenomic screening methods