Jeffries, JWE; (2017) Functional metagenomics: metagenome mining for industrial biocatalysis. Doctoral thesis , UCL (University College London).

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Abstract

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.

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