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Trevor Charles

Publications and source records attributed to Trevor Charles.

2 recordsLinked to original sources
bioRxiv2016

Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences

A soil metagenomic library carried in pJC8 (an IncP cosmid) was used for functional complementation for {beta}-galactosidase activity in both -Proteobacteria (Sinorhizobium meliloti) and{gamma} -Proteobacteria (Escherichia coli). One {beta}-galactosidase, encoded by overlapping clones selected in both hosts, was identified as a member of glycoside hydrolase family 2. ORFs obviously encoding possible {beta}-galactosidases were not identified in 19 other clones that were only able to complement S. meliloti. Based on low sequence similarity to known glycoside hydrolases but not {beta}-galactosidases, three ORFs were examined further. Biochemical analysis confirmed that all encoded {beta}-galactosidase activity. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain.

Microbiology↗
bioRxiv2016

Functional metagenomics using Pseudomonas putida expands the known diversity of polyhydroxyalkanoate synthases and enables the production of novel polyhydroxyalkanoate copolymers

Bacterially produced biodegradable polyhydroxyalkanoates with versatile properties can be achieved using different PHA synthase enzymes. This work aims to expand the diversity of known PHA synthases via functional metagenomics, and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either Class I, Class II or unclassified PHA synthases, and many did not have close sequence matches to known PHA synthases. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of SCL-and MCL-PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics, and their use for the production of a variety of PHA polymer and copolymer mixtures.

Microbiology↗