Expansion of the functional diversity of PHA synthase enzymes through functional metagenomics

Improvements to the bacterial production of PHA bioplastics would benefit from the availability of a diverse collection of PHA synthesis enzymes. We have developed functional screens for isolation of novel PHA synthesis genes from metagenomic cosmid libraries. These screens are based on phenotypic complementation of Sinorhizobium meliloti and Pseudomonas putida PHA synthesis mutants, and rely on changes to colony appearance, exopolysaccharide production, and growth on certain carbon sources. Advantages of using phenotypic screening rather than sequence­ based screening include the potential for isolating genes that would not be predicted through sequence, and the reassurance that the genes on the isolated clones are able to be expressed in the heterologous background. From P. putida complementation, several clones were isolated. Examination of the polymers produced by expression in the heterologous backgrounds revealed a broad diversity, including both short­-chain­-length and medium­-chain­-length forms. The composition varied according to the feedstock provided, and in some cases multiple polymers were produced in the same strain. DNA sequence analysis of the clones indicated that they carried genes encoding Class I, II or III PHA synthase enzymes, as well as other genes known to be involved in the PHA synthesis pathway. Through this work, we have demonstrated the ability to efficiently screen for clones expressing novel PHA synthesis genes that can direct the production of different PHA polymer mixes. The resulting collection of genes represents a resource for the construction of new PHA production strains. Future libraries will be constructed using new vectors that block transcriptional readthrough from insert DNA that likely disrupts replication.