Abstract
Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs 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 PhaCs. 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 short-chain-length (SCL) and medium-chain-length (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.
Highlights
Polyhydroxyalkanoates (PHAs) are natural polyesters biosynthesized by a variety of bacteria under unbalanced growth conditions
Following selection of P. putida PpUW2 transconjugants (~4 million) on LB Km Tc plates supplemented with 0.5% Na octanoate, we obtained 72 clones that exhibited greater opacity than the PpUW2 PHA- recipient strain
Rm11476 containing PHB+ clones pCX92, pCX9M1, pCX9M3 or pCX9M5 (Fig. 4). These data indicated that the phaC116 gene was expressed and its product was functional. These results suggest that P. putida is a more permissive surrogate host than S. meliloti for screening of novel PHA
Summary
Polyhydroxyalkanoates (PHAs) are natural polyesters biosynthesized by a variety of bacteria under unbalanced growth conditions. They serve as reserves of carbon and reducing power and aid in survival during starvation or stress conditions (Verlinden et al 2007). These biodegradable and environmentally friendly polymers can be used as alternative materials to conventional petrochemical-based plastics. MCL-PHA is generally more useful than SCL-PHA due to it being less brittle and more flexible. To produce PHA with versatile properties cost-effectively, strategies have involved mining new PHA synthase enzymes (PhaC), engineering PhaC proteins and modifying the metabolic pathways of the production strains (Keshavarz and Roy 2010; Schallmey et al 2011; Cheema et al 2012; Park et al 2012; Tripathi et al 2013; Meng et al 2014)
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