(499b) Metabolic Engineering of Paraburkholderia Sacchari for Improved Bioplastic Co-Polymer Production.

Recent research endeavors have turned to generating useful chemicals from biological platforms as an environmentally responsible alternative to non-sustainable sources. Here we share development of the non-model bacterium, Paraburkholderia sacchari (hereafter P. sacchari), as a microbial factory for production of polyhydroxyalkanoates (PHAs), which serve as precursors for biodegradable plastic. The particular PHAs of interest produced by P. sacchari include poly(3-hydroxybutyrate) (hereafter PHB) and the co-polymer produced by the combination of PHB and 3-hydroxyvalerate (hereafter 3HV) called poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (hereafter PHBV). PHBV is particularly interesting due to its improved toughness and elasticity over other PHAs, making it more competitive with petroleum-based plastics. While growth studies have shown high PHB production by P. sacchari in the presence of single carbon sources, PHBV production can only be achieved with additional co-fed substrates such as proprionate. When proprionate is added to the media, P. sacchari produces PHBV with a 3HV fraction of 28%, however, this is also accompanied by a significant drop in overall PHA accumulation (from 75% of cell dry weight to 45%) and inhibition to growth. Strategies to either engineer an increase in the efficiency of converting proprionate to 3HV could produce PHBV with less growth inhibition thus resulting in a more industrially useful strain. To address this short-coming, we have improved incorporation of proprionate by engineering P. sacchari to upregulate propionyl-CoA transferase, the enzyme which converts proprionate and acetyl-CoA into propionyl-CoA. We have done so using the propionyl-CoA transferase gene (pct) from Cupriavidus necator H16. This strategy in combination with overexpression of acetyl-CoA reductase along the PHB production pathway (using the phaB gene from C. necator) has shown a drastic increase in PHBV production over wild type. Through these methods, we have shown promising progress toward an industrially useful strain of P. sacchari for PHBV production.