In vivo production of medium-chain-length poly(3-hydroxyalkanoates) (MCL-PHA) containing a side chain carboxyl group from azelaic acid (AzA), a nine-carbon α,ω-dicarboxylic acid, was investigated using Pseudomonas citronellolis DSM 50332 in a phosphate (P)-limited chemostat. Co-feeding with nonanoic acid (NA) and inhibition of β-oxidation with acrylic acid (AA) were strategies that were used to stimulate the incorporation of carboxylated monomers, but both were unsuccessful. P. citronellolis DSM50332 was capable of growing on AzA as a sole source of carbon and energy, indicating that enzymes in β-oxidation utilized AzA and its derivatives. However, the MCL-PHA produced from AzA comprised 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) monomers, which was consistent with precursor supplied via the de novo fatty acid biosynthesis pathway. This evidence suggests that one or more of 3-ketoacyl-CoA reductase (FabG), enoyl-CoA hydratase (PhaJ) and PHA synthase (PhaC) of this organism do not have the low specificity required to utilize a carboxylated substrate. Future work involving mutations may broaden the substrate specificity of these key enzymes to overcome this obstacle. Two-stage high-cell density carbon (C)-limited chemostat cultivation of P. putida KT2440 was examined for MCL-PHA production from nonanoic acid (NA) at high intracellular polymer content and volumetric productivity. Growth conditions stimulating good PHA production were first established in single-stage chemostat, which yielded 63.1 wt% PHA containing 90 mol% C9 units and a productivity of 1.52 g L-1h-1 at a dilution rate of 0.30 h-1. This productivity was higher than any value reported in literature for continuous MCL-PHA production systems and comparable to the upper range of fed-batch results. Two-stage production yielded promising results, notably the increase in polymer content from the first to second stage. However, complications involving foaming and an unexplained decline in PHA content adversely affected system performance. The best PHA content and overall productivity were 58.5 wt% and 0.76 g L-1h-1, respectively. Nonetheless, the results demonstrate the potential to achieve high PHA content without the need for pure oxygen at high dilution rates, warranting further investigation focusing on the optimization of growth conditions. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-12-19 15:48:21.808

The metabolism and physiology of Pseudomonas putida strain LS46 was investigated using biodiesel-derived waste streams as potential low cost substrates for production of medium chain length polyhydroxyalkanoates (mcl-PHA). Proteomic and trranscriptomic analyses were used to correlate specific gene and gene product expression patterns with differences in phenotypes of mcl-PHA biosynthesis by P. putida LS46. Growth and mcl-PHA content of P. putida LS46 were similar in cultures containing biodiesel-derived waste glycerol versus pure glycerol, and mcl-PHA synthesis occurred during stationary phase after nitrogen concentrations in the medium were exhausted. Waste glycerol cultures contained elevated concentrations of heavy metal ions, such as copper, which induced significant changes in gene expression levels related to heavy metal resistance. Several membrane-bound proteins, such as CusABC efflux and CopAB were identified and putatively play a role in regulating cellular copper concentrations. Cultures containing waste free fatty acids synthesized mcl-PHA throughout the exponential growth phase. Protein expression levels of two mcl-PHA synthases were suppressed during exponential phase growth in waste glycerol cultures, putatively via post-transcriptional regulation. Culture specific expression of monomer supplying proteins (PhaJ1 and PhaG), and sets of fatty acid oxidation enzymes were observed, and may have contributed to differences in the composition of polymers synthesized by P. putida LS46 cultured on the two substrates. Expression levels of the majority of mcl-PHA biosynthesis pathway genes were stable during active polymer synthesis in waste glycerol cultures. However, variations in protein expression levels, and in some cases their corresponding mRNAs, were observed in a number of other metabolic patheays, such as glycerol transportation, partial glycolysis, pyruvate metabolism, the TCA cycle, and fatty acid biosynthesis. These data suggest potential regulatory points that may determine carbon flux during mcl-PHA biosynthesis. Evaluation of identified genetic targets in P. putida LS46 that putatively influence mcl-PHA biosynthesis and monomer composition merit further studies. / February 2016