ABSTRACT
Glycolate (GL)-containing polyhydroxyalkanoate (PHA) was synthesized in Escherichia coli expressing the engineered chimeric PHA synthase PhaC AR and coenzyme A transferase. The cells produced poly[GL-co-3-hydroxybutyrate (3HB)] with the supplementation of GL and 3HB, thus demonstrating that PhaC AR is the first known class I PHA synthase that is capable of incorporating GL units. The triad sequence analysis using 1H nuclear magnetic resonance indicated that the obtained polymer was composed of two distinct regions, a P(GL-ran-3HB) random segment and P(3HB) homopolymer segment. The random segment was estimated to contain a 71 mol% GL molar ratio, which was much greater than the value (15 mol%) previously achieved by using PhaC1 P s STQK. Differential scanning calorimetry analysis of the polymer films supported the presence of random copolymer and homopolymer phases. The solvent fractionation of the polymer indicated the presence of a covalent linkage between these segments. Therefore, it was concluded that PhaC AR synthesized a novel random-homo block copolymer, P(GL-ran-3HB)-b-P(3HB).
ABSTRACT
A two-stage chemostat cultivation was used to investigate the biosynthesis of functionalized medium-chain-length polyhydroxyalkanoate (mcl-PHA) in the ß-oxidation weakened strain of Pseudomonas putida KTQQ20. Chemostats were linked in sequence and allowed separation of biomass production in the first stage from the PHA synthesis in the second stage. Four parallel reactors in the second stage provided identical growth conditions and ensured that the only variable was the ratio of decanoic acid (C10) to an unusual PHA monomer precursor, such as 10-undecenoic acid (C11:1) or phenylvaleric acid (PhVA). Obtained PHA content was in the range of 10 to 25 wt%. When different ratios of C10 and C11:1 were fed to P. putida, the produced PHA had a slightly higher molar ratio in favor of C11:1-based 3-hydroxy-10-undecenoate. However, in case of PhVA a significantly lower incorporation of 3-hydroxy-5-phenylvalerate over 3-hydroxydecanoate took place when compared to the ratio of their precursors in the feed medium. A result that is explained by a less efficient uptake of PhVA compared to C10 and a 24% lower yield of polymer from the aromatic fatty acid ( y P H A - M P h V A = 0.25). In addition, PHA isolated from cultivations with PhVA resulted in the number average molecular weight M n ¯ two times lower than the PHA produced from C10 alone. Detection of products from PhVA metabolism in the culture supernatant showed that uptaken PhVA was not entirely converted into PHA, thus explaining the difference in the yield polymer from substrate. It was concluded that PhVA or its related metabolites increased the chain transfer rate during PHA biosynthesis in P. putida KTQQ20, resulting in a reduction of the polymer molecular weight.
