Overproduction of MCL-PHA with high 3-Hydroxydecanoate Content (Biotechnology and Bioengineering)

Methods of producing medium-chain-length poly-3-hydroxyalkanoate (mcl-PHA) with high content of the dominant subunit, 3-hydroxydecanoate (HD), were examined with an emphasis on a high yield of polymer from decanoic acid. High HD content was achieved by using a ?-oxidation knockout mutant of Pseudomonas putida KT2440 (designated as P. putida DBA-F1) or by inhibiting ?-oxidation with addition of acrylic acid to wild type P. putida KT2440 in carbon-limited, fed-batch fermentations. At a substrate feed ratio of decanoic acid and acetic acid to glucose (DAA:G) of 6:4 g/g, P. putida DBA-F1 accumulated significantly higher HD (97 mol%), but much lower biomass (8.5 g/L) and PHA (42% of dry biomass) than the wild type. Both biomass and PHA concentrations were improved by decreasing the ratio of DAA:G to 4:6. Moreover, when the substrate feed ratio was further decreased to 2:8, 18 g/L biomass containing 59% mcl-PHA consisting of 100 mol% HD was achieved. The yield of PHA from decanoic acid was 1.24 (g/g) indicating that de novo synthesis had contributed to production. Yeast extract and tryptone addition allowed the mutant strain to accumulate 74% mcl-PHA by weight with 97 mol% HD at a production rate of 0.41 g/L/h, at least twice that of published data for any ?-oxidation knock-out mutant. Higher biomass concentration was achieved with acrylic acid inhibition of ?-oxidation in the wild type but the HD content (84 mol%) was less than that of the mutant. A carbon balance showed a marked increase in supernantant organic carbon for the mutant indicating overflow metabolism. Increasing the dominant monomer content (HD) greatly increased melting point, crystallinity and rate of crystallization. This article is protected by copyright. All rights reserved