1 3 J Ind Microbiol Biotechnol (2015) 42:637–646 DOI 10.1007/s10295-014-1574-5 METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates by a phaZ-knockout strain of Pseudomonas putida KT2440 Minh Tri Vo · Kenton Ko · Bruce Ramsay Received: 3 November 2014 / Accepted: 19 December 2014 / Published online: 7 January 2015 © Society for Industrial Microbiology and Biotechnology 2015 commercial interest due to their biocompatibility and biodegradability [14, 18]. PHAs are classified as short- chain-length PHAs (SCL-PHAs) when the pendant group of monomer varies from 0 to 2 carbons, medium-chain- length PHAs (MCL-PHAs) when there are three or more carbons on the pendant group and SCL–MCL-PHAs when polymers consist of both SCL and MCL monomers [19, 22]. MCL-PHAs are of increasing interest because of their low crystallinity, high elasticity [13, 25] and the possibility of having different functional groups, such as alkenes [21], aromatic groups [12], halogen [9, 16, 20], esters [30] and phenoxy groups [27] on their side chains. Since more than 100 different MCL-PHA monomers can be incorporated, MCL-PHAs can exhibit a wide variety of properties with many possible applications including coatings, medical implants, drug delivery, water-based latex paints and others [39, 40]. PHAs are more expen- sive to produce than conventional plastics with expendi- tures almost evenly divided between carbon source, fer- mentation process and separation process [37]. Metabolic engineering may be used to achieve higher PHA cellu- lar content, more effective carbon source usage, and to obtain novel PHAs with valuable properties, thus increas- ing commercially viability. One such approach is the elimination of PhaZ activity. The phaZ gene encoding PhaZ is located between phaC1 and phaC2 genes of the MCL-PHA metabolism gene clus- ter which consists of phaC1, phaZ, phaC2, phaD, phaF and phaI genes in Pseudomonas putida KT2440 whose entire genome has been mapped [17, 24]. The PhaZ of P. putida KT2442, a spontaneous rif r mutant of P. putida KT2440 [2], is an intracellular MCL-PHA depolymerase and an MCL-PHA granule surface-associated protein [7, 8]. PhaZ is reported to play a crucial role in the turnover of MCL- PHAs under carbon starvation in P. putida KT2442 [5]. Abstract A medium-chain-length poly-3-hydroxyalkanote (MCL-PHA) depolymerase knockout mutant of Pseu- domonas putida KT2440 was produced by double homolo- gous recombination. A carbon-limited shake-flask study confirmed that depolymerase activity was eliminated. Lysis of both mutant and wild-type strains occurred under these conditions. In carbon-limited, fed-batch culture, the yield of unsaturated monomers from unsaturated substrate averaged only 0.62 mol mol -1 for the phaZ minus strain compared to 0.72 mol mol -1 for the wild type. The mutant strain also produced more CO 2 and less residual biomass from the same amount of carbon substrate. However, most results indicated that elimination of PHA depolymerase activity had little impact on the overall yield of biomass and PHA. Keywords phaZ · Knockout · PHA · putida · Depolymerase Introduction Poly-3-hydroxyalkanote (PHA) polyesters are synthe- sized by many bacteria and have attracted extensive M. T. Vo · B. Ramsay (*) Chemical Engineering, Queen’s University, Kingston, ON K7L 3N6, Canada e-mail: Bruce.Ramsay@polyferm.com; bruce.ramsay@queensu.ca K. Ko Biology, Queen’s University, 116 Barrie St., Kingston, ON K7L 3N6, Canada B. Ramsay Polyferm Canada, 4530 Camden-Portland Bdry Rd, RR#1, Harrowsmith, ON K0H 1V0, Canada Downloaded from https://academic.oup.com/jimb/article/42/4/637/5995501 by guest on 24 November 2022