Construction of pha‐Operon‐Defined Knockout Mutants of Pseudomonas putida KT2442 and their Applications in Poly(hydroxyalkanoate) Production

Abstract

Pseudomonas putida KT2442 could accumulate medium-chain-length poly(hydroxyalkanoate)s (PHA) consisting of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate from a wide range of carbon sources. In this study, the PHA synthase pha operon (phaC1-phaZ-phaC2) was knocked out and the vgb gene encoding vitreoscilla hemoglobin protein (VHb), which could enhance oxygen uptake rate especially at low oxygen concentration, was integrated into the P. putida KT2442 genome to replace the deleted fragment. The resulting mutant P. putida KTOY01 or gene-replaced mutant KTOY02 was used as the host to study PHA synthase properties and PHA production. Different PHA polymerase (PhaC) genes, phaC(Re) from Rastonia eutropha H16, phaC(Ac) from Aeromonas cavie, and phaC2(Ps) from Pseudomonas stutzeri 1317, were expressed in the mutant strains to test the PhaC enzyme substrate specificity. The result showed P. putida KTOY01 or KTOY02 could provide not only mcl PHA monomers but also 3-hydroxybutyrate from fatty acids, which may allow the production of copolyesters poly(3HB-co-mcl 3HA). Plasmid pCJY10 containing phaC2(Ps), phbA, and phbB genes encoding PHA polymerase, beta-ketothiolase, and acetoacetyl-CoA reductase, respectively, were transformed into P. putida KTOY01 and KTOY02. Shake-flask culture showed P. putida KTOY01 or KTOY02 (pCJY10) could accumulate poly(3HB-co-mcl 3HA) from glucose. The above result showed pha operon knockout mutant of P. putida KT2442 was a very useful host of great potential not only for studying PhaC synthase, but also for microbial production of copolyesters poly(3HB-co-mcl 3HA), which is very difficult to obtain.

A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate

“…A strong reduction of specific growth rate was observed for P. putida KT2442 as well as a smaller growth yield on carbon and difficulties to cope with nitrogen starvation. P. putida KT2442 is often preferred over P. putida KT2440 to generate metabolically engineered organisms with enhanced production of mcl-PHA because the procedure is simplified by the rifampicin resistance [17,35]. However, the productivity of mcl-PHA - which is correlated to its cost - depends not only on the polymer content in the cells but also on their growth rate.…”

Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production, biocompatibility, biodegradation, physical properties and applications

“…Second, the metabolic background is quite different between E. coli and native PHA production strains. Many PHA-producing strains can accumulate PHA from fatty acids, while E. coli grows poorly on fatty acid culture medium (289,290). Therefore, application of E. coli as the host to produce PHA has not been the best option.…”

Production of Polyhydroxyalkanoates Copolymers by Recombinant <b><i>Pseudomonas</i></b> in Plasmid- and Antibiotic-Free Cultures

“…LFM046 and LFM461 would lead to the same biopolymer production. A small amount of P(3HB) (0.14 g/L) and no HA MCL monomers were detected in the study of Ouyang et al [2007] when a plasmid-encoded copy of phaC Re was expressed in a PHA-negative strain of Pseudomonas putida KT2442. R. eutropha naturally accumulates P(3HB) when grown in a narrow range of carbohydrates [Sichwart et al, 2011] and can co-polymerize small fractions of 3HHx and 3HO; but only when supplied with related carbon sources including hexanoic or octanoic acids [Liebergesell et al, 1991].…”