Construction of pha‐Operon‐Defined Knockout Mutants of Pseudomonas putida KT2442 and their Applications in Poly(hydroxyalkanoate) Production
- 12 February 2007
- journal article
- research article
- Published by Wiley in Macromolecular Bioscience
- Vol. 7 (2), 227-233
- https://doi.org/10.1002/mabi.200600187
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, phaCRe from Rastonia eutropha H16, phaCAc from Aeromonas cavie, and phaC2Ps 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 phaC2Ps, phbA, and phbB genes encoding PHA polymerase, β‐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.Keywords
This publication has 22 references indexed in Scilit:
- Poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) Production in Recombinant Aeromonas hydrophila 4AK4 Harboring phbA, phbB and vgb GenesMacromolecular Symposia, 2005
- Biosynthesis of Polyhydroxyalkanoate (PHA) Copolymer from Fructose Using Wild‐Type and Laboratory‐Evolved PHA SynthasesMacromolecular Bioscience, 2005
- Molecular cloning and functional analysis of two polyhydroxyalkanoate synthases from two strains ofAeromonas hydrophilaspp.FEMS Microbiology Letters, 2005
- EngineeredAeromonas hydrophilafor enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with alterable monomers compositionFEMS Microbiology Letters, 2004
- Metabolic Engineering for the Production of Copolyesters Consisting of 3‐Hydroxybutyrate and 3‐Hydroxyhexanoate by Aeromonas hydrophilaMacromolecular Bioscience, 2004
- Evolution of Polyhydroxyalkanoate (PHA) Production System by “Enzyme Evolution”: Successful Case Studies of Directed EvolutionMacromolecular Bioscience, 2004
- Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) Copolymer from Sugars by Recombinant Ralstonia eutropha Harboring the phaC1Ps and the phaGPs Genes of Pseudomonas sp. 61-3Biomacromolecules, 2001
- Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettesGene, 1995
- Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicumGene, 1994
- A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative BacteriaBio/Technology, 1983