Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures

J Mol Microbiol Biotechnol. 2018;28(5):225-235. doi: 10.1159/000495752. Epub 2019 Feb 19.

Abstract

Three different polyhydroxyalkanoate (PHA) synthase genes (Ralstonia eutropha H16, Aeromonas sp. TSM81 or Aeromonas hydrophila ATCC7966 phaC) were introduced into the chromosome of two Pseudomonas strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHAMCL) producer (Pseudomonas sp. LFM046) and a UV-induced mutant strain unable to produce PHA (Pseudomonas sp. LFM461). We reported for the first time the insertion of a chromosomal copy of phaC using the transposon system mini-Tn7. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-co-3HAMCL) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring A. hydrophila phaC produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of Pseudomonas sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HAMCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

Keywords: P(3HB-co-3HAMCL); PHA synthase; Polyhydroxyalkanoates; Pseudomonas sp..

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3-Hydroxybutyric Acid / metabolism
  • Acyltransferases / genetics
  • Aeromonas / genetics
  • Aeromonas hydrophila / genetics
  • Anti-Bacterial Agents
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Caproates / metabolism
  • Chromosomes, Bacterial
  • Culture Media / chemistry
  • Cupriavidus necator / genetics
  • Decanoic Acids / metabolism
  • Glucose / metabolism
  • Mutation
  • Plasmids / genetics*
  • Polyhydroxyalkanoates / biosynthesis*
  • Polyhydroxyalkanoates / genetics*
  • Pseudomonas / enzymology
  • Pseudomonas / genetics*
  • Pseudomonas / metabolism*
  • Transformation, Bacterial

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Caproates
  • Culture Media
  • Decanoic Acids
  • Polyhydroxyalkanoates
  • 3-hydroxyhexanoic acid
  • Acyltransferases
  • poly(3-hydroxyalkanoic acid) synthase
  • myrmicacin
  • Glucose
  • 3-Hydroxybutyric Acid