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

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 (PHA_MCL) 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-3HA_MCL) 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 3HA_MCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

Enhancing Production of Medium-Chain-Length Polyhydroxyalkanoates from Pseudomonas sp. SG4502 by tac Enhancer Insertion

“…Gomez et al reported that engineered Pseudomonas containing PHA synthases of Aeromonas spp. can successfully produce P(3HB- co -HA MCL ) [ 16 ]. Nevertheless, recombinant plasmid containing the target gene is unstable and easily lost during the subculture of the host cells.…”