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

Journal
Microbial Physiology
ISSN
2673-1673
Date Issued
2018
Author(s)
Edmar Ramos Oliveira-Filho
Thatiane Teixeira Mendonça
Paul F. Long
Marilda Keico Taciro
José Gregório Cabrera Gomez
Luiziana F. Silva
DOI
URL
Abstract
<jats:p>Three different polyhydroxyalkanoate (PHA) synthase genes (<i>Ralstonia eutropha</i> H16, <i>Aeromonas</i> sp. TSM81 or <i>Aeromonas hydrophila</i> ATCC7966 <i>phaC</i>) were introduced into the chromosome of two <i>Pseudomonas</i> strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHA<sub>MCL</sub>) producer (<i>Pseudomonas</i> sp. LFM046) and a UV-induced mutant strain unable to produce PHA (<i>Pseudomonas</i> sp. LFM461). We reported for the first time the insertion of a chromosomal copy of <i>phaC</i> using the transposon system mini-Tn<i>7</i>. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-<i>co</i>-3HA<sub>MCL</sub>) 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 <i>A. hydrophila phaC</i> 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 <i>Pseudomonas</i> sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HA<sub>MCL</sub> using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.</jats:p>