Pseudomonas sp. SG4502 screened from biodiesel fuel by-products can synthesize medium-chain-length polyhydroxyalkanoates (mcl-PHAs) using glycerol as a substrate. It contains a typical PHA class II synthase gene cluster. This study revealed two genetic engineering methods for improving the mcl-PHA accumulation capacity of
Pseudomonas sp. SG4502. One way was to knock out the PHA-depolymerase
phaZ gene, the other way was to insert a
tac enhancer into the upstream of the
phaC1/
phaC2 genes. Yields of mcl-PHAs produced from 1% sodium octanoate by +(
tac-phaC2) and ∆
phaZ strains were enhanced by 53.8% and 23.1%, respectively, compared with those produced by the wild-type strain. The increase in mcl-PHA yield from +(
tac-phaC2) and ∆
phaZ was due to the transcriptional level of the
phaC2 and
phaZ genes, as determined by RT-qPCR (the carbon source was sodium octanoate).
1H-NMR results showed that the synthesized products contained 3-hydroxyoctanoic acid (3HO), 3-hydroxydecanoic acid (3HD) and 3-hydroxydodecanoic acid (3HDD) units, which is consistent with those synthesized by the wild-type strain. The size-exclusion chromatography by GPC of mcl-PHAs from the (∆
phaZ), +(
tac-phaC1) and +(
tac-phaC2) strains were 2.67, 2.52 and 2.60, respectively, all of which were lower than that of the wild-type strain (4.56). DSC analysis showed that the melting temperature of mcl-PHAs produced by recombinant strains ranged from 60 °C to 65 °C, which was lower than that of the wild-type strain. Finally, TG analysis showed that the decomposition temperature of mcl-PHAs synthesized by the (∆
phaZ), +(
tac-phaC1) and +(
tac-phaC2) strains was 8.4 °C, 14.7 °C and 10.1 °C higher than that of the wild-type strain, respectively.
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