Polyhydroxybutyrate production by recombinant Escherichia coli based on genes related to synthesis pathway of PHB from Massilia sp. UMI-21.

BACKGROUND: Polyhydroxybutyrate (PHB) is currently the most common polymer produced by natural bacteria and alternative to conventional petrochemical-based plastics due to its similar material properties and biodegradability. Massilia sp. UMI-21, a newly found bacterium, could produce PHB from starch, maltotriose, or maltose, etc. and could serve as a candidate for seaweed-degrading bioplastic producers. However, the genes involved in PHB metabolism in Massilia sp. UMI-21 are still unclear. RESULTS: In the present study, we assembled and annotated the genome of Massilia sp. UMI-21, identified genes related to the metabolism of PHB, and successfully constructed recombinant Escherichia coli harboring PHB-related genes (phaA2, phaB1 and phaC1) of Massilia sp. UMI-21, which showed up to 139.41% more product. Also, the vgb gene (encoding Vitreoscilla hemoglobin) was introduced into the genetically engineered E. coli and gained up to 117.42% more cell dry weight, 213.30% more PHB-like production and 44.09% more product content. Fermentation products extracted from recombinant E. coli harboring pETDuet1-phaA2phaB1-phaC1 and pETDuet1-phaA2phaB1-phaC1-vgb were identified as PHB by Fourier Transform Infrared and Proton nuclear magnetic resonance spectroscopy analysis. Furthermore, the decomposition temperature at 10% weight loss of PHB extracted from Massilia sp. UMI-21, recombinant E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb was 276.5, 278.7 and 286.3 °C, respectively, showing good thermal stability. CONCLUSIONS: Herein, we presented the whole genome information of PHB-producing Massilia sp. UMI-21 and constructed novel recombinant strains using key genes in PHB synthesis of strain UMI-21 and the vgb gene. This genetically engineered E. coli strain can serve as an effective novel candidate in E. coli cell factory for PHB production by the rapid cell growth and high PHB production.

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

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.