Genomic study of polyhydroxyalkanoates producing Aeromonas hydrophila 4AK4.

The complete genome of Gram-negative Aeromonas hydrophila 4AK4 that has been used for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) was sequenced and annotated. Its chromosome is 4,527,993 bp in size encoding 4,272 genes, including 28 rRNA genes and 104 tRNA genes. Comparative analysis indicated that genome of A. hydrophila 4AK4 was similar to that of the A. hydrophila ATCC 7966(T), an intensively studied aeromonad for its pathogenicity related to its genomic information. Genes possibly coming from other species or even other genus were identified in A. hydrophila 4AK4. A large number of putative virulent genes were predicted. However, a cytotonic enterotoxin (Ast) is absent in A. hydrophila 4AK4, allowing the industrial strain to be different from other A. hydrophila strains, indicating possible reduced virulence of strain 4AK4, which is very important for industrial fermentation. Genes involved in polyhydroxyalkanoate (PHA) metabolism were predicted and analyzed. The resulting genomic information is useful for improved production of PHA via metabolic engineering of A. hydrophila 4AK4.

Production of copolyesters of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by E. coli containing an optimized PHA synthase gene.

BACKGROUND: Microbial polyhydroxyalkanoates (PHA) are biopolyesters consisting of diverse monomers. PHA synthase PhaC2Ps cloned from Pseudomonas stutzeri 1317 is able to polymerize short-chain-length (scl) 3-hydroxybutyrate (3HB) monomers and medium-chain-length (mcl) 3-hydroxyalkanoates (3HA) with carbon chain lengths ranging from C6 to C12. However, the scl and mcl PHA production in Escherichia coli expressing PhaC2Ps is limited with very low PHA yield. RESULTS: To improve the production of PHA with a wide range of monomer compositions in E. coli, a series of optimization strategies were applied on the PHA synthase PhaC2Ps. Codon optimization of the gene and mRNA stabilization with a hairpin structure were conducted and the function of the optimized PHA synthase was tested in E. coli. The transcript was more stable after the hairpin structure was introduced, and western blot analysis showed that both codon optimization and hairpin introduction increased the protein expression level. Compared with the wild type PhaC2Ps, the optimized PhaC2Ps increased poly-3-hydroxybutyrate (PHB) production by approximately 16-fold to 30% of the cell dry weight. When grown on dodecanoate, the recombinant E. coli harboring the optimized gene phaC2PsO with a hairpin structure in the 5' untranslated region was able to synthesize 4-fold more PHA consisting of 3HB and medium-chain-length 3HA compared to the recombinant harboring the wild type phaC2Ps. CONCLUSIONS: The levels of both PHB and scl-mcl PHA in E. coli were significantly increased by series of optimization strategies applied on PHA synthase PhaC2Ps. These results indicate that strategies including codon optimization and mRNA stabilization are useful for heterologous PHA synthase expression and therefore enhance PHA production.

Engineering of polyhydroxyalkanoate (PHA) synthase PhaC2Ps of Pseudomonas stutzeri via site-specific mutation for efficient production of PHA copolymers.

The site-specific mutagenesis for PHA synthase PhaC2(Ps1317) from Pseudomonas stutzeri 1317 was conducted for optimizing production of short-chain-length and medium-chain-length polyhydroxyalkanoates (scl-mcl PHA). Recombinant Ralstonia eutropha PHB-4 harboring double mutated phaC2 (Ps1317) gene (phaC2 (Ps) QKST) produced 42 wt.% PHA content in the cell dry weight (CDW) with 93 mol% 3-hydroxybutyrate (HB) as monomer in the PHA copolymer. Compared to that of wild-type phaC2 (Ps1317), the higher PHA content indicated the effectiveness of the specific point mutations for improvement on PhaC2(Ps1317) activity and PHA production. The physical characterization revealed that the PHA produced by the recombinant strain was scl-mcl PHA copolymers with molecular weights and polydispersity reasonable for practical applications. Recombinant R. eutropha PHB-4 containing mutated phaC2 (Ps1317) termed phaC2 (Ps) QKST was demonstrated to be able to produce scl-mcl PHA copolymers consisting of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers covering the carbon chain lengths from C4 to C12 when related substrates were provided. Recombinant R. eutropha PHB-4 containing phaC2PsQKST could be used as a strain for production of copolymers consisting of dominated HB and medium-chain-length 3-hydroxyalkanoates (HA) with better application properties.

Microbial production of meso-2,3-butanediol by metabolically engineered Escherichia coli under low oxygen condition.

A metabolically engineered Escherichia coli has been constructed for the production of meso-2,3-butanediol (2,3-BD) under low oxygen condition. Genes responsible for 2,3-BD formation from pyruvate were assembled together to generate a high-copy plasmid pEnBD, in which each gene was transcribed with a constitutive promoter. To eliminate by-product formation under low oxygen condition, genes including ldhA, pta, adhE, and poxB which functioned for the mixed acid fermentation pathways were deleted in E. coli JM109. Compared with the wild type, the quadruple gene deletion mutant produced smaller amounts of acetate, succinate, and ethanol from glucose when cultivated in LB medium in shake flasks under low-aeration. When 2,3-BD producing pathway was introduced via pEnBD into the mutant, higher glucose consumption and faster 2,3-BD production rate compared with that of the wild-type control were observed under aerobic condition in shake flasks. In a 6-L fermentor supplied with only 3% dissolved oxygen (DO), the mutant harboring pEnBD converted glucose to 2,3-BD much faster than the control did. When DO supply was further lowered to 1% DO, the recombinant mutant grew much slower but produced 2,3-BD as a major fermentation metabolic product. In addition, the 2,3-BD yield showed an increase from 0.20 g BD/g glucose for the control to 0.43 g BD/g glucose for the mixed acid pathway deleted mutant grown in fermentors under 1% DO. These results reveals the potential of production of enantiomerically pure 2,3-BD isomer by recombinant E. coli under low oxygen condition.

Production and characterization of homopolymer poly(3-hydroxyvalerate) (PHV) accumulated by wild type and recombinant Aeromonas hydrophila strain 4AK4.

Aeromonas hydrophila 4AK4 normally produces copolyesters (PHBHHx) consisting of 3-hydroxybutyrate (C4) and 3-hydroxyhexanoate (C6). Wild type and recombinant A. hydrophila 4AK4 (pSXW02) expressing vgb and fadD genes encoding Vitreoscilla haemoglobin and Escherichia coli acyl-CoA synthase respectively, were found able to produce homopolyester poly(3-hydroxyvalerate) (PHV) (C5) on undecanoic acid as a single carbon source. The recombinant grew to 5.59 g/L cell dry weight (CDW) containing 47.74 wt% PHV in shake flasks when growth was conducted in LB medium and PHV production in undecanoic acid. The cells grew to 47.12 g/L CDW containing 60.08 wt% PHV in a 6 L fermentor study. Physical characterization of PHV produced by recombinant A. hydrophila 4AK4 (pSXW02) in fermentor showed a weight average molecular weight (M(w)) of 230,000 Da, a polydispersity of 3.52, a melting temperature of 103 degrees C and a glass transition temperature of -15.8 degrees C. The degradation temperature at 5% weight loss of the PHV was around 258 degrees C.