Hydrophila 4AK4 Research Articles

Production of polyhydroxyalkanoate from acetate by metabolically engineered Aeromonas hydrophilia

Aeromonas hydrophila 4AK4 normally produces the copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) using lauric acid as the carbon source. In this study we reported the metabolic engineering of A.hydrophila 4AK4 for the production of polyhydroxyalkanoate (PHA) using acetate as a main carbon source. Recombinant A.hydrophila overexpressing β-ketothiolase and acetoacetyl-CoA reductase could accumulate poly-3-hydroxybutyrate (PHB) from acetate with a polymer content of 1.39wt%. Further overexpression of acetate kinase/phosphotransacetylase and acetyl-CoA synthetase improved PHB content to 8.75wt% and 19.82wt%, respectively. When acetate and propionate were simultaneously supplied as carbon sources, the engineered A.hydrophila overexpressing β-ketothiolase, acetoacetyl-CoA reductase, and acetyl-CoA synthetase was found able to produce the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV). The recombinant grew to 3.79g/L cell dry weight (CDW) containing 15.02wt% PHBV. Our proposed metabolic engineering strategies illustrate the feasibility for producing PHA from acetate by A.hydrophila.

Biosynthesis of functional polyhydroxyalkanoates by engineered Halomonas bluephagenesis

Polyhydroxyalkanoates (PHA) have found widespread medical applications due to their biocompatibility and biodegradability, while further chemical modification requires functional groups on PHA. Halomonas bluephagenesis, a non-model halophilic bacterium serving as a chassis for the Next Generation Industrial Biotechnology (NGIB), was successfully engineered to express heterologous PHA synthase (PhaC) and enoyl coenzyme-A hydratase (PhaJ) from Aeromonas hydrophila 4AK4, along with a deletion of its native phaC gene to synthesize the short chain-co-medium chain-length PHA copolymers, namely poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-3-hydroxyhex-5-enoate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyhex-5-enoate). After optimizations of the expression cassette and ribosomal binding site combined with introduction of endogenous acyl-CoA synthetase (fadD), the resulting recombinant strain H. bluephagenesis TDR4 achieved a remarkably high 3-hydroxyhexenoate (3HHxE) molar ratio of 35% when grown on glucose and 5-hexenoic acid as co-substrates. The total ratio of side chain consisting of 3HHx and 3HHxE monomers in the terpolymer can approach 44 mol%. H. bluephagenesis TDR4 was grown to a cell dry mass (CDM) of 30 g/L containing approximately 20% poly(3-hydroxybutyrate-co-22.75 mol% 3-hydroxy-5-hexenoate) in a 48-h of open and unsterile fermentation with a 5-hexenoic acid conversion efficiency of 91%. The resulted functional PHA containing 12.5 mol% 3-hydroxy-5-hexenoate exhibits more than 1000% elongation at break. The engineered H. bluephagenesis TDR4 can be used as an experimental platform to produce functional PHA.

Synthesis of Medium-Chain-Length Polyhydroxyalkanoate Homopolymers, Random Copolymers, and Block Copolymers by an Engineered Strain of Pseudomonas entomophila.

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs), widely used in medical area, are commonly synthesized by Pseudomonas spp. This study tries to use β-oxidation pathways engineered P. entomophila to achieve single source of a series of mcl-monomers for microbial production of PHA homopolymers. The effort is proven successful for the first time to obtain a wide range of mcl-PHA homopolymers from engineered P. entomophila LAC23 grown on various fatty acids, respectively, ranging from poly(3-hydroxyheptanoate) to poly(3-hydroxytetradecanoate). Effects of a PHA monomer chain length on thermal and crystallization properties including the changes of Tm , Tg , and Td5% are investigated. Additionally, strain LAC23 is used to synthesize random copolymers of 3-hydroxyoctanoate (3HO) and 3-hydroxydodecanoate (3HDD) or 3-hydroxytetradecanoates, their compositions could be controlled by adjusting the ratios of two related fatty acids. Meanwhile, block copolymer P(3HO)-b-P(3HDD) is synthesized by the same strain. It is found for the first time that even- and odd number mcl-PHA homopolymers have different physical properties. When the gene of the PHA synthase in the engineered P. entomophila is replaced by phaC from Aeromonas hydrophila 4AK4, poly(3-hydroxybutyrate-co-30 mol%-3-hydroxyhexanoate) is synthesized. Therefore, P. entomophila can be used to synthesize the whole range of PHA (C7-C14) homopolymers, random- and block copolymers.

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 and characterization of poly(3-hydroxybutyrateco-3-hydroxyhexanoate)-poly(ethylene glycol) hybird copolymer with adjustable molecular weight

A novel natural-synthetic hybrid block copolymer was synthesized by Aeromonas hydrophila 4AK4 in poly(ethylene glycol) (PEG, Mn = 200) modified fermentation. This hybrid biomaterial consists of the natural hydrophobic polymer poly(3-hydroxybutyrat-co-3-hydroxyhexanoate) (PHBHHx) end-capped with hydrophilic PEG, which has the increased flexibility as well as the improved thermal stability. Addition of diethylene glycol (DEG) and ethylene glycol could not result in the accumulation of hybrid block copolymer. DEG and ethylene glycol, together with PEG-200, could cause a reduction of molar mass of PHBHHx, resulting in a series of low molecular weight polymer and the reduction of the polymer yield as well as the cellular productivity. In vitro degradation of PHBHHx and PHBHHx-PEG with different molecular weight showed that the decrease of molecular weight accelerated the degradation of copolymers, but PEG modification has little effect on its degradation rate. The results in this study provided a convenient and direct method to produce a series of PHBHHx and PHBHHx-PEG materials with adjustable molecular weight and broad molecular weight distribution which will be very useful for the biomedical applications.

Production of 3-hydroxypropionate homopolymer and poly(3-hydroxypropionate- co-4-hydroxybutyrate) copolymer by recombinant Escherichia coli

Conversion of 3-hydroxypropionate (3HP) from 1,3-propanediol (PDO) was improved by expressing dehydratase gene ( dhaT) and aldehyde dehydrogenase gene ( aldD) of Pseudomonas putida KT2442 under the promoter of phaCAB operon from Ralstonia eutropha H16. Expression of these genes in Aeromonas hydrophila 4AK4 produced up to 21 g/L 3HP in a fermentation process. To synthesize homopolymer poly(3-hydroxypropionate) (P3HP), and copolymer poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P3HP4HB), dhaT and aldD were expressed in E. coli together with the phaC1 gene encoding polyhydroxyalkanoate (PHA) synthase gene of Ralstonia eutropha, and pcs' gene encoding the ACS domain of the tri-functional propionyl-CoA ligase (PCS) of Chloroflexus aurantiacus. Up to 92 wt% P3HP and 42 wt% P3HP4HB were produced by the recombinant Escherichia coli grown on PDO and a mixture of PDO+1,4-butanediol (BD), respectively.

Metabolic engineering of Aeromonas hydrophila 4AK4 for production of copolymers of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoate

A mutant termed Aeromonas hydrophila AKLF was constructed by deleting acetic acid pathway related genes pta and ackA in A. hydrophila 4AK4. Accumulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) in A. hydrophila AKLF was increased by 47% from 2.11 to 3.10g/L associated with a reduction on acetic acid formation compared with A. hydrophila 4AK4 when lauric acid was used as carbon resource. A. hydrophila AKLF harboring pVGAB encoding Vitreoscilla hemoglobin, β-ketothiolase and acetoacetyl-CoA reductase was found to produce 85% more PHBHHx compared to its wild type. Expression of plasmid pDEcLPp harboring genes related to fatty acid metabolism in A. hydrophila AKLF led to 63% more PHBHHx production than A. hydrophila 4AK4. Replacing phaC in A. hydrophila AKLF with a mutant phaC2 from Pseudomonas stutzeri 1317 resulted in enhanced production of copolymers of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates compared to A. hydrophila 4AK4 harboring the mutant phaC2 in the chromosome as control.

Biosynthesis of polyhydroxyalkanoate homopolymers by Pseudomonas putida

Pseudomonas putida KT2442 has been a well-studied producer of medium-chain-length (mcl) polyhydroxyalkanoate (PHA) copolymers containing C6 ~ C14 monomer units. A mutant was constructed from P. putida KT2442 by deleting its phaG gene encoding R-3-hydroxyacyl-ACP-CoA transacylase and several other β-oxidation related genes including fadB, fadA, fadB2x, and fadAx. This mutant termed P. putida KTHH03 synthesized mcl homopolymers including poly(3-hydroxyhexanoate) (PHHx) and poly(3-hydroxyheptanoate) (PHHp), together with a near homopolymer poly(3-hydroxyoctanoate-co-2 mol% 3-hydroxyhexanoate) (PHO*) in presence of hexanoate, heptanoate, and octanoate, respectively. When deleted with its mcl PHA synthase genes phaC1 and phaC2, the recombinant mutant termed P. putida KTHH08 harboring pZWJ4-31 containing PHA synthesis operon phaPCJ from Aeromonas hydrophila 4AK4 accumulated homopolymer poly(3-hydroxyvalerate) (PHV) when valerate was used as carbon source. The phaC deleted recombinant mutant termed P. putida KTHH06 harboring pBHH01 holding PHA synthase PhbC from Ralstonia eutropha produced homopolymers poly(3-hydroxybutyrate) (PHB) and poly(4-hydroxybutyrate) using γ-butyrolactone was added as precursor. All the homopolymers were physically characterized. Their weight average molecular weights ranged from 1.8 x 10⁵ to 1.6 x 10⁶, their thermal stability changed with side chain lengths. The derivatives of P. putida KT2442 have been developed into a platform for production of various PHA homopolymers.

Poly(hydroxyalkanoate) synthase genes in pseudomonads strains, isolation and heterologous expression

A mutant termed Aeromonas hydrophila AKLF was constructed by deleting acetic acid pathway related genes pta and ackA in A. hydrophila 4AK4. Accumulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) in A. hydrophila AKLF was increased by 47% from 2.11 to 3.10 g/L associated with a reduction on acetic acid formation compared with A. hydrophila 4AK4 when lauric acid was used as carbon resource. A. hydrophila AKLF harboring pVGAB encoding Vitreoscilla hemoglobin, β-ketothiolase and acetoacetyl-CoA reductase was found to produce 85% more PHBHHx compared to its wild type. Expression of plasmid pDEcLPp harboring genes related to fatty acid metabolism in A. hydrophila AKLF led to 63% more PHBHHx production than A. hydrophila 4AK4. Replacing phaC in A. hydrophila AKLF with a mutant phaC2 from Pseudomonas stutzeri 1317 resulted in enhanced production of copolymers of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates compared to A. hydrophila 4AK4 harboring the mutant phaC2 in the chromosome as control.

Biosynthesis and characterization of 3‐hydroxyalkanoate terpolyesters with adjustable properties by Aeromonas hydrophila

Polyhydroxyalkanoates (PHA) terployesters P(3HB-co-3HV-co-3HHx) consisting of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) were produced by wild-type Aeromonas hydrophila 4AK4, its recombinant harboring PHA synthesis genes phaPCJ encoding PHA binding protein phasin, PHA synthase, and enoyl-CoA hydratase, and another its recombinant harboring phaAB encoding beta-ketothiolase and acetoacetyl-CoA reductase, respectively, when grown in lauric acid and/or valerate. The terpolyesters produced by A. hydrophila 4AK4 (phaAB) grown in velarate were found to produce copolymers P(3HB-co-3HV) containing high 3HV fractions with a maximum of 99 mol% 3HV. In terpolyesters, 3HV ranged from 9 to 32 mol% depending on the valerate concentration and strain used. A maximal terpolyester P(3HB-co-3HV-co-3HHx) content in dry cells was 71 wt%. Transmission electron microscopy study of A. hydrophila 4AK4 harboring phaPCJ revealed the full occupation of terpolyester P(3HB-co-3HV-co-HHx) in the cellular spaces. Terpolyesters with various monomer compositions showed changing thermal and mechanical properties. Those with higher 3HV fractions demonstrated an improved property over the lower HV containing ones.

Microbial production of 4-hydroxybutyrate, poly-4-hydroxybutyrate, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by recombinant microorganisms

4-Hydroxybutyrate (4HB) was produced by Aeromonas hydrophila 4AK4, Escherichia coli S17-1, or Pseudomonas putida KT2442 harboring 1,3-propanediol dehydrogenase gene dhaT and aldehyde dehydrogenase gene aldD from P. putida KT2442 which are capable of transforming 1,4-butanediol (1,4-BD) to 4HB. 4HB containing fermentation broth was used for production of homopolymer poly-4-hydroxybutyrate [P(4HB)] and copolymers poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)]. Recombinant A. hydrophila 4AK4 harboring plasmid pZL-dhaT-aldD containing dhaT and aldD was the most effective 4HB producer, achieving approximately 4 g/l 4HB from 10 g/l 1,4-BD after 48 h of incubation. The strain produced over 10 g/l 4HB from 20 g/l 1,4-BD after 52 h of cultivation in a 6-L fermenter. Recombinant E. coli S17-1 grown on 4HB containing fermentation broth was found to accumulate 83 wt.% of intracellular P(4HB) in shake flask study. Recombinant Ralstonia eutropha H16 grew to over 6 g/l cell dry weight containing 49 wt.% P(3HB-13%4HB) after 72 h.

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 °C and a glass transition temperature of −15.8 °C. The degradation temperature at 5% weight loss of the PHV was around 258 °C.

Biocompatibility of poly(3-hydroxybutyrate- co-3-hydroxyvalerate- co-3-hydroxyhexanoate) with bone marrow mesenchymal stem cells

As a new member of the polyhydroxyalkanoate (PHA) family, poly(3-hydroxybutyrate- co-3-hydroxyvalerate- co-3-hydroxyhexanoate) (PHBVHHx) was produced by recombinant Aeromonas hydrophila 4AK4. PHBVHHx showed a rougher surface and had higher hydrophobicity than the well-studied polymers poly( l-lactic acid) (PLA) and poly(3-hydroxybutyrate- co-3-hydroxyhexanoate) (PHBHHx). Human bone marrow mesenchymal stem cells (MSCs) adhered better on PHBVHHx film than on tissue culture plates (TCPs), PLA film and PHBHHx film. The cell number on the PHBVHHx film was 115% higher than that on the TCPs, 66% higher than on the PHBHHx film and 263% higher than on the PLA film ( p<0.01). PHBVHHx also supported the osteogenic differentiation of MSCs. Previous studies have shown that all PHA polymers tested were either poorer than or equal to TCPs for supporting cell growth. PHBVHHx is the only PHA polymer to significantly increase cell numbers compared with TCPs. These data demonstrate that PHBVHHx could be a promising biomaterial for bone tissue engineering.

Influence of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) on growth and osteogenic differentiation of human bone marrow‐derived mesenchymal stem cells

As a new member of polyhydroxyalkanoate (PHA) family, the novel polyester poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-4HB-3HHx)) was produced by recombinant Aeromonas hydrophila 4AK4 and used for the first time to test its biocompatibility. It was shown that P(3HB-4HB-3HHx) had higher hydrophobicity, surface energy, and rougher surface than the well-studied polymers poly(L-lactic acid) (PLA) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Human mesenchymal stem cells (MSCs) attached better on P(3HB-4HB-3HHx) film than on tissue culture plates (TCPs), PLA film, and PHBHHx film. MSC proliferation on P(3HB-4HB-3HHx) film was 126% higher than that on TCPs, 84% higher than that on PHBHHx film, and 312% higher than that on PLA film (p < 0.01). P(3HB-4HB-3HHx) also supported osteogenic differentiation of MSCs. Previous studies found that all PHA materials tested were either less than or equal to TCPs for supporting cell growth. Among all PHA materials tested, P(3HB-4HB-3HHx) was the only PHA material to significantly promote cell proliferation compared with TCPs. P(3HB-4HB-3HHx) could be exploited for applications in bone tissue engineering.

Disruption of the polyhydroxyalkanoate synthase gene inAeromonas hydrophilareduces its survival ability under stress conditions

Aeromonas hydrophila 4AK4 produces PHBHHx copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate. In this paper, the physiological functions of PHBHHx on the bacterial survival ability were systematically studied using a PHBHHx synthase PhaC-disrupted mutant termed A. hydrophila CQ4 and its wild type A. hydrophila 4AK4. It was found that PHBHHx synthesis in A. hydrophila 4AK4 provided improved resistance against environmental stress factors including heat and cold treatments, hydrogen peroxide, UV irradiation, ethanol and high osmotic pressure compared with its PHBHHx synthase negative mutant A. hydrophila CQ4. The above resistant abilities were further confirmed by the different expression levels of sigma factor sigma(S) encoded by rpoS via real-time PCR study. The results suggested that rpoS played an important role in the enhanced resistance of host strain to stresses conferred by PHBHHx.

Production and characterization of terpolyester poly(3-hydroxybutyrate- co-4-hydroxybutyrate- co-3-hydroxyhexanoate) by recombinant Aeromonas hydrophila 4AK4 harboring genes phaPCJ

Terpolyester consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyhexanoate (3HHx) abbreviated as P(3HB- co-4HB- co-3HHx), was produced by recombinant Aeromonas hydrophila 4AK4 harboring polyhydroxyalkanoate (PHA) synthesis genes phaPCJ. The monomer content of 4HB in the terpolyesters was dependent on the concentration of 1,4-butanediol, ranging from 4.3 to 8.5 mol% in the presence of 5–25 g/l 1,4-butanediol, respectively, accompanying by a decreasing 3HHx content from 22 to 17.6 mol% and a constant 3HB content of 74 mol% during growth in lauric acid containing mineral medium. Number average molecular weights of the terpolyesters were 440,000 to 550,000, much higher than 220,000 of the copolyester consisting of 3HB and 12 mol%3HHx commonly produced by wild type A. hydrophila 4AK4. Thermal and mechanical properties of the above terpolyesters were better than those of homopolyester poly(3-hydroxybutyrate) (PHB) and its copolymers P(3HB- co-7 mol%4HB), P(3HB- co-12 mol%3HHx). Moreover, physical properties of the terpolyesters changed with variations of monomer composition. The terpolyesters were characterized using nuclear magnetic resonance (NMR), gas chromatography (GC), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and stress–strain measurements. The introduction of 4HB and 3HHx monomers into PHB enhanced the thermal stability, changed its crystallinity and flexibility compared with homopolyester PHB and their two monomer containing copolyesters.

Production and characterization of terpolyester poly(3-hydroxybutyrate- co-3-hydroxyvalerate- co-3-hydroxyhexanoate) by recombinant Aeromonas hydrophila 4AK4 harboring genes phaAB

Recombinant Aeromonas hydrophila 4AK4 harboring phbA and phbB ( phaAB) genes encoding β-ketothiolase and acetoacetyl-CoA reductase of Ralstonia eutropha produced a terpolyester of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB- co-3HV- co-3HHx)] from mixtures of dodecanoic acid and propionic acid. Depending on the concentration of propionic acid in bacterial cultures, cell growth represented by cellular dry weight (CDW), P(3HB- co-3HV- co-3HHx) contents in dry cells and 3HV molar percentage in the terpolyester ranged from 0.43 g l −1 to 3.29 g l −1, 20.7% to 35.6%, 2.3 mol% to 7.1 mol%, respectively. Number average molecular ( M n) weights of the terpolyesters were 303,000–800,000, independent from monomer fraction content. This terpolyester was characterized by nuclear magnetic resonance (NMR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and stress–strain measurement studies. Results showed that the terpolyester had higher thermal stability and elongation at break compared with that of homopolymer poly(3-hydroxybutyrate) (PHB) and its copolymers P(3HB- co-5 mol%3HV) or P(3HB- co-12 mol%3HHx). In addition, the terpolyester had lower melting ( T m) temperatures and enthalpy of fusions (Δ H m) than PHB did.

Biosynthesis of polyhydroxyalkanoate copolyesters by Aeromonas hydrophila mutant expressing a low-substrate-specificity PHA synthase PhaC2 Ps

A polyhydroxyalkanoate (PHA) synthase negative mutant termed Aeromonas hydrophila CQ4 was constructed from its wild type strain A. hydrophila 4AK4. Heterologous expression of a low-substrate-specificity PHA synthase PhaC2 Ps cloned from Pseudomonas stutzeri 1317 in A. hydrophila CQ4 could copolymerize short-chain-length (SCL) 3-hydroxybutyrate (3HB) and medium-chain-length (MCL) 3-hydroxyalkanoates (3HA). Co-expressing (R)-specific enoyl-CoA hydratase and PHA synthase PhaC2 Ps in A. hydrophila CQ4 ( phaJ Ah , phaC2 Ps ) led to accumulation of 20.86 wt% PHA copolyesters containing 59 mol% 3HB, 37 mol% 3-hydroxyhexanoate (3HHx) and 4 mol% of other MCL 3HA in shake flask culture. When grown in fermentor, cellular dry weight (CDW) and PHA content were 22.75 g L −1 and 20.08 wt%, respectively, with 72.38 mol% 3HB, 25.18 mol% 3HHx and 2.45 mol% other MCL 3HA in the copolymer. If beta-ketothiolase and acetoacetyl-CoA reductase of Ralstonia eutropha were co-expressed with PhaC2 Ps, A. hydrophila CQ4 ( phaC2 Ps , phbA Re , phbB Re ) accumulated PHA copolyesters containing enhanced 3HB content up to 79.95 mol% and reduced 3HHx fraction of 8.55 mol%. When grown in fermentor it produced 16.28 g L −1 CDW containing 11.64 wt% PHA consisting of 87.38 mol% 3HB, 9.75 mol% 3HHx and 2.87 mol% other MCL 3HA. These results further demonstrate that A. hydrophila is a good candidate for metabolic engineering for diverse PHA production.

Microbial transformation of benzene to cis-3,5-cyclohexadien-1,2-diols by recombinant bacteria harboring toluene dioxygenase gene tod

Toluene dioxygenase (TDO) catalyzes asymmetric cis-dihydroxylation of aromatic compounds. To achieve high efficient biotransformation of benzene to benzene cis-diols, Pseudomonas putida KT2442, Pseudomonas stutzeri 1317, and Aeromonas hydrophila 4AK4 were used as hosts to express TDO gene tod. Plasmid pSPM01, a derivative of broad-host plasmid pBBR1MCS-2 harboring tod from plasmid pKST11, was constructed and introduced into the above three strains. Their abilities to catalyze the biotransformation of benzene to benzene cis-diols, namely, cis-3,5-cyclohexadien-1,2-diols abbreviated as DHCD, were examined. In shake-flask cultivation under optimized culture media and growth condition, benzene cis-diols production by recombinant P. putida KT2442 (pSPM01), P. stutzeri 1317 (pSPM01), and A. hydrophila 4AK4 (pSPM01) were 2.68, 2.13, and 1.17 g/l, respectively. In comparison, Escherichia coli JM109 (pSPM01) and E. coli JM109 (pKST11) produced 0.45 and 0.53 g/l of DHCD, respectively. When biotransformation was run in a 6-l fermenter, DHCD production in P. putida KT2442 (pSPM01) was approximately 60 g/l; this is the highest DHCD production yield reported so far.

Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Gluconate and Glucose by Recombinant Aeromonas hydrophila and Pseudomonas putida

Aeromonas hydrophila 4AK4 and Pseudomonas putida GPp104 were genetically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) using gluconate and glucose rather than fatty acids. A truncated tesA gene, encoding cytosolic thioesterase I of Escherichia coli which catalyzes the conversion of acyl-ACP into free fatty acids, was introduced into A. hydrophila 4AK4. When grown in gluconate, the recombinant A. hydrophila 4AK4 synthesized 10% (w/w) PHBHHx containing 14% (mol/mol) 3-hydroxyhexanoate. If additional PHBHHx synthesis genes, phaPCJ, were over-expressed with the truncated tesA in A. hydrophila 4AK4, the PHBHHx content increased to 15% (w/w) and contained 19% (mol/mol) 3-hydroxyhexanoate. Recombinant P. putida GPp104 harboring phaC encoding PHBHHx synthase of A. hydrophila, phaB encoding acetoacetyl-CoA reductase of Wautersia eutropha and phaG encoding 3-hydroxyacyl-ACP-CoA transferase of P. putida, synthesized 19% (w/w) PHBHHx containing 5% (mol/mol) 3-hydroxyhexanoate from glucose. The results suggest that the engineered pathways were applicable to synthesize PHBHHx from unrelated carbon sources such as gluconate and glucose.