Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: enhancement of PHA production and alteration of polymer molecular weight

In vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 61-3 (phaC1(Ps)) has been performed to generate highly active enzymes. In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro. Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) [P(3HB)] from glucose, compared to recombinant E. coli harboring the wild-type PHA synthase gene (0.1 wt %). Recombinant E. coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %). The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase. In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme. The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity. Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by metabolically engineered Escherichia coli strains

The recombinant Escherichia coli strain, equipped with the newly cloned Aeromonas PHA biosynthesis genes, could produce a terpolymer of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB-co-3HV-co-3HHx)] from dodecanoic acid plus odd carbon number fatty acid. In addition, the orf1 gene of Aeromonas hydrophila was found to play a critical role in assimilating the 3HV monomer and in regulating the monomer fraction in the terpolymer.     

Biosynthesis of polyhydroxyalkanoate copolymers from mixtures of plant oils and 3-hydroxyvalerate precursors

The combination of plant oils and 3-hydroxyvalerate (3HV) precursors were evaluated for the biosynthesis of polyhydroxyalkanoate (PHA) copolymers containing 3HV monomers by Cupriavidus necator H16. Among various mixtures of plant oils and 3HV-precursors, the mixture of palm kernel oil and sodium propionate was suitable for the biosynthesis of high concentration of PHA (6.8gL(-1)) containing 7mol% of 3HV. The 3HV monomer composition can be regulated in the range of 0-23mol% by changing culture parameters such as the initial pH, and the nitrogen source and its concentration. PHA copolymers with high weight-average molecular weights (Mw) ranging from 1,400,000 to 3,100,000Da were successfully produced from mixtures of plant oils and 3HV-precursors. The mixture of plant oils and sodium propionate resulted in PHA copolymers with higher M(w) compared to the mixture of plant oils and sodium valerate. DSC analysis on the PHA containing 3HV monomers showed the presence of two distinct melting temperature (Tm), which indicated that the PHA synthesized might be a blend of P(3HB) and P(3HB-co-3HV). Sodium propionate appears to be the better precursor of 3HV than sodium valerate.     

In vivo and in vitro characterization of Ser477X mutations in polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. 61-3: effects of beneficial mutations on enzymatic activity, substrate specificity, and molecular weight of PHA

Evolutionary engineered polyhydroxyalkanoate (PHA) synthases from Pseudomonas sp. 61-3 enhance PHA accumulation and enable the monomer composition of PHAs to be regulated. We characterized a newly screened Ser477Arg (S477R) mutant of PHA synthase by in vivo analyses of P(3-hydroxybutyrate) [P(3HB)] homopolymer and P(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] copolymer productions in the recombinants of Escherichia coli. The results indicated that the S477R mutation contributed to a shift in substrate specificity to smaller monomers containing a 3HB unit rather than to an enhancement in catalytic activity. Multiple mutations of S477R with other beneficial mutations, for example, Ser325Cys, exhibited synergistic effects on both an increase in PHA production (from 9 wt % to 21 wt %) and an alteration of substrate specificity. Furthermore, the effects of complete amino acid substitutions at position 477 were characterized in terms of in vivo PHA production and in vitro enzymatic activity. The five mutations, S477Ala(A)/Phe(F)/His(H)/Arg(R)/Tyr(Y), resulted in a shift in substrate specificity to smaller monomer units. The S477Gly(G) mutant greatly enhanced activity toward all different sizes of substrates with carbon numbers ranging from 4 to 12. These results indicated that the residue 477 contributes to both the catalytic activity and substrate specificity of PHA synthase. In recombinant E. coli, the S477A/F/G/H/R/Y mutations consistently led to increases (up to 6 times that of wild-type enzyme) in weight average molecular weights of P(3HB) homopolymers. On the basis of our studies, we created a structural feasibility accounting for the mutational effects on enzymatic activity and substrate specificity of PHA synthase.     

Biosynthesis and local sequence specific degradation of poly(3-hydroxyvalerate-co-4-hydroxybutyrate) in Hydrogenophaga pseudoflava

A novel copolymer that consisted of 3-hydroxyvalerate and 4-hydroxybutyrate, P(3HV-co-4HB), was synthesized in Hydrogenophaga pseudoflava by growing it in media containing gamma-valerolactone and gamma-butyrolactone as a carbon source. The monomer ratio in the copolymer was changed by altering the feed ratio of the two lactones. The cultivation technique was composed of three steps: the first-step for high cell production in Luria-Bertani medium, the second-step for intracellular degrading removal of poly(3-hydroxybutyrate) (P(3HB)), which was formed in the first step, by culturing the cells in carbon-source-free medium, and the final step for accumulation of P(3HV-co-4HB) in a mixed lactone medium. All the P(3HV-co-4HB) copolymers contained less than 1 mol % of 3HB unit. These copolymers were characterized by NMR spectroscopy, differential scanning calorimetry, wide-angle X-ray diffraction, and first-order kinetic analysis of intracellular degradation. The copolymer with an approximately equal ratio of the comonomers was found amorphous. The NMR microstructural analysis showed that the copolymers contained appreciable amounts of 3HV-rich or 4HB-rich chains. The (13)C NMR splitting patterns associated with the four carbons in the 4HB unit of P(3HV-co-4HB) bear close resemblance to those observed in the 4HB unit of P(3HB-co-4HB). The signals arising from the carbons in the 3HV unit of P(3HV-co-4HB) split in a manner similar to those in the 3HB unit of P(3HB-co-4HB). Thus the sequences were assigned by comparing the NMR splittings for P(3HV-co-4HB) with those for P(3HB-co-4HB) and P(3HB-co-3HV). The sequence assignment was further checked by comparing the signal intensities before and after degradation of the copolymers. This was considered reasonable because the H. pseudoflava intracellular PHA depolymerase is more specific to the 3HV unit than to the 4HB unit, which was also confirmed by the higher degradation rate constant for the 3HV unit in the first-order kinetic analysis.     

Expression of 3-ketoacyl-acyl carrier protein reductase (fabG) genes enhances production of polyhydroxyalkanoate copolymer from glucose in recombinant Escherichia coli JM109

Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.     

Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthase

By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC(Ac), was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC(Ac) gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) [by LS5218 [fadR601 atoC(Con)] strain]. The expression vector for the phaC(Ac) gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).     

Effective enhancement of short-chain-length-medium-chain-length polyhydroxyalkanoate copolymer production by coexpression of genetically engineered 3-ketoacyl-acyl-carrier-protein synthase III (fabH) and polyhydroxyalkanoate synthesis genes

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that have a wide variety of physical properties dependent on the lengths of the pendant groups of the monomer units in the polymer. PHAs composed of mostly short-chain-length (SCL) monomers are often stiff and brittle, whereas PHAs composed of mostly medium-chain-length (MCL) monomers are elastomeric in nature. SCL-MCL PHA copolymers can have properties between the two states, dependent on the ratio of SCL and MCL monomers in the copolymer. It is desirable to elucidate new and low cost ways to produce PHA composed of mostly SCL monomer units with a small mol % of MCL monomers from renewable resources, since this type of SCL-MCL PHA copolymer has superior qualities compared to SCL homopolymer. To address this issue, we have created strains of recombinant E. coli capable of producing beta-ketothiolase (PhbA) and acetoacetyl-CoA synthase (PhbB) from Ralstonia eutropha, genetically engineered 3-ketoacyl-ACP synthase III (FabH) from Escherichia coli, and genetically engineered PHA synthases (PhaC) from Pseudomonas sp. 61-3 to enhance the production of SCL-MCL PHA copolymers from glucose. The cumulative effect of having two monomer-supplying pathways and genetically engineered PHA synthases resulted in higher accumulated amounts of SCL-MCL PHA copolymer from glucose. Polymers were isolated from two recombinant E. coli strains, the first harboring the phbAB, fabH(F87T), and phaC1(SCQM) genes and the second harboring the phbAB, fabH(F87W), and phaC1(SCQM) genes. The thermal and physical properties of the isolated polymers were characterized. It was found that even a very low mol % of MCL monomer in a SCL-MCL PHA copolymer had dramatic effects on the thermal properties of the copolymers.     

Alteration of substrate chain-length specificity of type II synthase for polyhydroxyalkanoate biosynthesis by in vitro evolution: in vivo and in vitro enzyme assays

In our previous study, in vitro evolution of type II polyhydroxyalkanoate (PHA) synthase (PhaC1Ps) from Pseudomonas sp. 61-3 yielded eleven mutant enzymes capable of synthesizing homopolymer of (R)-3-hydroxybutyrate [P(3HB)] in recombinant Escherichia coli JM109. These recombinant strains were capable of accumulating up to approximately 400-fold more P(3HB) than strains expressing the wild-type enzyme. These mutations enhanced the ability of the enzyme to specifically incorporate the 3HB-coenzyme A (3HB-CoA) substrate or improved catalytic efficiency toward the various monomer substrates of C4 to C12 (R)-3-hydroxyacyl-CoAs which can intrinsically be channeled by PhaC1Ps into P(3HB-co-3HA) copolymerization. In this study, beneficial amino acid substitutions of PhaC1Ps were analyzed based on the accumulation level and the monomer composition of P(3HB-co-3HA) copolymers generated by E. coli LS5218 [fadR601 atoC(Con)] harboring the monomer supplying enzyme genes. Substitutions of Ser by Thr(Cys) at position 325 were found to lead to an increase in the total amount of P(3HB-co-3HA) accumulated, whereas 3HB fractions in the P(3HB-co-3HA) copolymer were enriched by substitutions of Gln by Lys(Arg, Met) at position 481. This strongly suggests that amino acid substitutions at positions 325 and 481 are responsible for synthase activity and/or substrate chain-length specificity of PhaC1Ps. These in vivo results were supported by the in vitro results obtained from synthase activity assays using representative single and double mutants and synthetic substrates, (R,S)-3HB-CoA and (R,S)-3-hydroxydecanoyl-CoA. Notably, the position 481 was found to be a determinant for substrate chain-length specificity of PhaC1Ps.     

补料培养提高真养产碱杆菌产3-羟基丁酸和3-羟基戊酸共聚物生产强度的研究

对Alcaligenes eutrophus进行高密度培养,研究表明在发酵过程中进行有效控制,可以较大幅度地提高3－羟基丁酸和3－羟基戊酸共聚物[P(3HB-co-3HV)]的生产强度。实验中选择使用限氮的方法积累P(3HB-co-3HV),分别采用丙酸和戊酸为3HV前体,对摇瓶种子生长状态,停氮时机对菌体生产P(3HB-co-3HV)的影响以及补酸（3HV前体）策略进行了研究,在6．6L罐中,以葡萄糖为碳源,以丙酸为3HV前体培养50h,细胞干重,PHA产量,PHA含量分别达到149．9g/L,149.9g/L,83.3%(其中3HV组分占PHA的12．4mol%),生产强度达到2．50（g.h^-1.L^-1);以戊酸为3HV前体培养45h,细胞干重,PHA产量,PHA含量分别达到160．2g/L,119.0g/L,74.2%（其中3HV组分占PHA的17．7mol%)生产强度达到2．64（g.h^-1.L^-1)。     

Analysis of in vivo substrate specificity of the PHA synthase from Ralstonia eutropha: formation of novel copolyesters in recombinant Escherichia coli

In order to investigate the in vivo substrate specificity of the type I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha, we functionally expressed the PHA synthase gene in various Escherichia coli mutants affected in fatty acid beta-oxidation and the wild-type. The PHA synthase gene was expressed either solely (pBHR70) or in addition to the R. eutropha genes encoding beta-ketothiolase and acetoacetyl-coenzyme A (CoA) reductase comprising the entire PHB operon (pBHR68) as well as in combination with the phaC1 gene (pBHR77) from Pseudomonas aeruginosa encoding type II PHA synthase. The fatty acid beta-oxidation route was employed to provide various 3-hydroxyacyl-CoA thioesters, depending on the carbon source, as in vivo substrate for the PHA synthase. In vivo PHA synthase activity was indicated by PHA accumulation and substrate specificity was revealed by analysis of the comonomer composition of the respective polyester. Only in recombinant E. coli fad mutants harboring plasmid pBHR68, the R. eutropha PHA synthase led to accumulation of poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (poly(3HB-co-3HO)) and poly(3HB-co-3HO-co-3-hydroxydodecanoate (3HDD)), when octanoate and decanoate or dodecanoate were provided as carbon source, respectively. Coexpression of phaC1 from P. aeruginosa indicated and confirmed the provision of PHA precursor via the beta-oxidation pathway and led to the accumulation of a blend of two different PHAs in the respective E. coli strain. These data strongly suggested that R. eutropha PHA synthase accepts, besides the main substrate 3-hydroxybutyryl-CoA, also the CoA thioesters of 3HO and 3HDD.     

Enzymatic hydrolysis of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s by poly(3-hydroxyalkanoate) depolymerase from Acidovorax Sp. TP4

Enzymatic degradability has been investigated for a series of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s (P(3HB-co-3HP)s) with 3-hydroxypropionate (3HP) unit contents from 11 to 86 mol % as well as poly(3-hydroxybutyrate) (P(3HB)) and chemosynthesized poly(3-hydroxypropionate) (P(3HP)). The behavior of degradation by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pikettii T1 and Acidovorax Sp. TP4, defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were compared in relation to the thermal properties and crystalline structures of the PHA samples elucidated by differential scanning calorimetry and wide-angle X-ray diffraction. The degradation products were characterized by high-performance liquid chromatography and one- (1D) and two-dimension (2D) (1)H NMR spectroscopy. It was found that the PHA depolymerase of Acidovorax Sp. TP4 showed degradation behavior different from that shown by depolymerase of R. pikettii T1. PHA depolymerase from Acidovorax Sp. TP4 degraded the P(3HB-co-3HP) films with lower crystallinity in higher rates than those with higher crystallinity, no matter what kinds of crystalline structures they formed. In contrast, PHA depolymerase from R. pikettii T1 degraded P(3HB-co-3HP) films forming P(3HB) crystalline structure in higher rates than those forming P(3HP)s. The increase in amorphous nature of the P(3HB-co-3HP) films with P(3HB)-homopolymer-like crystalline structure increases and then decreases the rate of degradation by depolymerase from R. pikettii T1. The 3-hydroxybutyrate (3HB) monomer was produced as a major product by the hydrolysis of P(3HB) film by PHA depolymerase from Acidovorax Sp. TP4. The P(3HB-co-3HP) films could be degraded into 3HB and 3-hydroxypropionate (3HP) monomer at last, indicating that the catalytic domain of the enzyme recognized at least two monomeric units as substrates. While the PHA depolymerase from R. pikettii T1 hydrolyzed P(3HB) film into 3HB dimer as a major product, and the catalytic domain recognized at least three monomeric units. The degradation behavior of P(3HB-co-3HP) films by the PHA depolymerase of Acidovorax Sp. TP4 could be distinguished from that by the depolymerase of R. pikettii T1.     

Expression of 3-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Genes Enhances Production of Polyhydroxyalkanoate Copolymer from Glucose in Recombinant Escherichia coli JM109

Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.     

Role of fadR and atoC(Con) mutations in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli.

Recombinant Escherichia coli fadR atoC(Con) mutants containing the polyhydroxyalkanoate (PHA) biosynthesis genes from Alcaligenes eutrophus are able to incorporate significant levels of 3-hydroxyvalerate (3HV) into the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]. We have used E. coli fadR (FadR is a negative regulator of fatty acid oxidation) and E. coli atoC(Con) (AtoC is a positive regulator of fatty acid uptake) mutants to demonstrate that either one of these mutations alone can facilitate copolymer synthesis but that 3HV levels in single mutant strains are much lower than in the fadR atoC(Con) strain. E. coli atoC(Con) mutants were used alone and in conjunction with atoA and atoD mutants to determine that the function of the atoC(Con) mutation is to increase the uptake of propionate and that this uptake is mediated, at least in part, by atoD+. Similarly, E. coli fadR mutants were used alone and in conjunction with fadA, fadB, and fadL mutants to show that the effect of the fadR mutation is dependent on fadB+ and fadA+ gene products. Strains that were mutant in the fadB or fadA locus were unable to complement a PHA biosynthesis pathway that was mutant at the phaA locus (thiolase), but a strain containing a fadR mutation and which was fadA+ fadB+ was able to complement the phaA mutation and incorporated 3HV into P(3HB-co-3HV) to a level of 29 mol%.     

Production of poly(3-hydroxybutyric-co-3-hydroxyvaleric acid) having a high hydroxyvalerate content with valeric acid feeding

The capability of different organic acids to produce a derivative of PHB [poly(3-hydroxybutyric-co-3-hydroxyvaleric acid), P(3HB-co-3HV)] was examined in shake flask cultivations. Propionic and valeric acids demonstrated the potential to produce P(3HB-co-3HV) under nitrogen limiting conditions at 30°C. The addition time and the initial concentration of valeric acid needed for a high cellular HV content were identified by extensive experimentation. Fed-batch cultivation in 7-l bioreactor with valeric acid feeding resulted in the production of PHA containing 54% HV units.     

Coexpression of genetically engineered 3-ketoacyl-ACP synthase III (fabH) and polyhydroxyalkanoate synthase (phaC) genes leads to short-chain-length-medium-chain-length polyhydroxyalkanoate copolymer production from glucose in Escherichia coli JM109

Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C3 to C5, medium-chain-length (MCL) PHAs consist of monomer units of C6 to C14, and SCL-MCL PHAs consist of monomers ranging in size from C4 to C14. Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PHA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C4 to C10 and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production.     

Synthesis of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichia coli

Several recombinant Escherichia coli strains, including XL1-Blue, JM109, HB101, and DH5alpha harboring a stable high-copynumber plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were constructed. These recombinant strains were examined for their ability to synthesize and accumulate poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from glucose and either propionate or valerate. All recombinant E. coli strains could synthesize the P(3HB-co-3HV) copolymer in the medium containing glucose and propionate. However, only the homopolymer poly-(3-hydroxybutyrate) [P(3HB)] was synthesized from glucose and valerate. The PHA concentration and the 3HV fraction could be increased by inducing with acetate and/or oleate. When supplemented with oleate, the 3HV fraction increased by fourfold compared with that obtained without induction. Induction with propionate resulted in lower PHA concentration due to the inhibitory effect, but an 3HV fraction of as high as 33.0% could be obtained. These results suggest that P(3HB-co-3HV) can be efficiently produced from propionate by recombinant E. coli by inducing with acetate, propionate, or oleate. (c) 1996 John Wiley & Sons, Inc.     

Enhanced biosynthesis of P(3HB-3HV) and P(3HB-4HB) by amplification of.the cloned PHB biosynthesis genes in Alcaligenes eutrophus

The biosynthesis of P(3HB-3HV) and P(3HB-4HB) was carried out using transformants of Alcaligenes eutrophus harboring the cloned phbCAB, phbAB, and phbC genes. The molar fractions and yields of 3HV and 4HB increased significantly by enhancing enzymes related to PHB biosynthesis compared to the parent strain. Especially, PHB synthase was the most critical enzyme that regulated monomer compositions of P(3HB-3HV) and P(3HB-4HB) in the transformant. Even at the lower propionate or 4-hydroxybutyrate concentrations, the high molar fractions of 3HV or 4HB could be accumulated. The enforcement of PHB biosynthetic enzymes through the transformation of corresponding genes was identified to be an excellent method for modification of monomer composition of copolymer of A. eutrophus.     

Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae.

A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.     

Estimation of the number of polyhydroxyalkanoate (PHA)-degraders in soil and isolation of degraders based on the method of most probable number (MPN) using PHA-film.

A new method to estimate the number of polyhydroxyalkanoates (PHA)-degraders in soil and to isolate degraders, called the film-MPN method, is proposed. The incubation time was measured by the first order reaction (FOR) model. This method was used to estimate numbers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)[P(3HB-co-3HV)]- and poly(3-hydroxyvalerate-co-4-hydroxybutyrate)[P(3HB-co-4HB)]-degraders in garden soil (4.30 x 10(5) and 2.15 x 10(5) aerobic degraders per gram of dry soil, respectively). The number of P(3HB-co-3HV)-degraders in paddy field soil was 5.06 x 10(5) aerobic degraders per gram dry soil. Also, several P(3HB-co-3HV)-degraders were isolated directly from positive-growth tubes of high dilution.