Poly(hydroxyalkanoic acid) Biosynthesis in Ectothiorhodospirashaposhnikovii: Characterization and Reactivity of a Type III PHA Synthase

Ectothiorhodospira shaposhnikovii is able to accumulate polyhydroxybutyrate (PHB) photoautotrophically during nitrogen-limited growth. The activity of polyhydroxyalkanoate (PHA) synthase in the cells correlates with PHB accumulation. PHA synthase samples collected during the light period do not show a lag phase during in vitro polymerization. Synthase samples collected in the dark period displays a significant lag phase during in vitro polymerization. The lag phase can be eliminated by reacting the PHA synthase with the monomer, 3-hydroxybutyryl-CoA (3HBCoA). The PHA synthase genes (phaC and phaE) were cloned by screening a genomic library for PHA accumulation in E. coli cells. The PHA synthase expressed in the recombinant E. coli cells was purified to homogeneity. Both sequence analysis and biochemical studies indicated that this PHA synthase consists of two subunits, PhaE and PhaC and, therefore, belongs to the type III PHA synthases. Two major complexes were identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer), whereas the small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of 3HBCoA and a negligible lag period. The results from in vitro polymerizations of 3HBCoA suggest that the PHA synthase from E. shaposhnikovii may catalyze a living polymerization and demonstrate that two PhaC and two PhaE subunits comprise a single catalytic site in the synthase complex.     

Genetic and biochemical characterization of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthase in Haloferax mediterranei

The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaEC(Hme)) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaE(Hme) and phaC(Hme) genes were constitutively expressed, and both the PhaE(Hme) and PhaC(Hme) proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaEC(Hme) genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaEC(Hme) genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaC(Hme) was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaE(Hme)/PhaC(Hme) (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaC(Hme) and PhaE(Hme), accounted for the PHBV synthesis in H. mediterranei.     

Wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type III synthases

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaC(Hm)) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaC(Hm) were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in haloarchaea and appears to be involved in PHA biosynthesis.     

Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum

This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the Rhodospirillum rubrum genome revealed by the occurrence of three homologous PHA polymerase genes (phaC1, phaC2, and phaC3). In vitro biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple phaC deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that phaC2 contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast, phaC3 is an insignificant contributor to PHA productivity, and phaC1, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C₅ and C₆ monomers. The in vitro characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of R. rubrum was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer.     

Cloning, characterization and comparison of the Pseudomonas mendocina polyhydroxyalkanoate synthases PhaC1 and PhaC2

This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina. The P mendocina pha gene locus, encoding two PHA synthase genes [phaC1Pm and phaC2pm flanking a PHA depolymerase gene (phaZ)], was cloned, and the nucleotide sequences of phaC1Pm (1,677 bp), phaZ (1,034 bp), and phaC2pm (1,680 bp) were determined. The amino acid sequences deduced from phaC1Pm and phaC2pm showed highest similarities to the corresponding PHA synthases from other pseudomonads sensu stricto. The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants P. putida GPp104 and Ralstonia eutropha PHB-4. In P. putida GPp 104, phaC1Pm and phaC2Pm mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6-C12) as often reported for other pseudomonads. In contrast, in R. eutropha PHB-4, either PHA synthase gene also led to the incorporation of 3-hydroxybutyrate (3HB) into PHA. Recombinant strains of R. eutropha PHB-4 harboring either P. mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed. Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host. Furthermore, isogenic phaC1 and phaC2 knock-out mutants of P. mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in P. mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate.     

Studies on polyhydroxyalkanoate (PHA) accumulation in a PHA synthase I-negative mutant of Burkholderia cepacia generated by homogenotization

In the genome of Burkholderia cepacia strain IPT64, which accumulates a blend of the two homopolyesters poly(3-hydroxybutyrate), poly(3HB), and poly(3-hydroxy-4-pentenoic acid), poly(3H4PE), from sucrose or gluconate as single carbon source, the polyhydroxyalkanoate (PHA) synthase structural gene was disrupted by the insertion of a chloramphenicol-resistant gene cassette (phaC1::Cm). The suicide vector pSUP202 harboring phaC1::Cm was transferred to B. cepacia by conjugation. The inactivated gene was integrated into the chromosome of B. cepacia by homologous recombination. This mutant and also 15 N-methyl-N'-nitrosoguanidine (NMG)-induced mutants still accumulated low amounts of PHAs and expressed low PHA synthase activity. The analysis of the mutant phaC1::Cm showed that it accumulated about 1% of PHA consisting of 68.2 mol% 3HB and 31.8 mol% 3H4PE from gluconate. The wild-type, in contrast, accumulated 49.3% of PHA consisting of 96.5 mol% 3HB and 3. 5 mol% 3H4PE. Our results indicated that the genome of B. cepacia possesses at least two PHA synthase genes, which probably have different substrate specificities.     

Molecular weight change of polyhydroxyalkanoate (PHA) caused by the PhaC subunit of PHA synthase from Bacillus cereus YB-4 in recombinant Escherichia coli

Polyhydroxyalkanoate (PHA)-producing Bacillus strains possess class IV PHA synthases composed of two subunit types, namely, PhaR and PhaC. In the present study, PHA synthases from Bacillus megaterium NBRC15308(T) (PhaRC(Bm)), B. cereus YB-4 (PhaRC(YB4)), and hybrids (PhaR(Bm)C(YB4) and PhaR(YB4)C(Bm)) were expressed in Escherichia coli JM109 to characterize the molecular weight of the synthesized poly(3-hydroxybutyrate) [P(3HB)]. PhaRC(Bm) synthesized P(3HB) with a relatively high molecular weight (M(n) = 890 × 10(3)) during 72 h of cultivation, whereas PhaRC(YB4) synthesized low-molecular-weight P(3HB) (M(n) = 20 × 10(3)). The molecular weight of P(3HB) synthesized by PhaRC(YB4) decreased with increasing culture time and temperature. This time-dependent behavior was observed for hybrid synthase PhaR(Bm)C(YB4), but not for PhaR(YB4)C(Bm). These results suggest that the molecular weight change is caused by the PhaC(YB4) subunit. The homology between PhaCs from B. megaterium and B. cereus YB-4 is 71% (amino acid identity); however, PhaC(YB4) was found to have a previously unknown effect on the molecular weight of the P(3HB) synthesized in E. coli.     

Engineering of polyhydroxyalkanoate (PHA) synthase PhaC2<Subscript>Ps</Subscript> of <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> 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.     

A novel genetically engineered pathway for synthesis of poly(hydroxyalkanoic acids) in Escherichia coli

A new pathway to synthesize poly(hydroxyalkanoic acids) (PHA) was constructed by simultaneously expressing butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) genes of Clostridium acetobutylicum and the two PHA synthase genes (phaE and phaC) of Thiocapsa pfennigii in Escherichia coli. The four genes were cloned into the BamHI and EcoRI sites of pBR322, and the resulting hybrid plasmid, pBPP1, conferred activities of all three enzymes to E. coli JM109. Cells of this recombinant strain accumulated PHAs when hydroxyfatty acids were provided as carbon sources. Homopolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), or 4-hydroxyvalerate (4HV) were obtained from each of the corresponding hydroxyfatty acids. Various copolyesters of those hydroxyfatty acids were also obtained when two of these hydroxyfatty acids were fed at equal amounts: cells fed with 3HB and 4HB accumulated a copolyester consisting of 88 mol% 3HB and 12 mol% 4HB and contributing to 68.7% of the cell dry weight. Cells fed with 3HB and 4HV accumulated a copolyester consisting of 94 mol% 3HB and 6 mol% 4HV and contributing to 64.0% of the cell dry weight. Cells fed with 3HB, 4HB, and 4HV accumulated a terpolyester consisting of 85 mol% 3HB, 13 mol% 4HB, and 2 mol% 4HV and contributing to 68.4% of the cell dry weight.     

Location of functional region at N-terminus of polyhydroxyalkanoate (PHA) synthase by N-terminal mutation and its effects on PHA synthesis

Polyhydroxyalkanoate (PHA) synthase PhaC plays a very important role in biosynthesis of microbial polyesters PHA. Compared to the extensively analyzed C-terminus of PhaC, N-terminus of PhaC was less studied. In this paper, the N-terminus of two class I PHA synthases PhaC_Re and PhaC_Ah from Ralstonia eutropha and Aeromonas hydrophila, respectively, and one class II synthase PhaC2_Ps of Pseudomonas stutzeri strain 1317, were investigated for their effect on PHA synthesis. For PhaC_Re, deletion of 2–65 amino acid residues on the N-terminus led to enhanced PHB production with high PHB molecular weight of 2.50 × 10⁶ Da. For PhaC_Ah, the deletion of the N-terminal residues resulted in increasing molecular weights and widening polydispersity accompanied by a decreased PHA production. It was found that 3-hydroxybutyrate (3HB) monomer content in copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate (3HHx) increased when the first 2–9 and 2–13 amino acid residues in the N-terminus of PhaC2_Ps were deleted. However, deletion up to the 40th amino acid disrupted the PHA synthesis. This study confirmed that N-terminus in different types of PHA synthases showed significant roles in the PHA productivity and elongation activity. It was also indicated that N-terminal mutation was very effective for the location of functional regions at N-terminus.     

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.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil

High-yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha KCTC 2662 was investigated using soybean oil and γ-butyrolactone as carbon sources. In flask culture, it was shown that R. eutropha KCTC 2662 accumulated PHAs during the growth phase. The optimum carbon to nitrogen ratio (C/N ratio) giving the highest cell and PHA yield was 20 g-soybean oil/g-(NH(4))(2)SO(4). The 4-hydroxybutyrate (4HB) fraction in the copolymer was not strongly affected by the C/N ratio. In a 2.5-L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from soybean oil as the sole carbon source by batch and fed-batch cultures of R. eutropha with dry cell weights of 15-32 g/L, PHA contents of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. By co-feeding soybean oil and γ-butyrolactone as carbon sources, a copolymer of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] could be produced with dry cell weights of 10-21 g/L, yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50g-PHA/g-carbon sources used) and 4HB fractions of 6-10 mol%. Higher supplementation of γ-butyrolactone increased the 4HB fraction in the copolymer, but decreased cell and PHA yield.     

Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon sources by a newly characterized Bacillus spp

A newly acquired polyhydroxyalkanoate (PHA) producing Bacillus spp. was identified to be a strain of Bacillus cereus using a range of microbiological and molecular techniques. This strain, named B. cereus SPV, was found to be capable of using a wide range of carbon sources including glucose, fructose, sucrose, various fatty acids and gluconate for the production of PHAs, an advantage for the commercial production of the polymers. The media used for the polymer production was novel in the context of the genus Bacillus. The PHA, once produced, was found to remain at a constant maximal concentration, without any degradation, a great advantage for the commercial production of the PHAs. This particular strain of Bacillus spp. was able to synthesize various PHAs with 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB)-like monomer units from structurally unrelated carbon sources such as fructose, sucrose and gluconate. This is the first report of the incorporation of a 4HB related monomer containing PHA by the genus Bacillus and from structurally unrelated carbon sources. The PHAs isolated had molecular weights ranging between (0.4 and 0.8) x 10(6) and low polydispersity index values (M(W)/M(N)) ranging from 2.6 to 3.4.     

Engineering of chimeric class II polyhydroxyalkanoate synthases

PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.     

Biosynthesis of polyhydroxyalkanoates (PHA) by recombinant Ralstonia eutropha and effects of PHA synthase activity on in vivo PHA biosynthesis.

Recombinant strains of Ralstonia eutropha PHB 4, which harbored Aeromonas caviae polyhydroxyalkanoates (PHA) biosynthesis genes under the control of a promoter for R. eutropha phb operon, were examined for PHA production from various alkanoic acids. The recombinants produced poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from hexanoate and octanoate, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxypentano ate) [P(3HB-co-3HV-co-3HHp)] from pentanoate and nonanoate. One of the recombinant strains, R. eutropha PHB 4/pJRDBB39d3 harboring ORF1 and PHA synthase gene of A. caviae (phaC(Ac)) accumulated copolyesters with much more 3HHx or 3HHp fraction than the other recombinant strains. To investigate the relationship between PHA synthase activity and in vivo PHA biosynthesis in R. eutropha, the PHB- 4 strains harboring pJRDBB39d13 or pJRDEE32d13 were used, in which the heterologous expression of phaC(Ac) was controlled by promoters for R. eutropha phb operon and A. caviae pha operon, respectively. The PHA contents and PHA accumulation rates were similar between the two recombinant strains in spite of the quite different levels of PHA synthase activity, indicating that the polymerization step is not the rate-determining one in PHA biosynthesis by R. eutropha. The molecular weights of poly(3-hydroxybutyrate) produced by the recombinant strains were also independent of the levels of PHA synthase activity. It has been suggested that a chain-transfer agent is generated in R. eutopha cells to regulate the chain length of polymers.     

聚羟基烷酸酯 (PHA) 改性研究进展

本文简述了生物制造聚羟基烷酸酯(PHA),包括聚3-羟基丁酸酯(PHB)、聚(3-羟基丁酸酯-3-羟基戊酸酯)(PHBV)、聚(3-羟基丁酸酯-4-羟基丁酸酯)(P3/4HB)、聚(3-羟基丁酸酯-3-羟基己酸酯)(PHBH)的产业化现状,综述了针对PHA材料热稳定性差、加工窗口较窄等缺点而进行的一些改性研究。选用适当方法对PHA进行改性,可使其性能得到优化,应用领域得到拓展。     

Incremental truncation of PHA synthases results in altered product specificity

PHA synthase is the key enzyme involved in the biosynthesis of microbial polymers, polyhydroxyalkanoates (PHA). In this study, we created a hybrid library of PHA synthase gene with different crossover points by an incremental truncation method between the C-terminal fragments of the phaC(Cn) (phaC from Cupriavidus necator) and the N-terminal fragments of the phaC1(Pa) (phaC from Pseudomonas aeruginosa). As the truncation of the hybrid enzyme increased, the in vivo PHB synthesis ability of the hybrids declined gradually. PHA synthase PhaC(Cn) with a deletion on N-terminal up to 83 amino acid residues showed no synthase activity. While with the removal of up to 270 amino acids from the N-terminus, the activity of the truncated PhaC(Cn) could be complemented by the N-terminus of PhaC1(Pa). Three of the hybrid enzymes W188, W235 and W272 (named by the deleted nucleic acid number) were found to have altered product specificities.