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.     

PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-d-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.     

Detection of intermediates from the polymerization reaction catalyzed by a D302A mutant of class III polyhydroxyalkanoate (PHA) synthase

Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of (R)-3-hydroxybutyryl-CoA (HB-CoA) into high molecular weight PHB, biodegradable polymers. The class III PHB synthase from Allochromatium vinosum is composed of a 1:1 mixture of two approximately 40 kDa proteins: PhaC and PhaE. Previous studies using site-directed mutagenesis and a saturated trimer of hydroxybutyryl-CoA have suggested the importance of C149 (in covalent catalysis), H331 (in activation of C149), and D302 (in hydroxyl group activation for ester bond formation) in the polymerization process. All three residues are located on PhaC. We now report that incubation of D302A-PhaCPhaE with [14C]-HB-CoA results in detection, for the first time, of oligomeric HBs covalently bound to PhaC. The reaction products have been analyzed by SDS-PAGE, Westerns with PhaCPhaE antibodies, and autoradiography. Different migratory properties of D302A-PhaC on SDS-PAGE have been observed at [14C]-HB-CoA to enzyme (S/E) ratios between 5 and 100. Trypsin digestion and HPLC analysis of the D302A-PhaCPhaE (from a reaction with a S/E ratio of 5) allowed isolation of multiple radiolabeled peptides. N-Terminal sequencing, MALDI-TOF, and ESI mass spectrometric analysis of these peptides revealed that all of the peptides were identical but were modified by (HB)n ranging in size from n = 3 to n = 10. The in vitro results support the role of D302 in elongation rather than in activating the active site cysteine for acylation. This proposal has been further supported by our in vivo studies on a Wautersia eutropha strain in which the class I synthase gene has been replaced with the D302A-PhaCPhaE gene and the organism examined under PHB production conditions by transmission electron microscopy. Very small granules (<0.05 microm) were observed in contrast to the 0.2-0.5 microm granules observed with the wt strain. Use of the D302A synthase has allowed successful interrogation of the initiation and elongation steps catalyzed by the class III synthase.     

Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme

The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (M _r 40,950) and PhaC (M _r 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed. Received: 9 January 2000 / Received revision: 20 February 2000 / Accepted: 25 February 2000     

Polyhydroxyalkanoate (PHA) accumulation in sulfate-reducing bacteria and identification of a class III PHA synthase (PhaEC) in Desulfococcus multivorans

Seven strains of sulfate-reducing bacteria (SRB) were tested for the accumulation of polyhydroxyalkanoates (PHAs). During growth with benzoate Desulfonema magnum accumulated large amounts of poly(3-hydroxybutyrate) [poly(3HB)]. Desulfosarcina variabilis (during growth with benzoate), Desulfobotulus sapovorans (during growth with caproate), and Desulfobacterium autotrophicum (during growth with caproate) accumulated poly(3HB) that accounted for 20 to 43% of cell dry matter. Desulfobotulus sapovorans and Desulfobacterium autotrophicum also synthesized copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate when valerate was used as the growth substrate. Desulfovibrio vulgaris and Desulfotalea psychrophila were the only SRB tested in which PHAs were not detected. When total DNA isolated from Desulfococcus multivorans and specific primers deduced from highly conserved regions of known PHA synthases (PhaC) were used, a PCR product homologous to the central region of class III PHA synthases was obtained. The complete pha locus of Desulfococcus multivorans was subsequently obtained by inverse PCR, and it contained adjacent phaE(Dm) and phaC(Dm) genes. PhaC(Dm) and PhaE(Dm) were composed of 371 and 306 amino acid residues and showed up to 49 or 23% amino acid identity to the corresponding subunits of other class III PHA synthases. Constructs of phaC(Dm) alone (pBBRMCS-2::phaC(Dm)) and of phaE(Dm)C(Dm) (pBBRMCS-2::phaE(Dm)C(Dm)) in various vectors were obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB(-)4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively. In cells of the recombinant strains harboring phaE(Dm)C(Dm) small but significant amounts (up to 1.7% of cell dry matter) of poly(3HB) and of PHA synthase activity (up to 1.5 U/mg protein) were detected. This indicated that the cloned genes encode functionally active proteins. Hybrid synthases consisting of PhaC(Dm) and PhaE of Thiococcus pfennigii or Synechocystis sp. strain PCC 6308 were also constructed and were shown to be functionally active.     

Mutations derived from the thermophilic polyhydroxyalkanoate synthase PhaC enhance the thermostability and activity of PhaC from Cupriavidus necator H16

The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16β), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(β) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16β) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16β) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16β) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.     

Extracellular polymerization of 3-hydroxyalkanoate monomers with the polymerase of Alcaligenes eutrophus.

Previous investigations on the role of the polymerase in the synthesis of poly-3-hydroxybutyrate (PHB) are reviewed, and the results from earlier in vitro studies on the activity and selectivity of the polymerase of Alcaligenes eutrophus are discussed. In the present study the effect of glycerol on stabilizing the polymerase after purification and on eliminating the lag phase in in vitro polymerization reactions of 3-hydroxybutyl CoA (HBCoA), and 3-hydroxyvaleryl CoA (HVCoA) are described. K(M) values were determined for the activity of the polymerase with both HBCoA and HVCoA, and the rates of propagation for both monomers were estimated. With a racemic mixture of HBCoA, the enzyme polymerized only the [R] monomer.     

Purification of polyhydroxybutyrate synthase from its native organism, Ralstonia eutropha: implications for the initiation and elongation of polymer formation in vivo

Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaC(Ec)), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaC(Re)) was constructed and integrated into the R. eutropha genome in place of wild-type phaC. Strep2-PhaC(Re) was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (M(w) = 350 kDa) in a "high-molecular weight" (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaC(Re) (and PhaC(Ec)), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.     

Atomic force microscopic observation of in vitro polymerized poly[(R)-3-hydroxybutyrate]: insight into possible mechanism of granule formation

Atomic force microscopy (AFM) was used to study the formation and growth of poly[(R)-3-hydroxybutyrate] (PHB) structures formed in the enzymatic polymerization of (R)-3-hydroxybutyryl coenzyme A [(R)-3-HBCoA] in vitro. Poly(3-hydroxyalkanoate) (PHA) synthase (PhaC(Re)) from Ralstonia eutropha, a class I synthase, was purified by one-step purification and then used for in vitro reactions. Before the reaction, PhaC(Re) molecules were deposited on highly oriented pyrolytic graphite (HOPG) and observed as spherical particles with an average height of 2.7 +/- 0.6 nm and apparent width of 24 +/- 3 nm. AFM analysis during the initial stage of the reaction, that is, after a small amount of (R)-3-HBCoA had been consumed, showed that the enzyme molecules polymerize (R)-3-HBCoA and form flexible 3HB polymer chains that extend from the enzyme particles, resulting in the formation of an enzyme-nascent PHB conjugate. When a sufficient amount of (R)-3-HBCoA was used as substrate, the reaction rapidly increased after the first minute followed by a slow increase in rate, and substrate was completely consumed after 4 min. After 4 min, spherical granules continued to grow in size to form clusters over 10 um in width, and in later stages of cluster formation, the cluster developed small projections with a size of approximately 100-250 nm, suggesting qualitative changes of the PHB clusters. Moreover, the high-resolution AFM images suggested that globular structures of approximately 20-30 nm apparent width, which corresponds to the size of PhaC(Re), were located on the surface of the small PHB granule particles.     

Mechanism of the polymerization reaction initiated and catalyzed by the polyhydroxybutyrate synthase of Ralstonia eutropha

Polyhydroxybutyrate (PHB) synthases (polymerases) catalyze the polymerization of the coenzyme A thioester of 3-hydroxybutyrate to PHB. The Ralstonia eutropha PHB synthase purified from recombinant E. coli cells exists in aqueous solution in both monomeric (single subunit) and homodimeric (two subunits) forms in equilibrium. Several lines of evidence suggest that the homodimer is the active form of the synthase. The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site. The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis, but a second thiol group on the same protein molecule has not yet been identified. It is suggested that cysteines at 319 from each of the two molecules of a homodimer synthase provide two identical thiol groups to jointly form a single catalytic site. To verify this model using the strategy of in vitro reconstitution, heterodimers composed of the wild-type subunit and of the C(319) mutated subunit were constructed and the activities at various ratios of the wild-type subunit to the mutated subunit were measured. The experimental results indicate that the homodimer is the active form of the enzyme, that the heterodimer containing the mutated subunit has no activity, and that a single cysteine is not sufficient for catalysis. Two identical thiol groups from C(319) residues on each subunit of the homodimer are required to form the catalytic site for the initiation and propagation reactions. We further demonstrate that a dimer synthase that has initiated the polymerization reaction (primed synthase) is significantly more stable against dissociation than the unprimed (unreacted) dimer synthase. These two properties explain the nature of lag phenomenon during the in vitro polymerization reaction catalyzed by this enzyme     

地中海富盐菌PhaE蛋白乙酰化修饰对其功能的影响

【目的】研究地中海富盐菌PHA合酶(Pha EC)中Pha E亚基乙酰化修饰对其功能的影响,探讨乙酰化修饰对菌体生理代谢的调控作用。【方法】蔗糖密度梯度离心收集PHA颗粒,质谱鉴定颗粒结合蛋白Pha E的乙酰化位点。将乙酰化位点(赖氨酸,K)分别突变为精氨酸(R)(模拟去乙酰化)或谷氨酰胺(Q)(模拟乙酰化),利用同源双交换原理,将突变后的基因原位敲入基因组。以野生型为对照,检测突变对菌体生长、葡萄糖消耗和PHA合成能力的影响。利用Western blot检测PHA颗粒上Pha E的含量,进一步分析乙酰化修饰对蛋白功能的影响。【结果】在Pha E蛋白105位和170位赖氨酸(K)2个位点检测到乙酰化修饰。利用遗传操作系统将突变的基因原位敲入,共得到6种突变株。发酵结果表明,任何一种单突变对菌体生长及PHA合成的影响均不明显。但当2个位点同时突变成精氨酸(K105R/K170R)时,突变株生长及合成PHA的能力均受到明显抑制,2个位点同时突变成谷氨酰胺(K105Q/K170Q)则无明显影响。进一步的Western blot结果表明,突变成精氨酸的双突变株的PHA颗粒上,Pha E蛋白的含量相较于野生型约降低了一半。【结论】Pha E蛋白的去乙酰化能够导致菌株利用葡萄糖合成PHA的能力显著降低,其可能原因是降低了Pha E与PHA颗粒或PHA颗粒上Pha C的结合能力,从而降低了Pha EC合酶的活性。     

Characterization of the highly active polyhydroxyalkanoate synthase of Chromobacterium sp. strain USM2

The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.     

Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria

During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp. PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight. Our analysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis sp. PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB^–4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp. PCC6803 PHA synthase. PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A. eutrophus (PhaC) and of C. vinosum (PhaE and PhaC) revealed no immunoreaction. Received: 11 March 1998 / Accepted: 2 June 1998     

Polyhydroxyalkanoate film formation and synthase activity during in vitro and in situ polymerization on hydrophobic surfaces

In vitro and in situ enzymatic polymerization of polyhydroxyalkanoate (PHA) on two hydrophobic surfaces, a highly oriented pyrolytic graphite (HOPG) and an alkanethiol self-assembled monolayer (SAM), was studied by atomic force microscopy (AFM) and quartz crystal microbalance (QCM), using purified Ralstonia eutropha PHA synthase (PhaC(Re)) as a biocatalyst. (R)-Specific enoyl-CoA hydratase was used to prepare R-enantiomer monomers [(R)-3-hydroxyacyl-CoA] with an acyl chain length of 4-6 carbon atoms. PHA homopolymers with different side-chain lengths, poly[(R)-3-hydroxybutyrate] [P(3HB)] and poly[(R)-3-hydroxyvalerate] [P(3HV)] were successfully synthesized from such R-enantiomer monomers on HOPG substrates. After the reaction, the surface morphologies were analyzed by AFM, revealing a nanometer thick PHA film. The same biochemical polymerization process was observed on an alkanethiol (C18) SAM surface fabricated on a gold electrode using QCM. This analysis showed that a complex sequence of PhaC(Re) adsorption and PHA polymerization has occurred on the hydrophobic surface. On the basis of these observations, the possible mechanisms of the PhaC(Re)-catalyzed polymerization reaction on the surface of hydrophobic substrates are proposed.     

Polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas stutzeri 1317 had different substrate specificities

The whole polyhydroxyalkanoate (PHA) synthesis gene locus of Pseudomonas stutzeri strain 1317 containing PHA synthase genes phaC1Ps, phaC2Ps and PHA depolymerase gene phaZPs was cloned using a PCR cloning strategy. The sequence analysis results of the phaC1Ps, phaC2Ps and phaZPs showed high homology to the corresponding pha loci of the known Pseudomonas strains, respectively. PhaC1Ps and PhaC2Ps were functionally expressed in recombinant Escherichia coli strains and their substrate specificity was compared. The results demonstrated that PhaC1Ps and PhaC2Ps from P. stutzeri 1317 had different substrate specificities when expressed in E. coli. In details, PhaC2Ps could incorporate both short-chain-length 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates (mcl 3HA) into PHA, while PhaC1Ps only favored mcl 3HA for polymerization.     

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.     

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.     

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. SG4502 with acquired LA-polymerizing activity

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1_SG and PhaC2_SG). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1_SG to evaluate the potential of the resulting protein as a “thermostable LPE”. The mutated PhaC1_SG [PhaC1_SG(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1_SG(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).