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1.
Kazunori Ushimaru Yoko Motoda Keiji Numata Takeharu Tsuge 《Applied and environmental microbiology》2014,80(9):2867-2873
In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc. 相似文献
2.
《Bioscience, biotechnology, and biochemistry》2013,77(6):1262-1268
Short-chain-length medium-chain-length polyhydroxyalkanoate (SCL-MCL PHA) copolymers are promising as bio-plastics with properties ranging from thermoplastics to elastomers. In this study, the hybrid pathway for the biosynthesis of SCL-MCL PHA copolymers was established in recombinant Escherichia coli by co-expression of β-ketothiolase (PhaA Re ) and NADPH-dependent acetoacetyl-CoA reductase (PhaB Re ) from Ralstonia eutropha together with PHA synthases from R. eutropha (PhaC Re ), Aeromonas hydrophila (PhaC Ah ), and Pseudomonas putida (PhaC2 Pp ) and with (R)-specific enoyl-CoA hydratases from P. putida (PhaJ1 Pp and PhaJ4 Pp ), and A. hydrophila (PhaJ Ah ). When glycerol supplemented with dodecanoate was used as primary carbon source, E. coli harboring various combinations of PhaABCJ produced SCL-MCL PHA copolymers of various monomer compositions varying from C4 to C10. In addition, polymer property analysis suggested that the copolymers produced from this recombinant source have thermal properties (lower glass transition and melting temperatures) superior to polyhydroxybutyrate homopolymer. 相似文献
3.
The F420S substitution enhances the specific activity of Ralstonia eutropha PHA synthase (PhaCRe). We have now carried out site-directed saturation mutagenesis of F420 of PhaCRe and, amongst the F420 mutants, the F420S mutant gave the highest poly(3-hydroxybutyrate) (PHB) content. In vitro activity assay showed that the F420S enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. Enhancement of PHB accumulation was achieved by combination of the F420S mutation with a G4D mutation, which conferred high PHB content and high in vivo concentration of PhaCRe enzyme. The G4D/F420S mutant gave a higher PHB content and in vivo concentration of PhaCRe enzyme than the F420S mutant, while the molecular weight of the PHB polymer of the double mutant was similar to that of the F420S mutant. 相似文献
4.
Anna Ochi Ken’ichiro Matsumoto Takashi Ooba Kohei Sakai Takeharu Tsuge Seiichi Taguchi 《Applied microbiology and biotechnology》2013,97(8):3441-3447
Class I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (PhaCRe) was engineered so as to acquire an unusual lactate (LA)-polymerizing activity. To achieve this, the site-directed saturation mutagenesis of PhaCRe was conducted at position 510, which corresponds to position 481 in the initially discovered class II LA-polymerizing PHA synthase (PhaC1PsSTQK), a mutation in which (Gln481Lys) was shown to be essential to its LA-polymerizing activity (Taguchi et al., Proc Natl Acad Sci USA 105(45):17323–17327, 2008). The LA-polymerizing activity of the PhaCReA510X mutants was evaluated based on the incorporation of LA units into the P[3-hydroxybutyrate(3HB)] backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaCRe(A510X) mutants, 15 synthesized P (LA-co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaCReA510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The 13C and 1H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA-co-3HB) copolymer with a weight-averaged molecular weight of 3.2?×?105 Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA*-LA triad sequence, suggesting that the polymer synthesized by PhaCRe mutant may not be a random copolymer, but presumably had a block sequence. 相似文献
5.
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. 相似文献
6.
The site-specific mutagenesis for PHA synthase PhaC2Ps1317 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 PhaC2Ps1317 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. 相似文献
7.
A polyhydroxyalkanoate (PHA) synthase gene phaC2
Ps from Pseudomonas stutzeri strain 1317 was introduced into a PHA synthase gene phbC
Re negative mutant, Ralstonia eutropha PHB−4. It conferred on the host strain the ability to synthesize PHA, the monomer compositions of which varied widely when grown
on different carbon sources. During cultivation on gluconate, the presence of phaC2
Ps in R. eutropha PHB−4 led to the accumulation of polyhydroxybutyrate (PHB) homopolymer in an amount of 40.9 wt% in dry cells. With fatty acids,
the recombinant successfully produced PHA copolyesters containing both short-chain-length and medium-chain-length 3-hydroxyalkanoate
(3HA) of 4–12 carbon atoms in length. When cultivated on a mixture of gluconate and fatty acid, the monomer composition of
accumulated PHA was greatly affected and the monomer content was easily regulated by the addition of fatty acids in the cultivation
medium. After the (R)-3-hydroxydecanol-ACP:CoA transacylase gene phaG
Pp from Pseudomonas putida was introduced into phaC2
Ps-containing R. eutropha PHB−4, poly(3HB-co-3HA) copolyester with a very high 3-hydroxybutyrate (3HB) fraction (97.3 mol%) was produced from gluconate and the monomer
compositions of PHA synthesized from fatty acids were also altered. This study clearly demonstrated that PhaC2Ps cloned from P. stutzeri 1317 has extraordinarily low substrate specificity in vivo, though it has only 54% identity in comparison to a previously described low-substrate-specificity PHA synthase PhaC1Ps from Pseudomonas sp. 61–3. This study also indicated that the monomer composition and content of the synthesized PHA can be effectively modulated
by controlling the addition of carbon sources or by modifying metabolic pathways in the hosts. 相似文献
8.
A Pseudomonas strain, 3Y2, that produced polyhydroxyalkanoate (PHA) polymers consisting of 3-hydroxybutyric acid (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-HA) units, with up to 30% 3HB, was isolated. Two PHA biosynthesis loci (pha
Ps-1 and pha
Ps-2) from 3Y2 were cloned by polymerase chain reaction amplification techniques. The pha
Ps-2 locus was similar to the PHA biosynthesis loci of other PHA-producing Pseudomonas strains, with five tandem open reading frames (ORFs) located in the order ORF1
Ps-2-phaC1
Ps-2-phaZ
Ps-2-phaC2
Ps-2-phaD
Ps-2. The pha
Ps-1 locus that contains phaC1
Ps-1-phaZ
Ps-1 appears to have arisen by a duplication event that placed it downstream of a gene (ORF1
Ps-1), encoding a putative glucose-methanol-choline flavoprotein oxidoreductase. The PHA synthases 1 encoded by phaC1
Ps-1 and phaC1
Ps-2 were investigated by heterologous expression in Wautersia eutropha PHB−4. Both synthases displayed similar substrate specificities for incorporating 3HB and mcl-HA units into PHA. The ability of PhaC1
Ps-1 to confer PHA synthesis, however, appeared reduced compared to that of PhaC1
Ps-2, since cells harboring PhaC1
Ps-1 accumulated 2.5 to 4.6 times less PHA than cells expressing PhaC1
Ps-2. Primary sequence analysis revealed that PhaC1
Ps-1 had markedly diverged from the other PHA synthases with a relatively high substitution rate (14.9 vs 2% within PhaC1
Ps-2). The mutations affected a highly conserved C-terminal region and the surroundings of the essential active site cysteine (Cys296) with a loss of hydrophobicity. This led us to predict that if phaC1
Ps-1 produces a protein product in the native strain, it is likely that PhaC1
Ps-1 may be destined for elimination by the accumulation of inactivating mutations, although its specialization to accommodate different substrates cannot be eliminated. 相似文献
9.
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. 相似文献
10.
Fumi Shozui Ken’ichiro Matsumoto Takahiro Sasaki Seiichi Taguchi 《Applied microbiology and biotechnology》2009,84(6):1117-1124
Class II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1Ps) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1Ps that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction
in the copolymers (62 mol.%). However, polymer content was often largely decreased (0.2 wt.%) regardless of an enhancement
in 3HB fraction, compared to the wild-type enzyme (14 mol.% 3HB and 12 wt.%; Matsumoto et al. (2006) Biomacromolecules, 7:2436–2442).
In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise
saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1Ps was performed using single and double mutations at sites 130 and 325 as templates to increase 3HB fraction in the copolymer
without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1Ps mutants were screened based on enhanced P(3HB) content and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer
(63 mol.%) and also polymer content (18 wt.%), indicating that mutation scrambling was effective for obtaining the desired
mutants. 相似文献
11.
Yang TH Jung YK Kang HO Kim TW Park SJ Lee SY 《Applied microbiology and biotechnology》2011,90(2):603-614
Previously, we have developed metabolically engineered Escherichia coli strains capable of producing polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] by employing evolved Clostridium propionicum propionate CoA transferase (Pct
Cp
) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1
Ps6-19). Introduction of mutations four sites (E130, S325, S477, and Q481) of PhaC1
Ps6-19 have been found to affect the polymer content, lactate mole fraction, and molecular weight of P(3HB-co-LA). In this study, we have further engineered type II Pseudomonas PHA synthases 1 (PhaC1s) from Pseudomonas chlororaphis, Pseudomonas sp. 61-3, Pseudomonas putida KT2440, Pseudomonas resinovorans, and Pseudomonas aeruginosa PAO1 to accept short-chain-length hydroxyacyl-CoAs including lactyl-CoA and 3-hydroxybutyryl-CoA as substrates by site-directed
mutagenesis of four sites (E130, S325, S477, and Q481). All PhaC1s having mutations in these four sites were able to accept
lactyl-CoA as a substrate and supported the synthesis of P(3HB-co-LA) in recombinant E. coli, whereas the wild-type PhaC1s could not accumulate polymers in detectable levels. The contents, lactate mole fractions, and
the molecular weights of P(3HB-co-LA) synthesized by recombinant E. coli varied depending upon the source of the PHA synthase and the mutants used. PLA homopolymer could also be produced at ca.
7 wt.% by employing the several PhaC1 variants containing E130D/S325T/S477G/Q481K quadruple mutations in wild-type E. coli XL1-Blue. 相似文献
12.
Jo-Ann Chuah Satoshi Tomizawa Miwa Yamada Takeharu Tsuge Yoshiharu Doi Kumar Sudesh Keiji Numata 《Applied and environmental microbiology》2013,79(12):3813-3821
Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases. 相似文献
13.
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. 相似文献
14.
Tajima K Han X Satoh Y Ishii A Araki Y Munekata M Taguchi S 《Applied microbiology and biotechnology》2012,94(2):365-376
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 (PhaC1SG and PhaC2SG). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic
Pseudomonas sp. 61-3 were introduced into PhaC1SG to evaluate the potential of the resulting protein as a “thermostable LPE”. The mutated PhaC1SG [PhaC1SG(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 PhaC1SG(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). 相似文献
15.
Han J Hou J Liu H Cai S Feng B Zhou J Xiang H 《Applied and environmental microbiology》2010,76(23):7811-7819
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. 相似文献
16.
The aim of this study is to enhance 3-hydroxyhexanoate (3HHx) fractions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), abbreviated as PHBHHx, through site-directed mutagenesis of Aeromonas hydrophila enoyl Coenzyme A hydratase (PhaJAh). Two amino acids (Leu-65 and Val-130) were selected as a substitutional site based on the structural information of PhaJAh. The purified proteins from the wild-type enzyme and mutants were used to determine hydratase activities. Hydratase activities
of four single-mutation enzymes were similar to those of the wild type PhaJAh, while hydratase activities of two double-mutation enzymes were much lower. In addition, the mutated phaJ
Ah was individually co-transformed into E. coli BL21 (DE3) together with pFH21, which carried the PHA synthase (PhaCAh) gene from A. hydrophila. The recombinant E. coli harboring plasmid pETJ1 (L65A), pETJ2 (L65V) or plasmid pETJ3 (V130A) synthesized the enhanced 3HHx fractions of PHBHHx from
dodecanoate, indicating that Leu-65 and Val-130 of PhaJAh play an important role in determining the acyl chain length substrate specificity. The mutated PhaJAh (L65A, L65V, or V130A) provided higher 3HHx precursors for PHA synthase, resulting in the enhanced 3HHx fractions of PHBHHx.
It is possible to change the acyl chain length substrate specificity of PhaJ through site-directed mutagenesis and produce
PHBHHx with a wider range of alterable monomer composition. 相似文献
17.
Background
Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary. 相似文献18.
For the first time, the purification has been achieved of the type II polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2
from Pseudomonas aeruginosa applying N-terminal His6-tag fusions and metal chelate affinity chromatography. In vivo His6-tagged PHA synthase activity was confirmed by functional expression of the corresponding genes in Escherichia coli, and PHA synthase activity could also be measured in vitro with the enzymes. The specific enzyme activity of PHA synthases PhaC1 and PhaC2 was 0.039 U mg−1 and 0.035 U mg−1 protein, respectively. Kinetic studies showed a lag phase for both PHA synthases using (R,S)-3-hydroxydecanoyl-CoA as substrate. Specific enzyme activity was increased to 0.055 U mg−1 when the phasin GA24 from Ralstonia eutropha was added to the assay. CoA inhibited PHA synthase activity, and a K
i of 85 μM was determined. A two-enzyme system was established, employing commercially available acyl-CoA synthetase and PHA
synthase, which allowed the in vitro de novo PHA granule formation and the in vitro synthesis of poly(3-hydroxydecanoate) exhibiting a weight average molar mass of 9.8 × 104 g mol−1, and which occurred independently of pre-existing PHA granules.
Received: 3 December 1999 / Revision received: 10 January 2000 / Accepted: 14 January 2000 相似文献
19.
Enhanced Accumulation and Changed Monomer Composition in Polyhydroxyalkanoate (PHA) Copolyester by In Vitro Evolution of Aeromonas caviae PHA Synthase
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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 PhaCAc, 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 phaCAc 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 phaCAc 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%). 相似文献
20.
Han X Satoh Y Satoh T Matsumoto K Kakuchi T Taguchi S Dairi T Munekata M Tajima K 《Applied microbiology and biotechnology》2011,92(3):509-517
A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization
activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA)
transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating
2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products
were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was
1.00 × 105, and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating
9 mol% 2HB were analyzed by DSC and TG-DTA. T
g, T
m, and T
d (10%) were observed at −1.1°C, 158.8°C, and 252.7°C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates,
and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl
group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize
2HB. 相似文献