NADPH-dependent acetoacetyl-CoA Reductase Research Articles

Expression of His-tagged NADPH-dependent acetoacetyl-CoA reductase in recombinant Escherichia coli BL-21(DE3)

Poly-3-hydroxybutyrate (P(3HB)), a member of the polyhydroxyalkanoate (PHA) family, is a biodegradable polyester with diverse industrial applications. NADPH-dependent acetoacetyl-CoA reductase (phaB) is the enzyme which plays an essential role in P(3HB) synthesis by catalyzing the conversion of the intermediates. The expression of phaB enzyme using the recombinant Escherichia coli BL-21(DE3) and the purification of the synthesized enzyme were studied. The pET-B3 plasmid harbouring the phaB gene derived from Ralstonia eutropha H16, was driven by the lac promoter in E.coli BL-21(DE3). The enzyme was expressed with different induction time, temperatures and cell age. Results showed that the cell age of 4h, induction time of 12h at 37°C were identified as the optimal conditions for the enzyme reductase expression. A specific activity of 0.151 Umg-1 protein and total protein concentration of 0.518mgmg-1 of dry cell weight (DCW) were attained. Affinity chromatography was performed to purify the His-tagged phaB enzyme, in which enhanced the specific activity (14.44 Umg-1) and purification fold (38-fold), despite relative low yield (44.6%) of the enzyme was obtained. The purified phaB showed an optimal enzyme activity at 30°C and pH 8.0. The findings provide an alternative for the synthesis of the reductase enzyme which can be used in the industrial-scale production of the biodegradable polymers.

Isopropanol production with reutilization of glucose-derived CO2 by engineered Ralstonia eutropha

Chemolithoautotrophic bacterium Ralstonia eutropha is a versatile host for production of various useful compounds including polyhydroxyalkanoates (PHAs) under both heterotrophic and autotrophic conditions. In this bacterium, Calvin-Benson-Bassham (CBB) cycle is functional even under heterotrophic conditions on sugars and reutilizes CO2 emitted through sugar metabolisms into PHA, leading to increase in yield of the storage polyester. This study focused on isopropanol production from glucose by engineered strains of R.eutropha. The isopropanol-producing strains were constructed by introduction of codon-optimized genes of acetoacetate decarboxylase (adc) and primary-secondary alcohol dehydrogenase (adh) from clostridia into glucose-utilizing and PHA-negative (ΔphaC1) strain of R.eutropha. Several genetic modifications showed that high expression of the isopropanol synthesis genes by using a strong synthetic promoter and deletion of NAD+-dependent (S)-3-hydroxybutyryl-CoA dehydrogenase genes (paaH1 and had) in addition to NADPH-dependent acetoacetyl-CoA reductase genes (phaB1 and phaB3) were effective for improving isopropanol production with low by-production of acetone. Isopropanol titer of 4.13g/L was achieved by two-stage cultivation of the strain IP-007/pBj5c2-adh-adc, corresponding to overall yield of 0.6mol mol-glucose-1. The fixation of sugar-derived CO2 during isopropanol synthesis was evaluated by 13C-labelling of the isopropanol produced from [1-13C]-glucose. The 13C-abundance in isopropanol synthesized by the engineered strain was significantly increased up to 4.8%, demonstrating actual reassimilation of CO2 emitted from glucose moiety by decarboxylation and potential contribution towards increase in the carbon yield of isopropanol on glucose.

Evaluation of BP-M-CPF4 polyhydroxyalkanoate (PHA) synthase on the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil using Cupriavidus necator transformants

Among the various types of polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] has a high potential to serve as commercial bioplastic due to its striking resemblance to petroleum-based plastics. In this study, five different genotypes of Cupriavidusnecator transformants harbouring the phaCBP-M-CPF4 gene (including PHB¯4/pBBR1-CBP-M-CPF4) were developed to evaluate the efficiency of 3HHx monomer incorporation. The fraction of 3-hydroxyhexanoate (3HHx) monomer that was incorporated into the PHA synthesized by these C. necator transformants using palm oil as the sole carbon source, was examined. Overall, co-expression of enoyl-CoA hydratase gene (phaJ1) from Pseudomonas aeruginosa, along with PHA synthase (PhaC), increased the 3HHx composition in the PHA copolymer. The differences in the enzyme activities of β-ketothiolase (PhaACn) and NADPH-dependent acetoacetyl-CoA reductase (PhaBCn) of the C. necator mutant hosts used in this study, were observed to alter the 3HHx composition and molecular weight of the PHA copolymer produced. The 3HHx fractions in the P(3HB-co-3HHx) produced by these C. necator transformants ranged between 1 and 18 mol%, while the weight-average molecular weight ranged from 0.7 × 106 to 1.8 × 106 Da. PhaCBP-M-CPF4 displayed a typical initial lag-phase and a relatively low synthase activity in the in vitro enzyme assay, which is thought to be the reason for the higher molecular weights of PHA obtained in this study.

Biosynthesis of novel lactate-based polymers containing medium-chain-length 3-hydroxyalkanoates by recombinant Escherichia coli strains from glucose

Novel lactate (LA)-based polymers containing medium-chain-length 3-hydroxyalkanoates (MCL-3HA) were produced in fadR-deficient Escherichia coli strains from glucose as the sole carbon source. The genes encoding LA and 3-hydroxybutyrate (3HB) monomers supplying enzymes [propionyl-CoA transferase (PCT), d-lactate dehydrogenase (D-LDH), β-ketothiolase (PhaA), and NADPH-dependent acetoacetyl-CoA reductase (PhaB)], MCL-3HA monomers supplying enzymes [(R)-3-hydroxyacyl-ACP thioesterase (PhaG) and (R)-3-hydroxyacyl (3HA)-CoA ligase] via fatty acid biosynthesis pathway, and modified polyhydroxyalkanoate (PHA) synthase [PhaC1(STQK)] of Pseudomonas sp. 61-3 were introduced into E.coli LS5218. This resulted in the synthesis of a novel LA-based copolymer, P(LA-co-3HB-co-3HA). 1H-nuclear magnetic resonance (NMR) analysis revealed the composition of P(LA-co-3HB-co-3HA) to be 19.7mol% LA (C3), 74.9mol% 3HB (C4), and 5.4mol% MCL-3HA units of C8 and C10. Furthermore, the recombinant E.coli CAG18497 strain carrying these genes, excluding the phaAB genes, accumulated P(92.0% LA-co-3HA) with a novel monomer composition containing C3, C8, C10, and C12. 13C-NMR analysis showed the existence of LA-3HA sequence in the polymer. The solvent cast film of P(92.0% LA-co-3HA) exhibited transparency similar to poly(lactic acid).

Production of 1,3-diols in Escherichia coli

To expand the diversity of chemical compounds produced through microbial conversion, a platform pathway for the production of widely used industrial chemicals, 1,3-diols, was engineered in Escherichia coli. The pathway was designed by modifying the previously reported (R)-1,3-butanediol synthetic pathway to consist of pct (propionate CoA-transferase) from Megasphaera elsdenii, bktB (thiolase), phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, bld (butyraldehyde dehydrogenase) from Clostridium saccharoperbutylacetonicum, and the endogenous alcohol dehydrogenase(s) of E. coli. The recombinant E. coli strains produced 1,3-pentanediol, 4-methyl-1,3-pentanediol, and 1,2,4-butanetriol, together with 1,3-butanediol, from mixtures of glucose and propionate, isobutyrate, and glycolate, respectively, in shake flask cultures. To the best of our knowledge, this is the first report of microbial production of 1,3-pentanediol and 4-methyl-1,3-pentanediol.

Engineering Yarrowia lipolytica for poly-3-hydroxybutyrate production.

Strains of Yarrowia lipolytica were engineered to express the poly-3-hydroxybutyrate (PHB) biosynthetic pathway. The genes for β-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase were cloned and inserted into the chromosome of Y. lipolytica. In shake flasks, the engineered strain accumulated PHB to 1.50 and 3.84% of cell dry weight in complex medium supplemented with glucose and acetate as carbon source, respectively. In fed-batch fermentation using acetate as sole carbon source, 7.35g/l PHB (10.2% of cell dry weight) was produced. Selection of Y. lipolytica as host for PHB synthesis was motivated by the fact that this organism is a good lipids producer, which suggests robust acetyl-CoA supply also the precursor of the PHB pathway. Acetic acid could be supplied by gas fermentation, anaerobic digestion, and other low-cost supply route.

Effect of nitrogen availability on the poly-3-d-hydroxybutyrate accumulation by engineered Saccharomyces cerevisiae

Poly-3-d-hydroxybutyrate (or PHB) is a polyester which can be used in the production of biodegradable plastics from renewable resources. It is naturally produced by several bacteria as a response to nutrient starvation in the excess of a carbon source. The yeast Saccharomyces cerevisiae could be an alternative production host as it offers good inhibitor tolerance towards weak acids and phenolic compounds and does not depolymerize the produced PHB. As nitrogen limitation is known to boost the accumulation of PHB in bacteria, the present study aimed at investigating the effect of nitrogen availability on PHB accumulation in two recombinant S. cerevisiae strains harboring different xylose consuming and PHB producing pathways: TMB4443 expressing an NADPH-dependent acetoacetyl-CoA reductase and a wild-type S. stipitis XR with preferential use of NADPH and TMB4425 which expresses an NADH-dependent acetoacetyl-CoA reductase and a mutated XR with a balanced affinity for NADPH/NADH. TMB4443 accumulated most PHB under aerobic conditions and with glucose as sole carbon source, whereas the highest PHB concentrations were obtained with TMB4425 under anaerobic conditions and xylose as carbon source. In both cases, the highest PHB contents were obtained with high availability of nitrogen. The major impact of nitrogen availability was observed in TMB4425, where a 2.7-fold increase in PHB content was obtained. In contrast to what was observed in natural PHB-producing bacteria, nitrogen deficiency did not improve PHB accumulation in S. cerevisiae. Instead the excess available carbon from xylose was shunted into glycogen, indicating a significant gluconeogenic activity on xylose.

Butyrate production under aerobic growth conditions by engineered Escherichia coli

Butyrate is an important industrial platform chemical. Although several groups have reported butyrate production under oxygen-limited conditions by a native producer, Clostridium tyrobutylicum, and by a metabolically engineered Escherichia coli, efforts to produce butyrate under aerobic growth conditions have met limited success. Here, we constructed a novel butyrate synthetic pathway that functions under aerobic growth conditions in E.coli, by modifying the 1-butanol synthetic pathway reported previously. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, and endogenous thioesterase(s) of E.coli. To evaluate the potential of this pathway for butyrate production, culture conditions, including pH, oxygen supply, and concentration of inorganic nitrogen sources, were optimized in a mini-jar fermentor. Under the optimal conditions, butyrate was produced at a concentration of up to 140mM (12.3g/L in terms of butyric acid) after 54h of fed-batch culture.

Enhanced poly(3-hydroxybutyrate) production in transgenic tobacco BY-2 cells using engineered acetoacetyl-CoA reductase.

Highly active mutant of NADPH-dependent acetoacetyl-CoA reductase (PhaB) was expressed in Nicotiana tabacum cv. Bright Yellow-2 cultured cells to produce poly(3-hydroxybutyrate) [P(3HB)]. The mutated PhaB increased P(3HB) content by three-fold over the control, indicating that the mutant was a versatile tool for P(3HB) production. Additionally, the PhaB-catalyzed reaction was suggested to be a rate-limiting step of P(3HB) biosynthesis in tobacco BY-2 cells.

Construction of CoA-dependent 1-butanol synthetic pathway functions under aerobic conditions in Escherichia coli

1-Butanol is an important industrial platform chemical and an advanced biofuel. While various groups have attempted to construct synthetic pathways for 1-butanol production, efforts to construct a pathway that functions under aerobic conditions have met with limited success. Here, we constructed a CoA-dependent 1-butanol synthetic pathway that functions under aerobic conditions in Escherichia coli, by expanding the previously reported (R)-1,3-butanediol synthetic pathway. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, bld (butylraldehyde dehydrogenase) from Clostridium saccharoperbutylacetonicum, and inherent alcohol dehydrogenase(s) from E. coli. To evaluate the potential of this pathway for 1-butanol production, culture conditions, including volumetric oxygen transfer coefficient (kLa) and pH were optimized in a mini-jar fermenter. Under optimal conditions, 1-butanol was produced at a concentration of up to 8.60gL−1 after 46h of fed-batch cultivation.

Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from unrelated carbon sources in engineered Rhodospirillum rubrum.

Different genes encoding pyridine nucleotide transhydrogenases (pntAB, udhA) and acetoacetyl-CoA reductases (phaB) were heterologously overexpressed in Rhodospirillum rubrum S1. A recombinant strain, which harbored the gene encoding the membrane-bound transhydrogenase PntAB from Escherichia coli MG1655 and the phaB1 gene coding for an NADPH-dependent acetoacetyl-CoA reductase from Ralstonia eutropha H16, accumulated poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [Poly(3HB-co-3HV)] with a 3HV fraction of up to 13 mol% from fructose. This was a 13-fold increase of the 3HV content when compared to the wild-type strain. Higher contents of 3HV are known to reduce the brittleness of this polymer, which is advantageous for most applications. The engineered R. rubrum strain was also able to synthesize this industrially relevant copolymer from CO2 and CO from artificial synthesis gas (syngas) with a 3HV content of 56 mol%. The increased incorporation of 3HV was attributed to an excess of propionyl-CoA, which was generated from threonine and related amino acids to compensate for the intracellular redox imbalance resulting from the transhydrogenase reaction. Thereby, our study presents a novel, molecular approach to alter the composition of bacterial PHAs independently from external precursor supply. Moreover, this study also provides a promising production strain for syngas-derived second-generation biopolymers.

EngineeredEscherichia colifor Short-Chain-Length Medium-Chain-Length Polyhydroxyalkanoate Copolymer Biosynthesis from Glycerol and Dodecanoate

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 (PhaARe) and NADPH-dependent acetoacetyl-CoA reductase (PhaBRe) from Ralstonia eutropha together with PHA synthases from R. eutropha (PhaCRe), Aeromonas hydrophila (PhaCAh), and Pseudomonas putida (PhaC2Pp) and with (R)-specific enoyl-CoA hydratases from P. putida (PhaJ1Pp and PhaJ4Pp), and A. hydrophila (PhaJAh). 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.

Engineered Corynebacterium glutamicum as an endotoxin-free platform strain for lactate-based polyester production

The first biosynthetic system for lactate (LA)-based polyesters was previously created in recombinant Escherichia coli (Taguchi et al. 2008). Here, we have begun efforts to upgrade the prototype polymer production system to a practical stage by using metabolically engineered Gram-positive bacterium Corynebacterium glutamicum as an endotoxin-free platform. We designed metabolic pathways in C. glutamicum to generate monomer substrates, lactyl-CoA (LA-CoA), and 3-hydroxybutyryl-CoA (3HB-CoA), for the copolymerization catalyzed by the LA-polymerizing enzyme (LPE). LA-CoA was synthesized by D: -lactate dehydrogenase and propionyl-CoA transferase, while 3HB-CoA was supplied by β-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB). The functional expression of these enzymes led to a production of P(LA-co-3HB) with high LA fractions (96.8mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.

Identification of the Polyhydroxyalkanoate (PHA)-Specific Acetoacetyl Coenzyme A Reductase among Multiple FabG Paralogs in Haloarcula hispanica and Reconstruction of the PHA Biosynthetic Pathway in Haloferax volcanii

Genome-wide analysis has revealed abundant FabG (beta-ketoacyl-ACP reductase) paralogs, with uncharacterized biological functions, in several halophilic archaea. In this study, we identified for the first time that the fabG1 gene, but not the other five fabG paralogs, encodes the polyhydroxyalkanoate (PHA)-specific acetoacetyl coenzyme A (acetoacetyl-CoA) reductase in Haloarcula hispanica. Although all of the paralogous fabG genes were actively transcribed, only disruption or knockout of fabG1 abolished PHA synthesis, and complementation of the DeltafabG1 mutant with the fabG1 gene restored both PHA synthesis capability and the NADPH-dependent acetoacetyl-CoA reductase activity. In addition, heterologous coexpression of the PHA synthase genes (phaEC) together with fabG1, but not its five paralogs, reconstructed the PHA biosynthetic pathway in Haloferax volcanii, a PHA-defective haloarchaeon. Taken together, our results indicate that FabG1 in H. hispanica, and possibly its counterpart in Haloarcula marismortui, has evolved the distinct function of supplying precursors for PHA biosynthesis, like PhaB in bacteria. Hence, we suggest the renaming of FabG1 in both genomes as PhaB, the PHA-specific acetoacetyl-CoA reductase of halophilic archaea.

Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil

Previous studies have demonstrated that heterologous expression of PHA synthase from Aeromonas caviae (PhaCAc), capable of accepting (R)-3-hydroxyacyl-CoA of C4–C7 as substrates, could confer the ability to PHA-negative mutant of Cupriavidus necator PHB-4 to synthesize poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from vegetable oils. The mutation point within pha operon in PHB-4 was determined to be a single nonsense mutation within the PHA synthase gene (phaCCn), suggesting the much lower β-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase activities observed in this strain would be a polar effect of the mutation. For further efficient biosynthesis of P(3HB-co-3HHx) copolyester, C. necator wild strain H16 was engineered by homologous recombination targeting the chromosomal phaCCn, and the PHA productivity was compared with previous PHB–4-derived strain harboring phaCAc on a multi-copy plasmid (PHB–4/pJRDEE32d13). A strain H16CAc, in which phaCCn was substituted for phaCAc on the chromosome, could produce P(3HB-co-3HHx) from soybean oil with high productivity, but the 3HHx fraction in the accumulated polymer was decreased. Meanwhile, H16ΔC/pJRDEE32d13, that lost region for the original synthase gene and expresses exochromosomal phaCAc, grew and accumulated PHA with similar properties to the PHB–4-derived strain. The results of enzyme assay suggested that low β-ketothiolase activity might be relevant for decrease of growth ability accompanied by increase of 3HHx composition when soybean oil was fed as a sole carbon source. Key words: poly(hydroxyalkanoates), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), PHA synthase, Cupriavidus necator, vegetable oil.

Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus

Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated beta-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28-34% cellular dry weight of PHA. Gas chromatography and (1)H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of beta-oxidation resulted in improved production (3-11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.

Purification and characterization of NADPH-dependent acetoacetyl-CoA reductase from Methylobacterium extorquens

NADPH-dependent acetoacetyl-CoA (AcAc-CoA) reductase (EC 1.1.1.36) was purified to electrophoretic homogeneity from the methylotroph Methylobacterium extorquens– a producer of poly-3-hydroxybutyrate. The enzyme has an Mr of 141 000 and consists of four identical subunits (Mr 31 000) and demonstrates absolute specificity for NADPH as cofactor. NADPH-AcAc-CoA reductase is inhibited by NADPH, AcAc-CoA, NADP and NAD but is activated by isocitrate and ATP. The calculated Km values were 11.6 and 41 μM for AcAc-CoA and NADPH, respectively. The results suggest that this enzyme plays a more important role in the coordinated operation of the tricarboxylic acid cycle and poly-3-hydroxybutyrate synthesis in M. extorquens than in the taxonomically related M. rhodesianum MB 126.

Metabolic engineering of Aeromonas hydrophila for the enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Wild-type Aeromonas hydrophila 4AK4 produced 35-45 wt.% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) consisting of 10-15 mol% 3-hydroxyhexanoate (3HHx). To enhance PHBHHx production, vgb gene encoding Vitreoscilla haemoglobin or fadD gene encoding Escherichia coli acyl-CoA synthase was co-expressed with polyhydroxyalkanoates (PHA) synthesis-related genes including phbAB from Wautersia eutropha and phaPCJ from A. hydrophila. Expression of vgb increased PHBHHx content from 46 to 53 wt.% without affecting the polymer monomers composition, whereas fadD increased both PHBHHx content from 46 to 64 wt.% and its 3HHx fraction from 15 to 24 mol%. Co-expression of vgb or fadD gene with PHA-synthesis-related genes generally increased PHBHHx content over 60 wt.%. Co-expression of phbAB with vgb increased PHBHHx content and concentration up to about 70 wt.% and 4.0 g l-1, respectively. Fermentor study also showed that in the recombinants harboring vgb, CDW, PHBHHx concentration and productivity were significantly elevated up to 54 g l-1, 28.5 g l-1 and 0.791 g l-1 h-1, respectively, suggesting that vgb could promote PHA synthesis. In this strain, lac promoter could be used to constitutively express foreign genes such as phbA and phbB encoding beta-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase of W. eutropha, respectively, without use of IPTG. The results showed that combined expression of different genes was a successful strategy to enhance PHA production, which could be useful for strain development to construct other recombinant PHA-producing strains.

Enhancement of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Production in the Transgenic Arabidopsis thaliana by the in Vitro Evolved Highly Active Mutants of Polyhydroxyalkanoate (PHA) Synthase from Aeromonas caviae

In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.

Enzyme-catalyzed poly(3-hydroxybutyrate) synthesis from acetate with CoA recycling and NADPH regeneration in Vitro

We established a novel enzyme-catalyzed poly(3-hydroxybutyrate) [P(3HB)] synthesis system capable of recycling CoA on the basis of the P(3HB) biosynthetic pathway in Ralstonia eutropha. The system includes purified β-ketothiolase (PhaA), NADPH-dependent acetoacetyl-CoA reductase (PhaB), PHA synthase (PhaC), acetyl-CoA synthetase (Acs) and glucose dehydrogenase (GDH). In this system, acetyl-CoA was synthesized from acetate and CoA by Acs and ATP, and then two molecules of acetyl-CoA were condensed by PhaA to synthesize acetoacetyl-CoA, which was converted to ( R)-3-hydroxybutyryl-CoA (3HBCoA) by PhaB and NADPH. The 3HBCoA was polymerized by PhaC and converted to P(3HB). In this system, the CoA molecules that were released during the condensation and polymerization reactions catalyzed by PhaA and PhaC, respectively, were reused successfully for the synthesis of acetyl-CoA. In addition, NADPH, which was consumed in the reduction of acetoacetyl-CoA, was regenerated by the action of GDH. In this system, the yield of P(3HB) synthesized from acetate as the substrate was 5.6 mg in a 5-ml reaction mixture, and the weight-average molecular weight and polydispersity were 6.64×10 6 and 1.36, respectively. Furthermore, CoA was reused at least 26 times, and NADPH was also regenerated at least 26 times during 24 h of reaction.