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Anthony J Sinskey - One of the best experts on this subject based on the ideXlab platform.
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biosynthesis of poly 3 hydroxybutyrate co 3 hydroxyhexanoate p hb co hhx from butyrate using engineered ralstonia eutropha
Applied Microbiology and Biotechnology, 2014Co-Authors: Jongmin Jeon, Chokyun Rha, Anthony J Sinskey, Christopher J Brigham, Yonghyun Kim, Hyunjoong Kim, Hyungsup Kim, Yunghun YangAbstract:Polyhydroxyalkanoates (PHAs), a promising family of bio-based polymers, are considered to be alternatives to traditional petroleum-based plastics. Copolymers like poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) have been shown to exhibit favorable physical and mechanical properties, due to decreased crystallinity resulting from the presence of medium-chain-length 3-hydroxyhexanoate (3HHx) monomers. In this study, we produced P(HB-co-HHx) using engineered Ralstonia eutropha strains containing deletions of the Acetoacetyl-CoA Reductase (phaB) genes and replacing the native PHA synthase with phaC2 from Rhodococcus aetherivorans I24 and by using butyrate, a short-chain organic acid, as the carbon source. Although the wild-type R. eutropha did not produce P(HB-co-HHx) when grown on mixed acids or on butyrate as the sole carbon source, we are able to produce polymer containing up to 40 wt% 3HHx monomer with the aforementioned engineered R. eutropha strains using various concentrations of just butyrate as the sole carbon source. This is the first report for the production of P(HB-co-HHx) copolymer in R. eutropha using butyrate.
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engineered corynebacterium glutamicum as an endotoxin free platform strain for lactate based polyester production
Applied Microbiology and Biotechnology, 2012Co-Authors: Yuyang Song, Anthony J Sinskey, Kenichiro Matsumoto, Miwa Yamada, Aoi Gohda, Christopher J Brigham, Seiichi TaguchiAbstract: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 (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.
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Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains
Applied and Environmental Microbiology, 2011Co-Authors: Charles F. Budde, Chokyun Rha, Laura B Willis, Sebastian L. Riedel, Anthony J SinskeyAbstract:The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of Acetoacetyl-CoA Reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate Reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.
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roles of multiple acetoacetyl coenzyme a Reductases in polyhydroxybutyrate biosynthesis in ralstonia eutropha h16
Journal of Bacteriology, 2010Co-Authors: Charles F. Budde, Chokyun Rha, Alison E Mahan, Anthony J SinskeyAbstract:The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a β-ketothiolase (PhaA), an Acetoacetyl-CoA Reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential Acetoacetyl-CoA Reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active Reductase. Addition of the individual Reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.
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pha synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo
Nature Biotechnology, 1997Co-Authors: Sang Jun Sim, Chokyun Rha, Kristi D Snell, Scott A Hogan, Joanne Stubbe, Anthony J SinskeyAbstract:A synthetic operon for polyhydroxyalkanoate (PHA) biosynthesis designed to yield high levels of PHA synthase activity in vivo was constructed by positioning a genetic fragment encoding beta-ketothiolase and Acetoacetyl-CoA Reductase behind a modified synthase gene containing an Escherichia coli promoter and ribosome binding site. Plasmids containing the synthetic operon and the native Alcaligenes eutrophus PHA operon were transformed into E. coli DH5 alpha and analyzed for polyhydroxybutyrate production. The molecular weight of polymer isolated from recombinant E. coli containing the modified synthase construct, determined by multiangle light scattering, was lower than that of the polymer from E. coli containing the native A. eutrophus operon. A further decrease in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon. Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain encoding the native operon with a strain possessing the synthetic operon indicates that the amount of polyhydroxyalkanoate synthase in a host organism plays a key role in controlling the molecular weight and the polydispersity of polymer.
Seiichi Taguchi - One of the best experts on this subject based on the ideXlab platform.
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directed evolution and structural analysis of nadph dependent acetoacetyl coenzyme a acetoacetyl coa Reductase from ralstonia eutropha reveals two mutations responsible for enhanced kinetics
Applied and Environmental Microbiology, 2013Co-Authors: Kenichiro Matsumoto, Yoshikazu Tanaka, Tsuyoshi Watanabe, Ren Motohashi, Koji Ikeda, Kota Tobitani, Min Yao, Isao Tanaka, Seiichi TaguchiAbstract:NADPH-dependent acetoacetyl-coenzyme A (Acetoacetyl-CoA) Reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and Acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.
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efficient r 3 hydroxybutyrate production using acetyl coa regenerating pathway catalyzed by coenzyme a transferase
Applied Microbiology and Biotechnology, 2013Co-Authors: Kenichiro Matsumoto, Takehiro Okei, Inori Honma, Toshihiko Ooi, Hirobumi Aoki, Seiichi TaguchiAbstract:(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by β-ketothiolase (PhaA) and acetoacetyl CoA Reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L−1 (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L−1, with a productivity of 0.22 g L−1 h−1. The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of 13C carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.
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engineered corynebacterium glutamicum as an endotoxin free platform strain for lactate based polyester production
Applied Microbiology and Biotechnology, 2012Co-Authors: Yuyang Song, Anthony J Sinskey, Kenichiro Matsumoto, Miwa Yamada, Aoi Gohda, Christopher J Brigham, Seiichi TaguchiAbstract: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 (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.
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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
Biomacromolecules, 2005Co-Authors: Kenichiro Matsumoto, Seiichi Taguchi, Rina Nagao, Takaaki Murata, Yuko Arai, Tomoyasu Kichise, Hideo Nakashita, Hiroaki Shimada, Yoshiharu DoiAbstract: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 specifi...
Hua Xiang - One of the best experts on this subject based on the ideXlab platform.
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Xiang H: Identification of the polyhydroxyalkanoate (PHA)-specific Acetoacetyl-CoA Reductase from multiple FabG paralogs in Haloarcula hispanica and reconstruction of PHA biosynthetic pathway in Haloferax volcanii
2013Co-Authors: Jing Han, Hailong Liu, Ligang Zhou, Hua XiangAbstract:Genome-wide analysis has revealed abundant FabG (�-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 �fabG1 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. Several haloarchaeal species belonging to the genera Haloferax, Haloarcula, Natrialba, and Haloquadratum are capable of synthesizing short-chain-length polyhydroxyalkanoates (SCL-PHAs) (6, 8, 14, 16), a large family of biopolymers with desirable biodegradability, biocompatibility, and thermoplastic features (31). Although the metabolic pathways of PHAs in bacteri
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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
Applied and environmental microbiology, 2009Co-Authors: Jing Han, Hailong Liu, Ligang Zhou, Hua XiangAbstract: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.
Kenichiro Matsumoto - One of the best experts on this subject based on the ideXlab platform.
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directed evolution and structural analysis of nadph dependent acetoacetyl coenzyme a acetoacetyl coa Reductase from ralstonia eutropha reveals two mutations responsible for enhanced kinetics
Applied and Environmental Microbiology, 2013Co-Authors: Kenichiro Matsumoto, Yoshikazu Tanaka, Tsuyoshi Watanabe, Ren Motohashi, Koji Ikeda, Kota Tobitani, Min Yao, Isao Tanaka, Seiichi TaguchiAbstract:NADPH-dependent acetoacetyl-coenzyme A (Acetoacetyl-CoA) Reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and Acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.
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efficient r 3 hydroxybutyrate production using acetyl coa regenerating pathway catalyzed by coenzyme a transferase
Applied Microbiology and Biotechnology, 2013Co-Authors: Kenichiro Matsumoto, Takehiro Okei, Inori Honma, Toshihiko Ooi, Hirobumi Aoki, Seiichi TaguchiAbstract:(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by β-ketothiolase (PhaA) and acetoacetyl CoA Reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g L−1 (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g L−1, with a productivity of 0.22 g L−1 h−1. The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of 13C carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.
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engineered corynebacterium glutamicum as an endotoxin free platform strain for lactate based polyester production
Applied Microbiology and Biotechnology, 2012Co-Authors: Yuyang Song, Anthony J Sinskey, Kenichiro Matsumoto, Miwa Yamada, Aoi Gohda, Christopher J Brigham, Seiichi TaguchiAbstract: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 (96.8 mol%). The omission of PhaA and PhaB from this pathway led to a further increase in LA fraction up to 99.3 mol%. The newly engineered C. glutamicum potentially serves as a food-grade and biomedically applicable platform for the production of poly(lactic acid)-like polyester.
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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
Biomacromolecules, 2005Co-Authors: Kenichiro Matsumoto, Seiichi Taguchi, Rina Nagao, Takaaki Murata, Yuko Arai, Tomoyasu Kichise, Hideo Nakashita, Hiroaki Shimada, Yoshiharu DoiAbstract: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 specifi...
Alexander Steinbuchel - One of the best experts on this subject based on the ideXlab platform.
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synthesis of poly 3 hydroxybutyrate co 3 hydroxyvalerate from unrelated carbon sources in engineered rhodospirillum rubrum
Fems Microbiology Letters, 2015Co-Authors: Daniel Heinrich, Alexander Steinbuchel, Matthias RabergAbstract: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.
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Central metabolism of R. eutropha H16 and mutant PHB-4 with regard to the results of proteome analyses.
2014Co-Authors: Matthias Raberg, Birgit Voigt, Michael Hecker, Alexander SteinbuchelAbstract:The numbers in the scheme indicate the following involved enzymes: 1, glucokinase; 2, phosphogluconate dehydratase; 3, phospho-2-keto-3-desoxygluconate aldolase; 4, glyceraldehyde-3-phosphate dehydrogenase; 5, phosphoglycerate dehydrogenase; 6, phosphoglyceromutase; 7, enolase; 8, pyruvate kinase; 9, pyruvate dehydrogenase/decarboxylase (E1 of PDHC); 10, dihydrolipoamide acetyltransferase (E1 of PDHC); 11, dihydrolipoamide dehydrogenase (E3 of PDHC); 12, acetoin dehydrogenase enzyme system; 13, acetyl-CoA acetyltransferase; 14, Acetoacetyl-CoA Reductase; 15, PHB synthase; 16, 3-oxoacid-CoA transferase; 17, 3-hydroxybutyrate dehydrogenase; 18, citrate synthase; 19, aconitase; 20, isocitrate dehydrogenase; 21, 2-oxoacid dehydrogenase multienzyme complex; 22, succinyl-CoA synthetase; 23, succinate dehydrogenase; 24, fumarase; 25, malate dehydrogenase; 26, citrate lyase.
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expression of polyhydroxyalkanoic acid biosynthesis genes in methylotrophic bacteria relying on the ribulose monophosphate pathway
Applied Microbiology and Biotechnology, 1993Co-Authors: Christina Follner, Wolfgang Babel, Henry E. Valentin, Alexander SteinbuchelAbstract:Four representatives of methylotrophic bacteria relying on the ribulose monophosphate (RMP) pathway were investigated for their capability to synthesize polyhydroxyalkanoic acids (PHA). In Methylophilus methylotrophus B115, Methylobacillus glycogenes strains B121 and B53 and Acetobacter methanolicus B58 no \-ketothiolase, acetoacetyl-coenzyme A (CoA) Reductase or PHA synthase could be detected, and hybridization experiments using heterologous DNA probes derived from PHA-biosynthesis genes of Methylobacterium extorquens or Alcaligenes eutrophus gave no evidence for the presence of the corresponding genes in these PHA-negative methylotrophic bacteria. Fragments harbouring a cluster of PHA-biosynthesis genes of A. eutrophus or Chromatium vinosum or isolated PHA synthase structural genes of M. extorquens, Rhodospirillum rubrum or Rhodobacter sphaeroides were mobilized into the RMP pathway bacteria mentioned above. Only transconjugants, which harboured the PHA-biosynthesis genes of A. eutrophus or C. vinosum, expressed active \-ketothiolase, Acetoacetyl-CoA Reductase and PHA synthase and accumulated poly(3-hydroxybutyric acid) (PHB). Highest amounts of PHB (up to 15% of the cellular dry weight) were accumulated in transconjugants of Methylophilus methylotrophus B115 or of Methylobacillus glycogenes strains B121 and B53 harbouring the PHA-biosynthesis genes of C. vinosum.
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cloning and nucleotide sequences of genes relevant for biosynthesis of poly 3 hydroxybutyric acid in chromatium vinosum strain d
FEBS Journal, 1992Co-Authors: Matthias Liebergesell, Alexander SteinbuchelAbstract:From a genomic library of Chromatium vinosum strain D in λL47, a 16.5-kbp EcoRI-restriction fragment was identified by hybridization with a DNA fragment harboring the operon for Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthesis. This fragment and subfragments thereof restored the ability to synthesize and accumulate PHA in PHA-negative mutants of A. eutrophus. A region of 6977 bp was sequenced; seven open reading frames (ORFs) were identified which probably represent coding regions; six of these are most probably relevant for PHA biosynthesis in C. vinosum. The structural genes for biosynthetic acetyl-CoA acyltransferase (β-ketothiolase; phbACv, 1188 bp) and NADH-dependent Acetoacetyl-CoA Reductase (phbBCv, 741 bp) were separated by ORF4 (462 bp) and ORF5 (369 bp). Downstream of phbBCv ORF7 (471 pb) was identified which was not completed at the 3′ terminus. The functions of ORF4, ORF5, and ORF7 are not known. The amino acid sequences of β-ketothiolase and Acetoacetyl-CoA Reductase deduced from phbACv and phbBCv, exhibited a similarity of 68.2% and 56.4% identical amino acids, respectively, to the corresponding enzymes of A. eutrophus. Antilinear to and upstream of the genes mentioned above, two genes were identified which were transcribed from a σ70-dependent promoter. This promoter overlapped with and was divergent to the phbACv promoter; the transcriptional start sites were mapped by S1 nuclease protection assays. These genes were ORF2 (1074 bp), whose function is not known but whose presence in Escherichia coli is essential for expression of PHA synthase activity, and the structural gene for a PHA synthase of low Mr (phbCCv, 1068 bp). The gene products of ORF2 and phbCCv, with Mr of 40525 and 39730, respectively, were expressed in E. coli applying the T7 RNA polymerase/promoter system. Although the amino acid sequence of PHA synthase deduced from phbCCv exhibited only 24.7% overall similarity with the PHA synthase of A. eutrophus, highly conserved regions were identified.
