Co-expression of 3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHA production in Escherichia coli HB101 strain

Taguchi, Kenichi

doi:10.1016/s0378-1097(99)00215-3

Cited by 37 publications

(61 citation statements)

References 16 publications

(30 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Recently, the MCL-PHA biosynthesis pathway was successfully established in recombinant Escherichia coli by expressing the MCL-PHA synthase gene. The ␤-oxidation pathway has been engineered by the overexpression of enoyl-CoA hydratase (34, 35) or 3-ketoacyl-ACP reductase (22,27,33), and/or by the disruption of FadB or FadA (15,22,24,25,27). In the former case, the metabolic connection of ␤-oxidation pathway to PHA biosynthesis is quite clear.…”

mentioning

confidence: 99%

“…The metabolic links between the fatty acid metabolism and PHA biosynthesis are mediated by various enzymes such as enoyl-CoA hydratase (7,8,9,23,34,35), 3-ketoacyl-ACP reductase (22,27,33), epimerase (20), and 3-hydroxyacyl-ACP:CoA transacylase (12,26). The genes encoding these enzymes have been cloned from various bacteria and characterized in detail at molecular level.…”

mentioning

confidence: 99%

“…Enoyl-CoA hydratase, epimerase and 3-ketoacyl-CoA or ACP reductase have been employed to supply PHA precursors. The following letters in parentheses represent the indicated enzymes: A, enzymes that have not been elucidated in E. coli; B, amplified enzymes to supply PHA precursors FabG (22,27,33), RhlG (22), and PhaJ (9,23,34,35); C, enzymes that are activated when the FadB is inactivated Ϫ YfcX (30), MaoC (this study); X, enzymes that should be inactivated to supply PHA precursors from fatty acid (15,22,24,25,27,30 2 and 4 compared with those of supplied fatty acid, was produced by fadB mutant E. coli, even though the key enzyme, FadB, involved in cycling intermediates of ␤-oxidation was inactivated. These results might have resulted from the presence of various FadB homologous enzymes mentioned above.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

Park

Lee

2003

J Bacteriol

View full text Add to dashboard Cite

The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoC Ec gene in E. coli WB108. Also, E. coli W3110 possessing fully functional ␤-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoC Ec gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His 6 -Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the ␤-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.Polyhydroxyalkanoates (PHAs) are polyesters of (R)-hydroxyalkanoic acids accumulated in numerous bacteria as an energy and carbon storage material under nutrient limiting condition in the presence of excess carbon source (1,17,20). PHAs have been attracting much attention as they can be used as biodegradable polymers (20) and as the sources of chiral pools for the synthesis of fine chemicals (18). The metabolic pathways for the biosynthesis and degradation of PHAs have been well examined in many bacteria (17,20). For example, in short-chain-length-PHA-producing bacteria such as Ralstonia eutropha and Alcaligenes latus, two acetyl coenzyme A (acetylCoA) moieties derived from various carbon sources are condensed to acetoacetyl-CoA by 3-ketothiolase (PhaA) and sequentially converted to (R)-3-hydroxybutyryl-CoA (R3HB-CoA) by acetoacetyl-CoA reductase (PhaB). Then, R3HB-CoA is added to the growing chain of poly(3-hydroxybutyrate) [P(3HB)] by the short-chain-length PHA synthase (PhaC) (29).In pseudomonads belonging to the rRNA homology group I, the intermediates of fatty acid metabolism including enoylCoA, (S)-3-hydroxyacyl-CoA, 3-ketoacyl-CoA, and 3-hydroxyacyl-acyl carrier protein (ACP) are major precursors for medium-chain-length (MCL) PHAs (17,20,36). The metabolic links between the fatty acid metabolism and PHA biosynthesis are mediated by various enzymes such as enoyl-CoA hydratase (7,8,9,23,34,35), 3-ketoacyl-ACP reductase (22, 27, 33), epimerase (20), and 3-hydroxyacyl-ACP:CoA transacylase (12, 26). The genes encoding the...

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

Park

Lee

2003

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…Taguchi et al (23) provided evidence that the overexpression of the E. coli fabG gene, which encodes the 3-ketoacyl-[acylcarrier-protein (ACP)] reductase, in E. coli HB101 mediated the supply of (R)-3-hydroxyacyl-CoA via fatty acid ␤-oxidation. It has also been shown recently that coexpression of the cytosolic thioesterase I gene and a PHA synthase-encoding gene in E. coli (fadB fadR) results in the synthesis of PHA composed mainly of 3-hydroxyoctanoate from the carbon source gluconate (6).…”

mentioning

confidence: 99%

PhaG-Mediated Synthesis of Poly(3-Hydroxyalkanoates) Consisting of Medium-Chain-Length Constituents from Nonrelated Carbon Sources in Recombinant Pseudomonas fragi

Fiedler

Steinbüchel

Rehm

2000

Appl Environ Microbiol

View full text Add to dashboard Cite

Recently, a new metabolic link between fatty acid de novo biosynthesis and biosynthesis of poly(3-hydroxyalkanoate) consisting of medium-chain-length constituents (C 6 to C 14 ) (PHA MCL ), catalyzed by the 3-hydroxydecanoyl-[acyl-carrier-protein]:CoA transacylase (PhaG), has been identified in Pseudomonas putida (B. H. A. Rehm, N. Krüger, and A. Steinbüchel, J. Biol. Chem. 273:24044-24051, 1998). To establish this PHA-biosynthetic pathway in a non-PHA-accumulating bacterium, we functionally coexpressed phaC1 (encoding PHA synthase 1) from Pseudomonas aeruginosa and phaG (encoding the transacylase) from P. putida in Pseudomonas fragi. The recombinant strains of P. fragi were cultivated on gluconate as the sole carbon source, and PHA accumulation to about 14% of the total cellular dry weight was achieved. The respective polyester was isolated, and GPC analysis revealed a weight average molar mass of about 130,000 g mol ؊1 and a polydispersity of 2.2. The PHA was composed mainly (60 mol%) of 3-hydroxydecanoate. These data strongly suggested that functional expression of phaC1 and phaG established a new pathway for PHA MCL biosynthesis from nonrelated carbon sources in P. fragi. When fatty acids were used as the carbon source, no PHA accumulation was observed in PHA synthase-expressing P. fragi, whereas application of the ␤-oxidation inhibitor acrylic acid mediated PHA MCL accumulation. The substrate for the PHA synthase PhaC1 is therefore presumably directly provided through the enzymatic activity of the transacylase PhaG by the conversion of (R)-3-hydroxydecanoyl-ACP to (R)-3-hydroxydecanoyl-CoA when the organism is cultivated on gluconate. Here we demonstrate for the first time the establishment of PHA MCL synthesis from nonrelated carbon sources in a non-PHA-accumulating bacterium, employing fatty acid de novo biosynthesis and the enzymes PhaG (a transacylase) and PhaC1 (a PHA synthase).Most fluorescent pseudomonads belonging to rRNA homology group I are able to synthesize and accumulate large amounts of polyhydroxyalkanoic acids (PHAs) consisting of various 3-hydroxy fatty acids with carbon chain lengths ranging from 6 to 14 carbon atoms (medium chain length [MCL]) as carbon and energy storage compounds (1,22). Pseudomonas fragi is an exception; it is not able to accumulate PHA from either fatty acids or other simple carbon sources such as gluconate (26). PHA composition depends on the PHA synthases present, the carbon source, and the metabolic routes involved (9,16,21). In Pseudomonas putida there are at least three different metabolic routes for the synthesis of 3-hydroxyacyl coenzyme A (CoA) thioesters, which are the substrates of PHA synthase (4, 15). ␤-Oxidation is the main pathway when fatty acids are used as the carbon source. Fatty acid de novo biosynthesis is the main route during growth on a carbon source which is metabolized to acetyl-CoA, like gluconate, acetate, or ethanol. The chain elongation reaction, in which acetyl-CoA moieties are condensed to 3-hydroxyacyl-CoA, is involved in PHA synthesis ...

show abstract

“…One commonly used engineering strain is E. coli. Recombinant E. coli strains harbouring a set of PHAs biosysthesis genes (phaCac from Aeromonas caviae, phaCABa1 from Alcaligenes latus, or phbCAB from Ralstonia eutropha) were constructed (Taguchi et al, 1999). Sufficient amount of PHAs can be produced, while using sugars or fatty acids as carbon sources.…”

Section: Synthetic Biological Approaches To Convert Biomass To Bioplamentioning

confidence: 99%

Conversion of Biomass into Bioplastics and Their Potential Environmental Impacts

Lei¹,

Schmidt²,

Wei³

2011

Biotechnology of Biopolymers

View full text Add to dashboard Cite

Co-expression of 3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHA production in Escherichia coli HB101 strain

Cited by 37 publications

References 16 publications

Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

PhaG-Mediated Synthesis of Poly(3-Hydroxyalkanoates) Consisting of Medium-Chain-Length Constituents from Nonrelated Carbon Sources in Recombinant Pseudomonas fragi

Conversion of Biomass into Bioplastics and Their Potential Environmental Impacts

Contact Info

Product

Resources

About