scholarly journals Expression of 3-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Genes Enhances Production of Polyhydroxyalkanoate Copolymer from Glucose in Recombinant Escherichia coli JM109

2005 ◽  
Vol 71 (8) ◽  
pp. 4297-4306 ◽  
Author(s):  
Christopher T. Nomura ◽  
Kazunori Taguchi ◽  
Zhihua Gan ◽  
Kazuhiro Kuwabara ◽  
Tomoyo Tanaka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chain Length ◽  
Acyl Carrier Protein ◽  
Carbon Sources ◽  
Pha Synthase ◽  
Carrier Protein ◽  
Substrate Specificities ◽  
E Coli ◽  
Pha Production ◽  
Pha Copolymers

ABSTRACT Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.

scholarly journals Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III (fabH) and Polyhydroxyalkanoate Synthase (phaC) Genes Leads to Short-Chain-Length-Medium-Chain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in Escherichia coli JM109

2004 ◽  
Vol 70 (2) ◽  
pp. 999-1007 ◽  
Author(s):  
Christopher T. Nomura ◽  
Kazunori Taguchi ◽  
Seiichi Taguchi ◽  
Yoshiharu Doi
Keyword(s):  
Escherichia Coli ◽  
Chain Length ◽  
Acyl Carrier Protein ◽  
Short Chain ◽  
Pha Synthase ◽  
Short Chain Length ◽  
E Coli ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Pha Copolymers

ABSTRACT Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C3 to C5, medium-chain-length (MCL) PHAs consist of monomer units of C6 to C14, and SCL-MCL PHAs consist of monomers ranging in size from C4 to C14. Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PHA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C4 to C10 and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production.

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

2003 ◽  
Vol 185 (18) ◽  
pp. 5391-5397 ◽  
Author(s):  
Si Jae Park ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Chain Length ◽  
Biosynthetic Pathway ◽  
Pha Synthase ◽  
Enzymatic Analysis ◽  
E Coli ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Enoyl Coa Hydratase

ABSTRACT 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 maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional β-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-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 β-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.

scholarly journals Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing

1993 ◽  
Vol 294 (2) ◽  
pp. 521-527 ◽  
Author(s):  
S A Morris ◽  
W P Revill ◽  
J Staunton ◽  
P F Leadlay
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Acyl Carrier Protein ◽  
Saccharopolyspora Erythraea ◽  
Carrier Protein ◽  
Freezing And Thawing ◽  
Acyl Carrier Proteins ◽  
Expression Plasmid ◽  
E Coli ◽  
Protein Dimer

Saccharopolyspora erythraea acyl-carrier protein, highly expressed from a T7-based expression plasmid in Escherichia coli, can be selectively released from the cells in near-quantitative yield by a single cycle of freezing and thawing in a neutral buffer. Electrospray mass spectrometry was used to confirm that the recombinant S. erythraea acyl-carrier protein over-expressed in E. coli is present predominantly as the holo-form, with variable amounts of apo-acyl-carrier protein, holo-acyl-carrier protein dimer and holo-acyl-carrier protein glutathione adduct. The holo- and apo-acyl-carrier proteins are both readily purified on a large scale from the freeze-thaw extracts and can be separated from one another by octyl-Sepharose chromatography. The holo-acyl-carrier protein obtained in this way was fully active in supporting the synthesis of acyl-acyl-carrier protein by extracts of S. erythraea.

scholarly journals Purification and characterization of [acyl-carrier-protein] acetyltransferase from Escherichia coli

1988 ◽  
Vol 250 (3) ◽  
pp. 789-796 ◽  
Author(s):  
P N Lowe ◽  
S Rhodes
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
E Coli ◽  
Step Procedure ◽  
Ping Pong ◽  
Protein Acetyltransferase ◽  
Enzyme Intermediate ◽  
Specific Reagent

A multi-step procedure has been developed for the purification of [acyl-carrier-protein] acetyltransferase from Escherichia coli, which allows the production of small amounts of homogeneous enzyme. The subunit Mr was estimated to be 29,000 and the native Mr was estimated to be 61,000, suggesting a homodimeric structure. The catalytic properties of the enzyme are consistent with a Bi Bi Ping Pong mechanism and the existence of an acetyl-enzyme intermediate in the catalytic cycle. The enzyme was inhibited by N-ethylmaleimide and more slowly by iodoacetamide in reactions protected by the substrate, acetyl-CoA. However, the enzyme was apparently only weakly inhibited by the thiol-specific reagent methyl methanethiosulphonate. The nature of the acetyl-enzyme intermediate is discussed in relationship to that found in other similar enzymes from E. coli, yeast and vertebrates.

scholarly journals Development of a New Strategy for Production of Medium-Chain-Length Polyhydroxyalkanoates by Recombinant Escherichia coli via Inexpensive Non-Fatty Acid Feedstocks

2011 ◽  
Vol 78 (2) ◽  
pp. 519-527 ◽  
Author(s):  
Qin Wang ◽  
Ryan C. Tappel ◽  
Chengjun Zhu ◽  
Christopher T. Nomura
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Chain Length ◽  
Acyl Carrier Protein ◽  
Carbon Sources ◽  
Content Type ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Coa Transferase ◽  
Fatty Acid Carbon

ABSTRACTPseudomonas putidaKT2440 is capable of producing medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when grown on unrelated carbon sources during nutrient limitation. Transcription levels of genes putatively involved in PHA biosynthesis were assessed by quantitative real-time PCR (qRT-PCR) inP. putidagrown on glycerol as a sole carbon source. The results showed that two genes,phaGand the PP0763 gene, were highly upregulated among genes potentially involved in the biosynthesis of MCL-PHAs from unrelated carbon sources. Previous studies have describedphaGas a 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase, and based on homology, the PP0763 gene was predicted to encode a medium-chain-fatty-acid CoA ligase. High expression levels of these genes during PHA production inP. putidaled to the hypothesis that these two genes are involved in PHA biosynthesis from non-fatty acid carbon sources, such as glucose and glycerol. ThephaGppand PP0763 genes fromP. putidawere cloned and coexpressed with the engineeredPseudomonassp. 61-3 PHA synthase genephaCl(STQK)psin recombinantEscherichia coli. Up to 400 mg liter−1MCL-PHAs was successfully produced from glucose. This study has produced the largest amount of MCL-PHAs reported from non-fatty acid carbon sources in recombinantE. colito date and opens up the possibility of using inexpensive feedstocks to produce MCL-PHA polymers.

scholarly journals The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase

2003 ◽  
Vol 185 (5) ◽  
pp. 1582-1589 ◽  
Author(s):  
Sean W. Jordan ◽  
John E. Cronan,
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Octanoic Acid ◽  
Acyl Carrier Protein ◽  
Temperature Sensitive ◽  
Carrier Protein ◽  
Genomic Databases ◽  
E Coli ◽  
Lipoyl Domain

ABSTRACT In an earlier study (S. W. Jordan and J. E. Cronan, Jr., J. Biol. Chem. 272:17903-17906, 1997) we reported a new enzyme, lipoyl-[acyl carrier protein]-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that E. coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase. However, it remained possible that lipB encoded a positive regulator required for lipoyltransferase expression or action. We now report genetic and biochemical evidence demonstrating that lipB encodes the lipoyltransferase. A lipB temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these experiments the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E. coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein. Prior genetic results (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Bacteriol. 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions.

scholarly journals Thioesterase II of Escherichia coli Plays an Important Role in 3-Hydroxydecanoic Acid Production

2004 ◽  
Vol 70 (7) ◽  
pp. 3807-3813 ◽  
Author(s):  
Zhong Zheng ◽  
Qiang Gong ◽  
Tao Liu ◽  
Ying Deng ◽  
Jin-Chun Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Coenzyme A ◽  
Acyl Carrier Protein ◽  
Physiological Role ◽  
Carrier Protein ◽  
E Coli ◽  
Thioesterase Ii ◽  
Abnormal Accumulation ◽  
Acyl Coenzyme A

ABSTRACT 3-Hydroxydecanoic acid (3HD) was produced in Escherichia coli by mobilizing (R)-3-hydroxydecanoyl-acyl carrier protein-coenzyme A transacylase (PhaG, encoded by the phaG gene). By employing an isogenic tesB (encoding thioesterase II)-negative knockout E. coli strain, CH01, it was found that the expressions of tesB and phaG can up-regulate each other. In addition, 3HD was synthesized from glucose or fructose by recombinant E. coli harboring phaG and tesB. This study supports the hypothesis that the physiological role of thioesterase II in E. coli is to prevent the abnormal accumulation of intracellular acyl-coenzyme A.

scholarly journals A Novel Genetically Engineered Pathway for Synthesis of Poly(Hydroxyalkanoic Acids) in Escherichia coli

2000 ◽  
Vol 66 (2) ◽  
pp. 739-743 ◽  
Author(s):  
Shuang-Jiang Liu ◽  
Alexander Steinbüchel
Keyword(s):  
Escherichia Coli ◽  
Clostridium Acetobutylicum ◽  
Recombinant Strain ◽  
Carbon Sources ◽  
Dry Weight ◽  
Genetically Engineered ◽  
Hybrid Plasmid ◽  
Pha Synthase ◽  
E Coli ◽  
Butyrate Kinase

ABSTRACT A new pathway to synthesize poly(hydroxyalkanoic acids) (PHA) was constructed by simultaneously expressing butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) genes of Clostridium acetobutylicum and the two PHA synthase genes (phaE and phaC) of Thiocapsa pfennigii inEscherichia coli. The four genes were cloned into theBamHI and EcoRI sites of pBR322, and the resulting hybrid plasmid, pBPP1, conferred activities of all three enzymes to E. coli JM109. Cells of this recombinant strain accumulated PHAs when hydroxyfatty acids were provided as carbon sources. Homopolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), or 4-hydroxyvalerate (4HV) were obtained from each of the corresponding hydroxyfatty acids. Various copolyesters of those hydroxyfatty acids were also obtained when two of these hydroxyfatty acids were fed at equal amounts: cells fed with 3HB and 4HB accumulated a copolyester consisting of 88 mol% 3HB and 12 mol% 4HB and contributing to 68.7% of the cell dry weight. Cells fed with 3HB and 4HV accumulated a copolyester consisting of 94 mol% 3HB and 6 mol% 4HV and contributing to 64.0% of the cell dry weight. Cells fed with 3HB, 4HB, and 4HV accumulated a terpolyester consisting of 85 mol% 3HB, 13 mol% 4HB, and 2 mol% 4HV and contributing to 68.4% of the cell dry weight.

scholarly journals Triple knockout of frdC gltA and pta genes enhanced pHA production in Escherichia coli

2018 ◽  
pp. 11-18
Author(s):  
Nurhajirah Mohamed Biran ◽  
Mohd Zulkhairi Mohd Yusoff ◽  
Toshinari Maeda ◽  
Mohd Rafein Zakaria ◽  
Lian-Ngit Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pha Synthase ◽  
Control Strain ◽  
E Coli ◽  
Pha Production ◽  
Coa Reductase ◽  
Genome Information ◽  
Acetoacetyl Coa Reductase ◽  
P1 Transduction ◽  
Engineered Strain

Polyhydroxyalkanoate (PHA) is a linear polyester produced through the fermentation of sugar or lipid. Biosynthesis of PHA comprises three enzymes known as acetyl-CoA acetyltransferase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthase (phaC). Comamonas sp. is one of the strains commonly used for PHA production. In order to develop higher PHA production from bacterial respond strategy, PHA biosynthesis operon of Comamonas sp. EB172 was introduced into Escherichia coli BW25113 through a pGEM-T vector. E. coli was chosen due to the complete genome information available and the absence of depolymerisation gene, phaZ. In this study, the deletion of several single genes, which are frdC, gltA, and pta, was found to be associated with PHA metabolism activity in E. coli BW25113. P1 transduction was performed to construct multiple genes knockout. The engineered strain, E. coli BW25113 frdCgltApta::kan/pGEM’-phaCABCo, yielded the highest PHA production at 64 wt.% with 1.4 fold higher than that of control strain of E. coli BW25113/pGEM’-phaCABCo. This strain is potential for industrial application for higher PHA production from E. coli.

scholarly journals Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

2014 ◽  
Vol 81 (4) ◽  
pp. 1406-1416 ◽  
Author(s):  
Jacob E. Vick ◽  
James M. Clomburg ◽  
Matthew D. Blankschien ◽  
Alexander Chou ◽  
Seohyoung Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Reductase Activity ◽  
Carrier Protein ◽  
Chemical Production ◽  
Efficient Operation ◽  
Content Type ◽  
E Coli ◽  
Coa Reductase

ABSTRACTWe recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production inEscherichia coli(J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012,http://dx.doi.org/10.1021/sb3000782). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as thetrans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (theEuglena gracilistrans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several nativeE. colienzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a β-oxidation reversal. Overexpression of FabI proved as effective asEgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature offabI, we investigated whether bacterial ENRs from other families were able to complement afabIdeletion without promiscuous reduction of crotonyl-CoA. These characteristics fromBacillus subtilisFabL enabled ΔfabIcomplementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal inE. coli.

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