scholarly journals YfcX Enables Medium-Chain-Length Poly(3-Hydroxyalkanoate) Formation from Fatty Acids in Recombinant Escherichia coli fadB Strains

2002 ◽  
Vol 184 (20) ◽  
pp. 5696-5705 ◽  
Author(s):  
Kristi D. Snell ◽  
Feng Feng ◽  
Luhua Zhong ◽  
David Martin ◽  
Lara L. Madison
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Chain Length ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Insertional Inactivation

ABSTRACT Expression of Escherichia coli open reading frame yfcX is shown to be required for medium-chain-length polyhydroxyalkanoate (PHAMCL) formation from fatty acids in an E. coli fadB mutant. The open reading frame encodes a protein, YfcX, with significant similarity to the large subunit of multifunctional β-oxidation enzymes. E. coli fadB strains modified to contain an inactivated copy of yfcX and to express a medium-chain-length synthase are unable to form PHAMCLs when grown in the presence of fatty acids. Plasmid-based expression of yfcX in the FadB− YfcX− PhaC+ strain restores polymer formation. YfcX is shown to be a multifunctional enzyme that minimally encodes hydratase and dehydrogenase activities. The gene encoding YfcX is located downstream from yfcY, a gene encoding thiolase activity. Results of insertional inactivation studies and enzyme activity analyses suggest a role for yfcX in PHA monomer unit formation in recombinant E. coli fadB mutant strains. Further studies are required to determine the natural role of YfcX in the metabolism of E. coli.

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 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 FabG, an NADPH-Dependent 3-Ketoacyl Reductase ofPseudomonas aeruginosa, Provides Precursors for Medium-Chain-Length Poly-3-Hydroxyalkanoate Biosynthesis inEscherichia coli

2000 ◽  
Vol 182 (10) ◽  
pp. 2978-2981 ◽  
Author(s):  
Qun Ren ◽  
Nicolas Sierro ◽  
Bernard Witholt ◽  
Birgit Kessler
Keyword(s):  
Escherichia Coli ◽  
Chain Length ◽  
Reductase Activity ◽  
Inescherichia Coli ◽  
E Coli ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Encoding Gene ◽  
Coa Reductase ◽  
Formation Step

ABSTRACT Escherichia coli hosts expressing fabG ofPseudomonas aeruginosa showed 3-ketoacyl coenzyme A (CoA) reductase activity toward R-3-hydroxyoctanoyl-CoA. Furthermore, E. coli recombinants carrying the poly-3-hydroxyalkanoate (PHA) polymerase-encoding gene phaCin addition to fabG accumulated medium-chain-length PHAs (mcl-PHAs) from alkanoates. When E. coli fadB orfadA mutants, which are deficient in steps downstream or upstream of the 3-ketoacyl-CoA formation step during β-oxidation, respectively, were transformed with fabG, higher levels of PHA were synthesized in E. coli fadA, whereas similar levels of PHA were found in E. coli fadB, compared with those of the corresponding mutants carrying phaC alone. These results strongly suggest that FabG of P. aeruginosais able to reduce mcl-3-ketoacyl-CoAs generated by the β-oxidation to 3-hydroxyacyl-CoAs to provide precursors for the PHA polymerase.

scholarly journals Production of Medium-Chain-Length Poly(3-Hydroxyalkanoates) from Gluconate by Recombinant Escherichia coli

1999 ◽  
Vol 65 (2) ◽  
pp. 540-548 ◽  
Author(s):  
Stefan Klinke ◽  
Qun Ren ◽  
Bernard Witholt ◽  
Birgit Kessler
Keyword(s):  
Escherichia Coli ◽  
Chain Length ◽  
De Novo ◽  
Dry Weight ◽  
Acid Synthesis ◽  
E Coli ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Encoding Gene ◽  
Encoding Genes

ABSTRACT It was shown recently that recombinant Escherichia coli, defective in the β-oxidation cycle and harboring a medium-chain-length (MCL) poly(3-hydroxyalkanoate) (PHA) polymerase-encoding gene of Pseudomonas, is able to produce MCL PHA from fatty acids but not from sugars or gluconate (S. Langenbach, B. H. A. Rehm, and A. Steinbüchel, FEMS Microbiol. Lett. 150:303–309, 1997; Q. Ren, Ph.D. thesis, ETH Zürich, Zürich, Switzerland, 1997). In this study, we report the formation of MCL PHA from gluconate by recombinant E. coli. By introduction of genes coding for an MCL PHA polymerase and the cytosolic thioesterase I (′thioesterase I) into E. coli JMU193, we were able to engineer a pathway for the synthesis of MCL PHA from gluconate. We used two expression systems, i.e., thebad promoter and alk promoter, for the ′thioesterase I- and PHA polymerase-encoding genes, respectively, which enabled us to modulate their expression independently over a range of inducer concentrations, which resulted in a maximum MCL PHA accumulation of 2.3% of cell dry weight from gluconate. We found that the amount of PHA and the ′thioesterase I activity are directly correlated. Moreover, the polymer accumulated in the recombinantE. coli consisted mainly of 3-hydroxyoctanoate monomers. On the basis of our data, we propose an MCL PHA biosynthesis pathway scheme for recombinant E. coli JMU193, harboring PHA polymerase and ′thioesterase I, when grown on gluconate, which involves both de novo fatty acid synthesis and β-oxidation.

scholarly journals Effects of propionate and carnitine on the hepatic oxidation of short- and medium-chain-length fatty acids

1988 ◽  
Vol 250 (3) ◽  
pp. 819-825 ◽  
Author(s):  
E P Brass ◽  
R A Beyerinck
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Organic Acid ◽  
Chain Length ◽  
Ketone Body ◽  
Acetyl Coa ◽  
Body Formation ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Hepatic Oxidation

Accumulation of propionate, or its metabolic product propionyl-CoA, can disrupt normal cellular metabolism. The present study examined the effects of propionate, or propionyl-CoA generated during the oxidation of odd-chain-length fatty acids, on hepatic oxidation of short- and medium-chain-length fatty acids. In isolated hepatocytes, ketone-body formation from odd-chain-length fatty acids was slow as compared with even-chain-length fatty acid substrates, and increased as the carbon chain length was increased from five to seven to nine. In contrast, rates of ketogenesis from butyrate, hexonoate and octanoate were all approximately equal. Propionate (10 mM) inhibited ketogenesis from butyrate, hexanoate and octanoate by 81%, 53% and 18% respectively. Addition of carnitine had no effect on ketogenesis from the even-chain-length fatty acids, but increased the rate of ketone-body formation from pentanoate (by 53%), heptanoate (by 28%) and from butyrate or hexanoate in the presence of propionate. The inhibitory effect of propionate could not be explained by shunting acetyl-CoA into the tricarboxylic acid cycle, as CO2 formation from butyrate was also decreased by propionate. Examination of the hepatocyte CoA pool during oxidation of butyrate demonstrated that addition of propionate decreased acetyl-CoA and CoA as propionyl-CoA accumulated. Addition of carnitine decreased propionyl-CoA by 50% (associated with production of propionylcarnitine) and increased acetyl-CoA and CoA. Similar changes in the CoA pool were seen during the oxidation of pentanoate. These results demonstrate that accumulation of propionyl-CoA results in inhibition of short-chain fatty acid oxidation. Carnitine can partially reverse this inhibition. Changes in the hepatocyte CoA pool are consistent with carnitine acting by generating propionylcarnitine, thereby decreasing propionyl-CoA and increasing availability of free CoA. The data provide further evidence of the potential cellular toxicity from organic acid accretion, and supports the concept that carnitine's interaction with the cellular CoA pool can have a beneficial effect on cellular metabolism and function under conditions of unusual organic acid accumulation.

scholarly journals Quantitative ‘Omics Analyses of Medium Chain Length Polyhydroxyalkanaote Metabolism in Pseudomonas putida LS46 Cultured with Waste Glycerol and Waste Fatty Acids

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0142322 ◽  
Author(s):  
Jilagamazhi Fu ◽  
Parveen Sharma ◽  
Vic Spicer ◽  
Oleg V. Krokhin ◽  
Xiangli Zhang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Pseudomonas Putida ◽  
Chain Length ◽  
Medium Chain ◽  
Waste Glycerol ◽  
Medium Chain Length

Biosynthesis of medium chain length alkanes for bio-aviation fuel by metabolic engineered Escherichia coli

2017 ◽  
Vol 239 ◽  
pp. 542-545 ◽  
Author(s):  
Meng Wang ◽  
Kaili Nie ◽  
Hao Cao ◽  
Haijun Xu ◽  
Yunming Fang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chain Length ◽  
Aviation Fuel ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Engineered Escherichia Coli

Engineering of Stable Recombinant Bacteria for Production of Chiral Medium-Chain-Length Poly-3-Hydroxyalkanoates

1999 ◽  
Vol 65 (8) ◽  
pp. 3265-3271 ◽  
Author(s):  
Maria A. Prieto ◽  
Michele B. Kellerhals ◽  
Gian B. Bozzato ◽  
Dragan Radnovic ◽  
Bernard Witholt ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Chain Length ◽  
Expression System ◽  
Average Molecular Weight ◽  
Polydispersity Index ◽  
Recombinant Bacteria ◽  
E Coli ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Pha Production

ABSTRACT In order to scale up medium-chain-length polyhydroxyalkanoate (mcl-PHA) production in recombinant microorganisms, we generated and investigated different recombinant bacteria containing a stable regulated expression system for phaC1, which encodes one of the mcl-PHA polymerases of Pseudomonas oleovorans. We used the mini-Tn5 system as a tool to constructEscherichia coli 193MC1 and P. oleovoransPOMC1, which had stable antibiotic resistance and PHA production phenotypes when they were cultured in a bioreactor in the absence of antibiotic selection. The molecular weight and the polydispersity index of the polymer varied, depending on the inducer level. E. coli 193MC1 produced considerably shorter polyesters thanP. oleovorans produced; the weight average molecular weight ranged from 67,000 to 70,000, and the polydispersity index was 2.7. Lower amounts of inducer added to the media shifted the molecular weight to a higher value and resulted in a broader molecular mass distribution. In addition, we found that E. coli 193MC1 incorporated exclusively the R configuration of the 3-hydroxyoctanoate monomer into the polymer, which corroborated the enantioselectivity of the PhaC1 polymerase enzyme.

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