YfcX Enables Medium-Chain-Length Poly(3-Hydroxyalkanoate) Formation from Fatty Acids in Recombinant Escherichia coli fadB Strains (original) (raw)
Related papers
Antonie van Leeuwenhoek, 2008
Four (R)-specific enoyl CoA hydratases (PhaJ) interconnect the b-oxidation pathway with PHA biosynthesis in Pseudomonas aeruginosa. The use of antisense technique and over-expression to delineate the role of two of these enzymes, PhaJ1 and PhaJ4 forms the basis of this study. It has been observed that P. aeruginosa recombinant with phaJ1 antisense construct, fed with different fatty acids, produces PHA with less hydroxy octanoate (7-11% reduction) and a proportionate increase in other monomer fractions, compared to that of the control. Recombinants bearing phaJ4 antisense construct are found to contain less hydroxy decanoate (10-11% reduction) and more or less equal amount of hydroxy octanoate, compared to that of the control. P. aeruginosa produced PHA with more hydroxy octanoate and decanoate (6-17% increase), respectively, when PhaJ1 and PhaJ4 have been overexpressed individually or along with PhaC1. PhaJ1 and PhaJ4 are found to be involved mainly in the production of hydroxy octanoyl CoA and hydroxy decanoyl CoA, respectively, in P. aeruginosa. The strongest accumulation of hydroxy octanoate and hydroxy decanoate has been observed along with hydroxy butyrate, in PHA, produced by E. coli, when PhaC1 has been co-expressed with PhaJ1 and PhaJ4, respectively. We have demonstrated, for the first time, the polymerization of hydroxy butyryl CoA monomers in recombinant E. coli by PhaC1 of P. aeruginosa.
Antonie van Leeuwenhoek, 2008
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 b-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 b-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.
Applied Microbiology and Biotechnology, 2000
An (R)-trans-2,3-enoylacyl-CoA hydratase was puri®ed to near-homogeneity from Rhodospirillum rubrum. Protein sequencing of enriched protein fractions allowed the construction of a degenerate oligonucleotide. The gene encoding the (R)-speci®c hydratase activity was cloned following three rounds of colony hybridization using the oligonucleotide, and overexpression of the gene in E. coli led to the puri®cation of the enzyme to homogeneity. The puri®ed enzyme used crotonyl-CoA, trans-2,3-pentenoyl-CoA, and trans-2,3hexenoyl-CoA with approximately equal speci®city as substrates in the hydration reaction. However, no activity was observed using trans-2,3-octenoyl-CoA as a substrate, but this compound did partially inhibit crotonyl-CoA hydration. Based on the nucleotide sequence, the protein has a monomeric molecular weight of 15.4 kDa and is a homotetramer in its native form as determined by gel ®ltration chromatography and native PAGE. The hydratase was expressed together with the PHA synthase from Thiocapsa pfennigii in E. coli strain DH5a. Growth of these strains on oleic acid resulted in the production of the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate).
Frontiers in Bioengineering and Biotechnology, 2021
Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaAA–04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaBA–04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaCA–04, 1770 bp), was identified. Sequence analysis of the phaAA–04, phaBA–04, and phaCA–04 genes revealed that phaCA–04 was 99% similar to phaCH16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaCA–04 was proline, whereas that of C. necator H16 was leucine. The intact phaCABA–04 operon ...
Applied Microbiology and Biotechnology, 2005
A Pseudomonas strain, 3Y2, that produced polyhydroxyalkanoate (PHA) polymers consisting of 3-hydroxybutyric acid (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-HA) units, with up to 30% 3HB, was isolated. Two PHA biosynthesis loci (pha Ps-1 and pha Ps-2 ) from 3Y2 were cloned by polymerase chain reaction amplification techniques. The pha Ps-2 locus was similar to the PHA biosynthesis loci of other PHA-producing Pseudomonas strains, with five tandem open reading frames (ORFs) located in the order ORF1 Ps-2 -phaC1 Ps-2 -phaZ Ps-2 -phaC2 Ps-2 -phaD Ps-2 . The pha Ps-1 locus that contains phaC1 Ps-1 -phaZ Ps-1 appears to have arisen by a duplication event that placed it downstream of a gene (ORF1 Ps-1 ), encoding a putative glucose-methanol-choline flavoprotein oxidoreductase. The PHA synthases 1 encoded by phaC1 Ps-1 and phaC1 Ps-2 were investigated by heterologous expression in Wautersia eutropha PHB − 4. Both synthases displayed similar substrate specificities for incorporating 3HB and mcl-HA units into PHA. The ability of PhaC1 Ps-1 to confer PHA synthesis, however, appeared reduced compared to that of PhaC1 Ps-2 , since cells harboring PhaC1 Ps-1 accumulated 2.5 to 4.6 times less PHA than cells expressing PhaC1 Ps-2 . Primary sequence analysis revealed that PhaC1 Ps-1 had markedly diverged from the other PHA synthases with a relatively high substitution rate (14.9 vs 2% within PhaC1 Ps-2 ). The mutations affected a highly conserved C-terminal region and the surroundings of the essential active site cysteine (Cys296) with a loss of hydrophobicity. This led us to predict that if phaC1 Ps-1 produces a protein product in the native strain, it is likely that PhaC1 Ps-1 may be destined for elimination by the accumulation of inactivating mutations, although its specialization to accommodate different substrates cannot be eliminated.
Journal of Bacteriology, 2009
A fatty acyl coenzyme A synthetase (FadD) from Pseudomonas putida CA-3 is capable of activating a wide range of phenylalkanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher k cat and K m values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of the fadD gene from P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3⌬fadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3⌬fadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3⌬fadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of fadD in P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longermonomer units into the PHA polymer.
FEMS Microbiology Letters, 2000
In order to investigate the in vivo substrate specificity of the type I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha, we functionally expressed the PHA synthase gene in various Escherichia coli mutants affected in fatty acid L-oxidation and the wild-type. The PHA synthase gene was expressed either solely (pBHR70) or in addition to the R. eutropha genes encoding L-ketothiolase and acetoacetylcoenzyme A (CoA) reductase comprising the entire PHB operon (pBHR68) as well as in combination with the phaC1 gene (pBHR77) from Pseudomonas aeruginosa encoding type II PHA synthase. The fatty acid L-oxidation route was employed to provide various 3-hydroxyacyl-CoA thioesters, depending on the carbon source, as in vivo substrate for the PHA synthase. In vivo PHA synthase activity was indicated by PHA accumulation and substrate specificity was revealed by analysis of the comonomer composition of the respective polyester. Only in recombinant E. coli fad mutants harboring plasmid pBHR68, the R. eutropha PHA synthase led to accumulation of poly(3hydroxybutyrate-co-3-hydroxyoctanoate) (poly(3HB-co-3HO)) and poly(3HB-co-3HO-co-3-hydroxydodecanoate (3HDD)), when octanoate and decanoate or dodecanoate were provided as carbon source, respectively. Coexpression of phaC1 from P. aeruginosa indicated and confirmed the provision of PHA precursor via the L-oxidation pathway and led to the accumulation of a blend of two different PHAs in the respective E. coli strain. These data strongly suggested that R. eutropha PHA synthase accepts, besides the main substrate 3-hydroxybutyryl-CoA, also the CoA thioesters of 3HO and 3HDD. ß
FEMS Microbiology Letters, 2003
Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli was expressed together with polyhydroxyalkanoate synthase gene (phaC Ac ) and (R)-enoyl-CoA hydratase gene (phaJ Ac ) from Aeromonas caviae. The expression plasmids were introduced into E. coli JM109, DH5K and XL1-blue, respectively. Compared with the strains harboring only phaC Ac and phaJ Ac , all recombinant E. coli strains harboring yafH, phaC Ac and phaJ Ac accumulated at least four times more poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Cell dry weights produced by all recombinants containing yafH were also considerably higher than that without yafH. The addition of acrylic acid which serves as inhibitor for L-oxidation and may lead to more precursor supply for PHA synthesis did not result in improved PHBHHx production compared with that of the overexpression of yafH. It appeared that the overexpression of acyl-CoA dehydrogenase gene (yafH) enhanced the supply of enoyl-CoA which is the substrate of (R)-enoyl-CoA hydratase. With the enhanced precursor supply, the recombinants accumulated more PHBHHx.