Identification of Amycolatopsis sp. strain HR167 genes, involved in the bioconversion of ferulic acid to vanillin.
The gene loci ech, encoding enoyl-CoA hydratase/aldolase, and fcs, encoding an unusual feruloyl-CoA synthetase, which are involved in the bioconversion of ferulic acid to vanillin by the gram-positive bacterium Amycolatopsis sp. strain HR167, were localized on a 4,000 bp PstI fragment (P40). The nucleotide sequence of P40 was determined, revealing open reading frames of 864 bp and 1,476 bp, representing ech and fcs, respectively. The deduced amino acid sequences of ech exhibited 62% amino acid identity to the enoyl-CoA hydratase/aldolase from Pseudomonas sp. strain HR199 and the enoyl-CoA hydratase/lyase from P. fluorescens strain AN103. The deduced amino acid sequences of fcs exhibited up to 37% amino acid identity to long-chain fatty acid coenzymeA ligases but no significant similarity to the feruloyl-CoA synthetase of Pseudomonas sp. strain HR199. Fragment P40 was cloned in pBluescript SK- and fcs and ech were expressed in Escherichia coli. Recombinant strains were able to transform ferulic acid to vanillin. In crude extracts of these recombinant strains, feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities were detected by photometric assay and high-performance liquid chromatography. The obtained data suggest that ferulic acid degradation in the gram-positive Amycolatopsis sp. strain HR167 proceeds via a pathway similar to that recently described for the gram-negative P. fluorescens strain AN103 and Pseudomonas sp. strain HR199.
Achterholt
S
S
Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, Germany.
Priefert
H
H
Steinbüchel
A
A
D000192:Actinomycetales / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D000595:Amino Acid Sequence; D001547:Benzaldehydes / Q000378:metabolism*; D001711:Biotransformation; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004746:Enoyl-CoA Hydratase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D001547:Benzaldehydes; D003373:Coumaric Acids; C004999:ferulic acid; C100058:vanillin; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D004746:Enoyl-CoA Hydratase; D003066:Coenzyme A Ligases; C400923:feruloyl-coenzyme A synthetase