Identification of Amycolatopsis sp. strain HR167 genes, involved in the bioconversion of ferulic acid to vanillin

• Published in: Applied microbiology and biotechnology Vol. 54; no. 6; pp. 799 - 807
• Main Authors: Achterholt, S, Priefert, H, Steinbuchel, A
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01-12-2000; Springer Nature B.V
• Subjects: Actinomycetales - genetics; Actinomycetales - growth & development; Actinomycetales - metabolism; Amino Acid Sequence; amino acid sequences; Amino acids; Amycolatopsis; Bacteria; Benzaldehydes - metabolism; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Biotransformation; Cloning, Molecular; Coenzyme A Ligases - chemistry; Coenzyme A Ligases - genetics; Coenzyme A Ligases - metabolism; Coumaric Acids - metabolism; Deoxyribonucleases, Type II Site-Specific - metabolism; E coli; ech gene; Enoyl-CoA Hydratase - genetics; Enoyl-CoA Hydratase - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; fcs gene; ferulic acid; feruloyl-CoA synthase; fructose-bisphosphate aldolase; Fundamental and applied biological sciences. Psychology; genes; Genetics; Gram-positive bacteria; high performance liquid chromatography; ligases; Liquid chromatography; loci; long chain fatty acids; Microbiology; Mission oriented research; Molecular Sequence Data; open reading frames; Pseudomonas; Pseudomonas fluorescens; Recombination, Genetic; Restriction Mapping; Sequence Analysis, DNA; vanillin; Recombinant microorganism; Nucleotide sequence; Enzyme; Industrial application; Escherichia coli; Amycolatopsis; Homology; Lyases; Pseudonocardiaceae; Gene expression; Cinnamic acid(4-hydroxy 3-methoxy); Enoyl-CoA hydratase; Enzymatic activity; Gene; Biotransformation; Bacteria; Actinomycetes; Aroma; Vanillin; Carbon-oxygen lyases; Enterobacteriaceae; Hydro-lyases
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530000431

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.