Organization and expression in Pseudomonas putida of the gene cluster involved in cephalosporin biosynthesis from Lysobacter lactamgenus YK90.

Applied microbiology and biotechnology ·Vol. 45 ·No. 4 ·1996-05-00 ·Pages 490-501

Kimura H, Miyashita H, Sumino Y

Abstract

The Lysobacter lactamgenus YK90 pcbAB gene encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase is located immediately upstream of the pcbC gene in the same orientation in the gene cluster involved in cephalosporin biosynthesis. The pcbAB gene encodes a large polypeptide composed of 3722 amino acid residues with a molecular mass of 411593 Da. The predicted amino acid sequence has a high degree of similarity with those of known ACV synthetases from fungi and actinomycetes. Within the pcbAB amino acid sequence, three conserved and repeated domains of about 600 amino acids were identified. the domains also share a high degree of similarity with non-ribosomal peptide synthetases such as gramicidin synthetase 2 of Bacillus brevis. The pcbAB gene was expressed under the control of the lac promoter in Pseudomonas putida. Expression of the gene cluster involved in cephalosporin biosynthesis in P. putida led to the accumulation of beta-lactam antibiotics. Deletion analysis of an open-reading frame located between the cefE and cefD genes from the gene cluster revealed that it encoded deacetylcephalosporin C synthetase (cefF). From the results presented here and those of previous studies, the genes involved in cephalosporin biosynthesis in L. lactamgenus appear to be clustered in the order pcbAB-pcbC- cefE-cefF-cefD-bla in the same orientation within a 17-kb region of DNA.

MeSH Terms

Amino Acid Isomerases/genetics Amino Acid Sequence Bacteria/genetics Bacterial Proteins/biosynthesis,genetics Base Sequence Cephalosporins/biosynthesis Gene Expression Regulation, Bacterial Genes, Bacterial Intramolecular Transferases Isomerases/genetics Molecular Sequence Data Open Reading Frames Oxidoreductases/genetics Oxygenases/genetics Penicillin-Binding Proteins Peptide Synthases/biosynthesis,genetics Pseudomonas putida/genetics Recombinant Fusion Proteins/biosynthesis,genetics Sequence Deletion

Chemicals

Bacterial Proteins Cephalosporins Penicillin-Binding Proteins Recombinant Fusion Proteins Oxidoreductases Oxygenases deacetoxycephalosporin C hydroxylase isopenicillin N synthetase Isomerases Amino Acid Isomerases isopenicillin N epimerase Intramolecular Transferases deacetoxycephalosporin C synthetase Peptide Synthases alpha-aminoadipyl-cysteinyl-valine synthetase

Authors & Affiliations

3 authors, click to expand affiliations / ORCID

Kimura H

Fermentation Center, Takeda Chemical Industries Ltd., Osaka, Japan.

Miyashita H

Sumino Y

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Article Info

Journal

Applied microbiology and biotechnology

Abbr.

Appl Microbiol Biotechnol

ISSN

0175-7598

DOI

10.1007/BF00578461

Published

1996-05-00

Pages

490-501

Language

English

Region

Germany

NLM ID

8406612

Databases

GENBANK

D50308

External Links

PubMed source DOI: 10.1007/BF00578461 View journal details

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Organization and expression in Pseudomonas putida of the gene cluster involved in cephalosporin biosynthesis from Lysobacter lactamgenus YK90.

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