Cloning, sequencing, overexpression in Escherichia coli, and inactivation of the valine dehydrogenase gene in the polyether antibiotic producer Streptomyces cinnamonensis.
The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide antibiotic biosynthesis. To determine for the first time the importance of valine dehydrogenase (vdh) in polyether antibiotic biosynthesis, the vdh gene from Streptomyces cinnamonensis has been cloned and sequenced. The enzyme (M(r)37,718 Da) has been produced in large amounts in an active form in the E. coli cytoplasm using a T7 RNA-polymerase expression system. Upon inactivation of the gene in S. cinnamonensis by a double-crossover mechanism, a hyg::vdh mutant was isolated that was devoid of vdh activity. Upon growth in chemically defined media, as well as a complex medium optimised for monensin production, the mutant and wild-type grew equally well and reached the same levels of monensin production. In both strains a valine transaminase activity could be detected that provides an alternative route for converting valine into 2-oxoisovaleric acid. The results show that vdh is not essential for normal growth of S. cinnamonensis, and its inactivation does not significantly affect normal levels of monensin production in this strain.
Leiser
A
A
Institute of Organic Chemistry, University of Zurich, Switzerland.
Birch
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A
Robinson
J A
JA
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001483:Base Sequence; D002934:Cinnamates; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli; D004987:Ethers; D005798:Genes, Bacterial; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D008985:Monensin / Q000096:biosynthesis*; D016254:Mutagenesis, Insertional; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D000900:Anti-Bacterial Agents; D002934:Cinnamates; D004269:DNA, Bacterial; D004987:Ethers; D006921:Hygromycin B; C026273:hygromycin A; D008985:Monensin; D000594:Amino Acid Oxidoreductases; C057545:valine dehydrogenase (NAD+)