THE CLAR GENE OF STREPTOMYCES-CLAVULIGERUS, ENCODING A LYSR-TYPE REGULATORY PROTEIN CONTROLLING CLAVULANIC ACID BIOSYNTHESIS, IS LINKED TO THE CLAVULANATE-9-ALDEHYDE REDUCTASE (CAR) GENE

Two genes, claR and car, encoding proteins involved in clavulanic acid biosynthesis, have been found in a 2.8-kb BglII-EcoRI DNA fragment of Streptomyces clavuligerus adjacent to the region containing the cepha mycin and clavulanic acid biosynthesis gene cluster. claR encoded a pr otein of 431 amino acids (deduced M-r 47 080), that showed a significa nt degree of homology with several transcriptional activators of the L ysR family. The ClaR protein contained two helix-turn-helix (HTH) moti fs in the amino and carboxyl terminal regions. The second gene, car, e ncoded a protein of 247 amino acids (M-r 26 629) that showed a strong similarity to oxydoreductases of the SDR family. Twelve amino acids of the amino-terminal region were identical to those previously obtained by Edman degradation of the purified clavulanic-9-aldehyde reductase of S. clavuligerus. Amplification of the claR gene in multicopy plasmi ds resulted in a threefold increase in clavulanic acid production and in a five- to sixfold increase of alanylclavam biosynthesis, whereas c ephamycin production was significantly reduced both in defined and in complex media. By contrast, amplification of the car gene had no signi ficant effect on clavulanic acid and alanylclavam or cephamycin produc tion. Both claR and car are expressed as monocistronic transcripts; th e level of transcript declined rapidly after 48 h in complex media, bu t low sustained levels of both transcripts were observed in defined GS PG medium until 96 h. claR and car were not significantly expressed in mutants disrupted in the ccaR gene, a regulatory gene that controls p ositively clavulanic acid and cephamycin biosynthesis. These results i ndicate that clavulanic acid and cephamycin biosynthesis in S. clavuli gerus is controlled by a cascade of regulatory proteins that include C caR and ClaR. (C) 1998 Elsevier Science B.V. All rights reserved.