EXPRESSION STUDIES WITH THE BIDIRECTIONAL PCBAB-PCBC PROMOTER REGION FROM ACREMONIUM-CHRYSOGENUM USING REPORTER GENE FUSIONS

Two cephalosporin genes from Acremonium chrysogenum, pcbAB and pcbC en code the ACV (alpha-aminoadipyl-cysteinyl-valine) synthetase and isope nicillin N-synthetase, respectively. The two adjacent genes are orient ated in opposite directions on the chromosomal DNA, separated by a 1.2 -kb non-translated sequence, carrying the putative promoter sequences. Complete sequencing of this intergenic region revealed differences fr om homologous sequences from other strains. To assess the putative pro moter strength, we constructed an expression vector carrying the beta- glucuronidase (gusA) and beta-galactosidase (lacZ) genes in opposite o rientation. Fusion of the pcbAB-pcbC promoter region resulted in recom binant Vector molecules, which were used for in-vivo expression studie s. Using the co-transformation procedure, the reporter gene fusions we re transferred into A. chrysogenum recipient strains together with vec tor pMW1. Individual transformants were used for protein preparations to measure specific activities of the enzymes coded by the reporter ge nes. The data provide in-vivo evidence that the pcbC promoter is at le ast five times stronger than the pcbAB promoter. Our approach should p rove useful in evaluating regulatory sequences that govern gene expres sion in A. chrysogenum.