Identification of a minimal cre1 promoter sequence promoting glucose-dependent gene expression in the β-lactam producer Acremonium chrysogenum

Abstract

The promoter of the cre1 gene, encoding the glucose-dependent regulator CRE1 from the beta-lactam producer Acremonium chrysogenum, carries 15 putative CRE1 binding sites (BS1 to BS15). For a detailed analysis, we fused cre1 promoter deletion derivatives with the DsRed reporter gene to perform a comparative gene expression analysis. Plate assays, Northern hybridizations, and spectrofluorometric measurements of DsRed identified the minimal D4 promoter sequence that promoted glucose-dependent expression. Truncated recombinant CRE1 interacted with D4 in electromobility shift analysis and these binding studies were further extended with two oligonucleotides, carrying putative CRE1 binding sites BS14 and BS15. Surface plasmon resonance analysis was performed using BS14 and BS15, along with four derivatives containing 2 or 4 bp substitutions within BS14 and BS15, respectively. Substitutions within BS14 abolished the high affinity interaction with CRE1, while mutations in BS15 only marginally diminished the affinity with CRE1. In vivo analysis of a modified D4 sequence with substitutions in the two binding sites confirmed the in vitro binding results and still promoted glucose-dependent gene expression. Our results will contribute to the construction of versatile expression vectors carrying a minimal cre1 promoter sequence that still confers glucose-dependent induction of gene expression.

Carbon source regulation of antibiotic production

“…Cre-binding sites are found in the promoters of cre genes from diverse filamentous fungi. 171 The promoters of many glucose-repressible target genes have Cre-binding consensus sequences. 172 The Cre1 protein (from the cre1 gene) sequence shows high similarity to other glucose-repressor proteins from filamentous fungi, suggesting a similar regulation and a related role in CCR.…”

“…172 The Cre1 protein (from the cre1 gene) sequence shows high similarity to other glucose-repressor proteins from filamentous fungi, suggesting a similar regulation and a related role in CCR. Janus et al 171 identified a 114-bp minimal promoter with two Cre1-binding sites, the first minimal regulatory promoter sequence for the b-lactam antibiotic-producing fungus. The identification of 114-bp CT-rich sequences in the nontranscribed cre1 upstream region suggests that it is required to trigger glucosedependent gene expression.…”

“…The identification of 114-bp CT-rich sequences in the nontranscribed cre1 upstream region suggests that it is required to trigger glucosedependent gene expression. 171 Mutations in creB and creC have shown little effect on carbon regulation of penicillin biosynthesis. All these results point to a second mechanism of carbon repression, which is creA independent.…”

“…Therefore, it is feasible that cre1 might be involved in glucose repression of cephalosporin C production and that during the program of strain improvement, this control mechanism could have been deregulated. 171,174 CT-rich regions are putative activator sequences in close proximity to transcription start sites. 175 In A. chrysogenum, the CT-rich region contains two closely spaced binding sites (namely BS14 and BS15) that cooperatively bind to Cre1.…”

“…171 Direct inhibition (a mechanism that restrains synthesis at the level of enzyme activity, therefore the effect is immediate) of the action of secondary metabolic enzymes may also be involved in carbon source control. Thus, glucose and a number of its phosphorylated metabolites inhibit ACVS, the first enzyme of cephalosporin synthesis, in crude extracts of A. chrysogenum and S. clavuligerus.…”