Summary top ↑
1) Northern analysis of pcbC transcript levels showed that this gene is subject to a strain-specific transcriptional regulation by glucose. This regulation takes place in the wild-type ATCC 14553, but not in the semi-producers A3/2 and Act. By raising specific antibodies against the pcbC gene product and subsequent western analysis, it could be shown that no additional regulation at the translational level is present.
2) By northern analysis of transcript levels, it was demonstrated that the cefEF gene is transcriptionally repressed by glucose in all strains tested. A glucose effect does not occur at the post-trancriptional level, as was shown by mRNA stability analysis. Western analysis ruled out further regulation at the translational level.
3) Regarding the expression of cefG, no glucose dependent regulation was observed at the transcriptional level. Western analysis, performed after previously raising specific antibodies, showed that there is no regulation on translational level, either. Quantification of produced B-lactam antibiotics did not suggest any effect on the post-translational level. Therefore, cefG is the only gene in the cephalosporin C biosynthesis not being regulated by glucose.
4) In many filamentous fungi, the transcriptional glucose repression is mediated by transcription factors of the CREA/CRE1 family. In the present work, the creA gene from A. chrysogenum was isolated from a gene-library. The gene product CREA is conserved in all functional domains and shows extended homology to CRE1 from Trichoderma reesei. In addition, there is a positive regulation of the creA transcription in presence of glucose, which suggests an auto-regulation and a yet unknown activator function.
5) By transforming A. chrysogenum A3/2 with additional copies of the creA gene, a wild-type-like transcriptional repression of the previously derepressed pcbC gene could be achieved. Simultaneously, the transcriptional repression of cefEF was increased. These findings suggest an involvement of CREA in the glucose regulation of these genes. A model of CREA function is given. The results of this work prove that glucose is an important factor in the negative regulation of the cephalosporin C biosynthesis in A. chrysogenum. So far, CREA is the first transcription factor in A. chrysogenum which has been identified as being involved in regulatory processes in this fungus. The investigation of nutrient-dependent and strain-specific regulatory mechanisms may serve to optimize the industrial cultivation and the targeted strain improvement by genetic engendering.