Functional Analysis Of A Novel Myb Encoding Gene AcmybA, APSES Transcription Factor Encoding Gene AcstuA And The Autophagy Related Gene Acatg8 In Acremonium Chrysogenum

Acremonium chrysogenum is an important industrial fungus which produces β-lactam antibiotic cephalosporin C (CPC). We got a mutant AC554 that showed less yellow pigment production by screening the A. chrysogenum T-DNA inserted mutant library which constructed previously in our laboratory. TAIL PCR combined with real-time RT-PCR demonstrated that the T-DNA insertion enhanced the transcription of a Myb protein encoding gene, designated as AcmybA. Further studies revealed that AC554 showed significant reduced conidiation and nearly lost the ability to produce CPC. Corespondence with the CPC production, the real-time RT-PCR result showed that the transcripton of cefEF and cefG reduced significantly. The full-size of AcmybA was 1019 base-pairs (bp) containig an intron of 80 bp. Its duduced protein (AcMybA) is constituted of 313 anomic acids with molecullar weitht of 34 KDa. A conserved Myb DNA binding doamin is located in the N-terminal of AcMybA. We could not find a konwn protein that showed end to end similarity with AcMybA, indicating that AcmybA encodes a novel protein. The expression of RFP-tagged AcMybA showed that AcMybA is naturally located in the nuclear of A. chrysogenum, suggesting that AcMybA is a putative transcriptional factor. To further analysis the function of AcmybA, we constructed the AcmybA overexpression strain (AcmybOE), the AcmybA disruption mutant (ΔAcmybA) and its complemented strain (AcmybAC). AcmybOE showed significantly reduced conidia formation as AC554, while the ΔAcmybA produced more conidia comparing with the wil-typle strain (WT). It has been demonstrated that brlA, abaA and wetA play a central regulatory role in conidiation of A. niger. Our real-time RT-PCR results indicated that AcbrlA, AcabaA and AcwetA, the homologous genes of brlA, abaA and wetA, were up-regulated in ΔAcmybA, while they were down regulated in AcmybOE. Together with the Electrophoretic Mobility Shift Assay (EMSA) results, we concluded that AcMybA inhibited the fungal conidiation by repressing the expression of AcbrlA indirectly. ΔAcmybA could form more and larger arthrospores ("yeast-like" cells) and more fragmented hypha than WT. In contrast, AC554 and AcmybOE formed less arthrospores and less fragmented hypha comparing with WT. When AcbrlA were overexpressed in AC554 and AcmybOE, more and larger arthrospores and more fragmented hypha were formed just as AAcmybA. These results demonstrated that AcmybA regulates the arthrospore formation and hypha fragmentation by regulating AcbrlA expression. During fermentation, overexpression of AcmybA extresmely impeded the biosynthesis of cephalosporin C. Real-time RT-PCR analysis verified that the transcriptions of all the six cephalosporin biosynthetic genes pcbAB, pcbC, cefDl, cefD2, cefEF and cefG declined significantly during the whole fermentation period. In contrast, the AcmybA disruption mutant showed an increased cephalosporin C production, and the transcription levels of pcbAB, pcbC and cefD1, were up-regulated. Together with the EMSA results, we concluded that AcMybA plays a negative control on cephalosporin biosynthesis indirectly. The AcmybAOE went on growth at the late stage of fermentation, and finally reached a higher amount of biomass weight while WT tend to die at the same time. In contrast, the dry cell weight of the ΔAcmybA rapidly decreased at the late stage of fermentation. Propidium iodide staining experiment validated a massive of cell death happened in ΔAcmybA during fermentation. In conclusion, we believe that AcMybA acts as a global regulator that plays a negative control on cellular differentiation. Overexpression of AcmybA could result in less differentiated cell that retains vegetative growth, while deletion of AcmybA could lead to over differentiation of the cell, and reduce its longevity.A transcriptional regulatory gene AcstuA was identified from A. chrysogenum. AcstuA encodes a basic helix-loop-helix protein with similarity to StuA which has been identified as a transcriptional factor in the core developmental processes of Aspergillus nidulans. Like disruption of stuA in A. nidulans, disruption of AcstuA blocked the conidiation of A. chrysogenum through severely down-regulating the expression of AcbrlA and AcabaA. Disruption of AcstuA also drastically reduced cephalosporin production during fermentation. In agreement, the transcriptions of pcbAB, pbcC, cefDl, cefD2, cefEF and cefG were remarkably decreased in the AcstuA disruption mutant (AAcstuA). In addition to defects in conidiation and cephalosporin biosynthesis, AAcstuA produced abnormal swollen and fragmented hyphal cells during fermentation and tended to autolysis. The phenotypic alterations caused by AcstuA deletion were restored by supplementation of NaCl, indicating that deficiency of AcstuA might have an influence on cell wall integrity. The transcriptions of two mannoprotein encoding genes Acmp2 and Acmp3 significantly reduced in ΔAcstuA, further indicating that the mutant has cell wall defects. These results strongly suggested that AcstuA is important for conidiation, cephalosporin production, hyphal fragmentation and cell wall integrity in A chrysogenum.Macroautophagy (hereafter autophagy) is a bulk degradative pathway by which cytoplasm (cytosol and organelles) is delivered to the lysosome (or its yeast analog, the vacuole) for recycling.In the present study, we isolated a homolog of Sacharomyces cerevisiae atg8, designated as Acatg8. The full-size of Acatg8 was 612 bp containig two introns. Its duduced protein (AcAtg8) is constituted of 119 anomic acids with molecullar weitht of 13.7 KDa. The Acatg8 could complete the atg8 disruption mutant (Δatg8) of yeast, indicating that AcAtg8 is functonal homolog of S. cerevisiae Atg8. The expression of EGFP-tagged AcAtg8 showed that AcAtg8 is located in the cytosol and autophagosome in A. chrysogenum, and can be induced by starvation. To further analysis the function of Acatg8, we constructed the Acatg8 disruption mutant (AAcatg8) and its complemented strain (Acatg8C). ΔAcatg8 could not form autophagosome, and showed autophagy defects. ΔAcatg8 showed significant reduced conidiation, but was partly restored by increasing the concentration of carbon source, while increasing the concentration of nitrogen source can not restore the conidiation of ΔAcatg8. This result indicated that the autophagy mediated carbon source balance is critical for conidia formation. We found that AAcatg8 showed a delayed conidial germination, but its pecentage of germination is not affected. Moreover, the lcoalization of EGFP-AcAtg8 during germination implied that autophagy is involved in the early stage of conidia germination. AAcatg8 showed a significant enhanced CPC producton, further results indicated that the accumulation of PcbC, CefD2 and peroxisome in AAcatg8 is responseable for its high CPC production. We found that the biomass of the AAcatg8 decreased drastically in the late stage of fermentation, sugesting that autophagy is critical for A. chrysogenum cell survival in nutrition deprived condition. Furthermore, the hypha cell of ΔAcatg8 showed a massive accumulation of mitochondria, these mitochondria especially the damaged mitochondria could produce a large amount of reactive oxygen species (ROS) which is a great threat to cell survival.