摘要
Laccases, multicopper oxidoreductases, are mainly produced in white-rot fungi and are considered as ideal green catalysts in industrial and biotechnological applications. However, the development of laccases is limited due to the slow growth of natural laccase producing strains and the low expression levels of laccases. In this study, we designed three regulation strategies for laccase gene expression in the model fungus Aspergillus nidulans. By introducing various promoters in front of the laccase gene pslcc from the white-rot fungus Pycnoporus sanguineus, we found that the laccase gene with the original promoter had effective expression in A. nidulans. Using the previously identified transcription factor RsmA regulatory mechanism, the aflR promoter was inserted into the pslcc expression vectors, and the laccase production was 15-fold higher in the strain overexpressing of RsmA compared to the control strain. To improve the laccase yield, the dipeptidyl-peptidase DppV, aspartic protease PepA and mannosyltransferase Mnn9 were successfully deleted in the A. nidulans host. The laccase activities were increased approximately 8-fold and 13-fold in the double deletions strains of Δmnn9ΔpepA and ΔdppVΔpepA over the control strains, respectively.Taken together, these results not only demonstrate an efficient system for heterologous protein production in the model fungus A.nidulans but also provide a general approach to applying regulatory methods to control gene expression.
Laccases, multicopper oxidoreductases, are mainly produced in white-rot fungi and are considered as ideal green catalysts in industrial and biotechnological applications. However, the development of laccases is limited due to the slow growth of natural laccase producing strains and the low expression levels of laccases. In this study, we designed three regulation strategies for laccase gene expression in the model fungus Aspergillus nidulans. By introducing various promoters in front of the laccase gene pslcc from the white-rot fungus Pycnoporus sanguineus, we found that the laccase gene with the original promoter had effective expression in A. nidulans. Using the previously identified transcription factor RsmA regulatory mechanism, the aflR promoter was inserted into the pslcc expression vectors, and the laccase production was 15-fold higher in the strain overexpressing of RsmA compared to the control strain. To improve the laccase yield, the dipeptidyl-peptidase DppV, aspartic protease PepA and mannosyltransferase Mnn9 were successfully deleted in the A. nidulans host. The laccase activities were increased approximately 8-fold and 13-fold in the double deletions strains of Δmnn9ΔpepA and ΔdppVΔpepA over the control strains, respectively.Taken together, these results not only demonstrate an efficient system for heterologous protein production in the model fungus A.nidulans but also provide a general approach to applying regulatory methods to control gene expression.
基金
supported by Beijing Natural Science Foundation (5152018)
the National Natural Science Foundation of China (31470178)
Wen-Bing Yin is a scholar of "the 100 Talents Project" of CAS
