Development Of Recombinant Saccharomyces Cerevisiae With Enhanced Cellulase Production

Saccharomyces cerevisiae can be used as a host to produce various pharmaceutical proteins and industrial enzymes.In recent years,it has been extensively studied as a host for the production of foreign proteins.However,low protein production and secretion levels remain a great challenge for practical applications.Hyperproduction of heterologous protein may cause metabolic burden,however,the regulatory mechanisms of heterologous protein production in S.cerevisiae remain unclear.During the study of environmental stress tolerance of yeast strains in our group,we found elevation of protein levels of Mdg1 p and Mhf1 p in the stress tolerant strains.Mdg1 p is a membrane protein,and Mhf1 p is a protein associated with maintenance of genome stability.However,the functions of these two proteins have been poorly studied.The effects of these two proteins on heterologous protein production are still unclear.In this study,CRISPR-Cas9-based genome editing technology was used to overexpress MHF1 and MDG1 genes in the recombinant S.cerevisiae strains that secrete cellobiohydrolase,endoglucanase and ?-glucosidase,respectively.The obtained recombinant yeast strains were evaluated for their ability to produce cellulase,and the underlying mechanisms were further explored.It was found that overexpression of both genes increased the activity of cellobiohydrolase in the recombinant yeast.In addition,overexpression of MDG1 also increased the secretion of endoglucanase and ?-glucosidase.Next,the molecular mechanisms underlying the promotion of cellulase production were explored.Real-time quantitative PCR analysis was used to analyze the transcription of protein-secreting pathway related genes.The results showed that the increase of cellobiohydrolase activity in recombinant yeast may be related to increased transcript levels of key genes in the vesicular transport pathway.Analysis of intracellular active oxygen accumulation revealed that the active oxygen content of the recombinant yeast overexpressing the MHF1 gene was not significantly different from that of the control.It was proposed that the recombinant yeast that overexpresses MHF1 had increased scavenging activity of the reactive oxygen species(ROS).There was no improvement in the growth of recombinant yeast on plates containing tunicamycin or dithiothreitol,suggesting that the increase in production of recombinant proteins was irrelevant to the relief of unfolded protein stress.The cell membrane permeability of three cellulase recombinant yeast strains overexpressed by MDG1 was significantly increased,which may be one of the key reasons for the increase of enzyme production.The high concentration of acetic acid,ethanol and stress tolerance analysis of the recombinant yeast were not found to increase the environmental stress tolerance of the strains that enhance the production of enzymes.Further analysis of the mechanisms underlying improved cellulase production by overexpression of MDG1 and MHF1 is helpful for understanding the regulatory mechanisms of the recombinant proteins synthetic secretion in S.cerevisiae and the development of new metabolic engineering strategies to improve recombinant S.cerevisiae secretory production of heterologous proteins.