Effect Of MRP8 Or MIG1 Overexpression On Cellulase Production By The Recombinant Saccharomyces Cerevisiae

Saccharomyces cerevisiae is widely used for ethanol production,and is also used as the host for heterologous protein expression.However,low efficiency of extracellular protein production is the bottleneck for practical application.Therefore,it is important to improve protein production levels of S.cerevisiae.However,it is still not clear on the underlying regulatory mechanisms of protein production and secretion.Protein production may lead to metabolic burden in S.cerevisiae.Therefore,it is of interest to explore novel genes involved in stress tolerance for their functions in protein production and secretion by S.cerevisiae.In this study,two genes,MRP8 and MIG1,which showed elevated levels in stress tolerant S.cerevisiae were focused.MRP8 encodes a mitochondrial ribosomal protein,whereas MIG1 encodes a transcription regulator that is involved in glucose repression,and we focused the MIG1 allele isolated from the self-flocculating yeast SPSC01,which contains mutations in amino acid sequences.In this study,MRP8 was overexpressed through CRISPR/Cas9-mediated genome editing in both the model yeast S.cerevisiae S288 C and the recombinant yeast expressing cellobiohydrolase I(CBH1)using S.cerevisiae Y294 as the host.It was found that overexpression of MRP8 in the model yeast S.cerevisiae S288 C improved acetic acid tolerance,but no significant effect was observed in the recombinant yeasts expressing CBH1 used in this study,suggesting that the effects of MRP8 are host dependent.In addition,the effects of overexpressing MRP8 on the production of CBH1 in the recombinant yeast Y294-CBH1 were investigated,and the underlying mechanisms for improved CBH1 production were explored.Compared with the control strain,the CBH1 activity was improved by 80% in the MRP8-overexpression strain,whereas no significant effect on growth ability was observed by MRP8 overexpression.Real-time quantitative PCR analysis showed that the transcription level of CBH1 was increased in the strain Y294-CBH1 overexpressing MRP8,whereas the transcription levels of key genes related to protein folding and secretion,such as IMH1,ERV25 and YPT32 exhibited no change.No difference on cell growth in the presence of Congo red,Tunicamycin(TM)or Dithiothreitol(DTT)was observed when overexpressing MRP8,as well as ATP content and reactive oxygen species(ROS)accumulation.Through the preliminary analysis of the secretome data of 96h sample,we found a lot of proteins enrichment related to translation,nitrogen metabolism,and oxidative stress tolerance were up-regulated significantly by MRP8 overexpression.Therefore,the potential mechanisms of MRP8-overexpression enhanced CBH1 production may be associated with improved protein glycosylation,leading to improved activity and stability,but not affected by cell-wall integrity or unfolded protein response(UPR).However,the detailed mechanisms still need to be further validated.In addition,effects of overexpression of MIG1SPSC01 on thermal tolerance and production of cellulase CBH1 in the strain Y294-CBH1 were evaluated by integration of MIG1SPSC01 into the HO site in the genome.It was found that overexpression of MIG1SPSC01 improved thermal tolerance and the production of CBH1.Compared with the control strain,CBH1 activity of the MIG1SPSC01-overexpressing strain was increased approximately by 80% at both 30 °C and 40 °C.The mechanisms underlying enhanced enzyme production were further investigated,and it was found that transcription levels of key genes associated with protein folding and secretion,such as ERV29,MDR1 and SEC18 were both up-regulated in the MIG1SPSC01-overexpression strain.In the preliminary secreted proteome data,a large number of proteins associated with cell wall synthesis were significantly down-regulated by MIG1SPSC01 overexpression,while the proteins related to hydrolase enzyme activity and carbohydrate metabolism were upregulated.Furthermore,cell growth in the Congo-red plates was improved by MIG1SPSC01 overexpression,which implies that the function of MIG1SPSC01 is indeed related to cell wall biogenesis.On the other hand,it was found that overexpression of the S288 C allele of MIG1 did not show comparable improvement of CBH1 production to that led by MIG1SPSC01-overexpression,suggesting that the mutations in MIG1SPSC01 may be responsible for the improvement phenotype,but the underlying mechanisms remain to be revealed.Our results will provide insights for further studies on the regulation mechanisms of protein production and secretion.The results in this study may benefit further exploration of novel genes for heterologous protein production in S.cerevisiae.