| Thermal tolerant Saccharomyces cerevisiae strains have significant roles in industrial application,owning to their advantages such as higher fermentation rate,shorter fermentation period and lower broth viscosity in SSF（Simultaneous Saccharification and Fermentation）.Their application in high-temperature fermentation could also reduce energy consumption in cooling system and thus lower production cost.To explore the thermal tolerance mechanism of thermal tolerant mutant of industrial S.cerevisiae 1015,back trace of the drive mutation gene was conducted based on whole-genomic and transcriptomic results,and spt23 was identified as a potential key gene that confers S.cerevisiae the thermal tolerance traits.Studies had found that spt23 affects thermal tolerance by modulating unsaturated lipid acid contents,through transcriptional regulation on ole1 that encodes forΔ9 lipid acid desaturase.Also,spt23 is a broader transcriptional regulator that influences thermal tolerance by involving in protein homeostasis and energy metabolism.Besides that,spt23 is a transposon suppressor,which may play a role in yeast thermal tolerance through adaptive evolution of transposons,but there are no adequate supporting reports.Therefore,we constructed a series of spt23-modified strains（deletion/Del,over-expression/OE and integrative expression/IE）and examined the growth and fermentation characteristics,thermal tolerance abilities,gene expression levels,cell membrane fatty acid contents and transposon activities of them,to explore the underlying mechanisms of spt23 in yeast thermal tolerance,as well as the weights of those mechanisms.Survival rate of recombinants after heat treatment showed that the expression level of spt23 displayed contrary tendency with thermal tolerance.The OE and IE strains had survival rate no higher than the WT strain.The survival rates of Del strains were significantly higher than the WT strains.The expression level of spt23 had a negative correlation with ethanol production in ethanol fermentation.Among all the recombinants,the Del type had highest peak ethanol production at 44 h（14.0±0.14）%,8.93%higher than the wild-type.Increase in saturated lipid acid contents was found under heat treatment,through investigation of total unsaturated lipid acids（TUSLA）contents in the cell membrane.By investigating the expression level of ole1,acc1,mga2 and erg1,the key genes in lipid acid synthesis,spt23 was found having a regulatory role on ole1 other than acc1,mag2 and erg1.A weak correlation between the expression level of spt23 and TUSLA could be attributed to the interference of mga2.With LTR amplicon sequencing of S.cerevisiae,the LTR sequence diversity was used as an evaluation of transposon activity.Results showed that heat treatment caused decreased OTU（t-test,p=0.043）and increased Shannon evenness（t-test,p=0.029）,indicating lower richness and higher evenness of LTR sequences.Ta2 of strain 101530 at 37℃was 0.653,higher than that at 30℃（0.627）,indicating higher transposon activity after heat treatment.Transposon activity did not have significant correlation with spt23 expression level,but it was one of the determinant factors of the survival rate after heat treatment.Transcriptomic analysis of S.cerevisiae 1015 and 101530 indicate proteostasis,maintained by proteosome,ribosome and RNA polymerase II,widely located in cell parts and highly associated with transcription regulation,was an important contributor to thermal tolerance.spt23’s regulation on spt3 affects the SAGA complex and eventually influences RNA polymerase II’s transcription which is highly related to environmental stress.No similar regulation on the SWI/SNF complex was observed.A linear regression model was built for survival rate after heat treatment with TUSLA and Ta2（Survival=-2.39·TUSLA+69.98·Ta2+152.43,R2=0.815,p=0.015）.With a total explanatory power of 81.5%,the relative importance of the two variables was 0.83:0.17(LMGTUSLA=0.83,LMGTa2=0.17).A new model describing S.cerevisiae thermal tolerance was proposed according to the results that both the transcriptional regulation of spt23 and adaptive evolution of transposons features S.cerevisiae thermal tolerance.In summary,spt23 contributes to yeast thermal tolerance through regulating cell membrane TUSLA.Deletion of spt23 increases ethanol production.as a global transcription regulator,spt23 had limited overall regulation on thermal tolerance;Thermal tolerance of S.cerevisiae was determined by TUSLA and transposon activity,with a relative weight of 83:17(LMGTUSLA=0.83,LMGTA2=0.17).However,transposon activity was not regulated by spt23.So,spt23 plays a wide but non-dominant role in yeast thermal tolerance.The results would be heuristic for the construction of thermal tolerant S.cerevisiae strains in the future. |
