| Fermentation with the brewing yeast Saccharomyces cerevisiae is the workhorse for fuel ethanol production.High titer ethanol achieved during the process can not only save energy consumption for ethanol recovery by distillation,but also reduce stillage discharge.However,high ethanol titer is inhibitory to yeast cells,resulting in stuck fermentation.On the other hand,temperature that can be tolerated by S.cerevisiae is in general below 34?.As a result,chilled water systems with intensive energy consumption and capital investment are needed for almost all fuel ethanol plants,since regular cooling water from the cooling tower with temperature only 2-3? lower than environmental temperature cannot cool down the fermentors in summer when temperature reaches as high as 35?,and apparently thermo-tolerant strains can address this challenge for ethanol fermentation to be operated at elevated temperature so that it can be cooled down by regular cooling water.Moreover,ethanol produced from sugar-and starch-based feedstocks is not sustainable,taking into account of increased population and consequent demand for food supply,and lignocellulosic biomass that is abundantly available is an alternative.Pretreatment is needed to deconstruct the carbohydrate-lignin complex for cellulose to be hydrolyzed by cellulases to release glucose for microbial conversion,during which toxic by-products are generated.High ethanol titer,elevated temperature and inhibitors are environmental stresses to yeast cells,which significantly compromise their growth and ethanol production.The self-flocculating S.cerevisiae SPSC01 was bred previously for fuel ethanol production in industry,and mechanism underlying its self-flocculating phenotype has been deciphered.However,impact of such a phenotype on environmental stress tolerance of yeast cells has not been explored.In this work,combined genome and transcriptome analysis of the SPSC01 was carried out,and candidate genes potentially responsible for stress tolerance were screened for functional exploration through their deletion and overexpression.1)Effect of the self-flocculating phenotype of the SPSC01 on its stress toleranceWith the model strain S288c and the de-flocculating mutant SPSC01?FLO1 as the control,ethanol fermentation was performed in flasks using medium containing 200 g/L glucose and supplemented with typical toxic by-products including formic acid,acetic acid,propionic acid,furfural,vanillin and vanillic acid,and ethanol to evaluate their impact on growth and ethanol production of SPSC01.A temperature of 42? was also exerted onto the fermentation.The experimental results indicated that SPSC01 grew better with more ethanol produced under the stressful conditions,indicating the contribution of its self-flocculating phenotype to stress tolerance.2)Omics analysis to screen genes for the stress tolerance of SPSC01The genome of SPSC01 was sequenced and compared to that of S288c,which contains 15.35 Mb,encompassing 5,315 genes.The SPSC01 has 80,388 SNPs,2,983 insertions,and 2,991 deletions.Among the 50,165 SNPs in the coding region,17,902 missense mutations corresponding to 4,667 mutated genes were found.Since some mutated genes filtered by the comparative genome analysis may be silent,transcriptome analysis was performed for SPSC01 and the de-flocculating mutant SPSC01?FLO1 during continuous ethanol fermentation.With the threshold-1?Log2R?1?801 differentially expressed genes(DEGs)were screened,in which 541 genes were up-regulated,and 260 genes were down-regulated.Based on KEGG pathways clustering,All the genes of SPSC01 with missense mutation followed by its differentially gene expression profiling(DGEp)were mutually mapped.Ultimately,a pool of stress tolerant genes involving thermal-,oxidative-,osmotic-stress,ethanol and acetic acid stresses,were generated for the SPSC01.Through qPCR verification or employing annotation information of SGD,MIGl and YCR049C were selected from the perspective of yeast genetic breeding and key-gene modification for further functional exploration.3)Impact of the expression of YCR049C on ethanol tolerance of S.cerevisiaeAs a putative gene,YCR049C is located at chromosome ? of S.cerevisiae with unknown function,which might encode a transmembrane protein,and was regulated in SPSC01 under ethanol stress conditions,implying that the gene might be related with ethanol tolerance.Since ethanol is a primary metabolite,and its production is tightly coupled with yeast growth,the growth of SPSC01?YCR049C was checked.In addition,SPSC01 and S288c mutants with YCR049C deleted,exhibited improved ethanol fermentation performance when very high gravity(VHG)medium containing 300 g/L glucose was used to produce more ethanol.The transcriptomics revealed that genes were differentially expressed in the S288c?YCR049C,in which 431 genes were up-regulated,and 583 genes were down-regulated compared to that wild-type S288c.While most up-regulated genes involve glycolysis and amino acid synthesis,genes for arginine metabolism and hexose transporters were down-regulated,particularly thiamine metabolism might be impaired under ethanol stress conditions.This progress not only confirmed the role of YCR049C in ethanol tolerance of S,cerevisiae,,but also indicated that such a regulation might be at global levels through interactions of multiple genes and metabolic pathways.4)Impact of the expression of MIGl-spscoi on thermal tolerance of S.cerevisiaeOverexpression of MIGl-SPSC01 distinctly enhanced thermal tolerance of SPSC01 and 6525 at 420C for their better growth on plates,but its contribution was observed to be host-specific,which was determined through ethanol fermentation at 42? using medium containing 200 g/L glucose.Further experiment indicated that the thermal tolerance was coordinated through intracellular heat shock proteins(HSPs),in particular Hsp30,since its expression was substantially up-regulated.Three SNPs were identified in MIGl_SPSC01:C212T,C409T and T1196C,which created two amino acid substitutions Pro71Leu and Phe399Ser.In-silico structural analysis indicated that the mutation Pro71Leu at the N-terminal is within the 2nd domain for binding with zinc finger proteins(ZFPs),through which the regulation of ZFPs on gene transcription would be affected,and on the other hand the mutation Phe399Ser at the C-terminal is between the two low complexity regions to alter space distance between key atoms to affect their interactions for structural functions.Experimental results verified that both of the two mutations are required for thermal tolerance of the yeast strains.5)Effect of MIGl-SPSC01 on xylose metabolism and cellulosic ethanol productionWhen MIGl-spscoi was overexpressed in YB-2625 engineered with xylose-metabolic pathway,xylitol accumulation was decreased,and consequently more biomass and ethanol were produced,illustrating potential role of MIGl-SPSC01 on xylose metabolism.Furthermore,overexpression of MIGl-SPSC01 in MNII/COC?BEC3 engineered with expression of cellulases for ethanol production from cellulose directly also improved its thermal tolerance,and more ethanol was produced from phosphoric acid-swollen cellulose(PASC)when the fermentation was performed at 40? due to enhanced hydrolysis of PASC by cellulases at the temperature.When Jerusalem artichoke stalk pretreated by steam explosion and alkali was used as the feedstock,15%and 42%improvement of cellulase activity and ethanol production were observed for the engineered strain in batch fermentation performed at 42?.With this work,correlation between its self-flocculating phenotype and stress tolerance improvement was confirmed,and the genetic background of SPSC01 was elucidated to a large extent.Through comparative genome and transcriptome analysis,genes related to stress tolerance were screened,and the functions of selected genes MIGl-SPSC01 and YCR049C were experimentally explored.The progress will lay a foundation for engineering yeast strains through rational designs to produce ethanol more efficiently with high titer and/or at elevated temperature as well as exploring functions for other genes. |
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