| Bioethanol is a potential alternative energy source for crude oil.Rich and inedible lignocellulose is used as a potential raw material for ethanol production.However,the inhibition of cell growth by furfural and acetic acid in ligoncellulose hydrolysate is the bottleneck of cellulosic ethanol production.Candida glycerinogenes is a multi-stress yeast which can tolerance high temperature,high concentration of glucose and acetic acid.In this paper,C.glycerinogenes was used as the research object.On the basis of discovering the production of ethanol under micro-oxygen conditions and high concentration of furfural tolerance,the research focused on the furfural tolerance mechanism and cellulosic ethanol production of C.glycerinogenes.The main results are as follows:?1?Under micro-oxygen condition,C.glycerinogenes produced 62.4 g·L-1 ethanol,and glycerol was the main by-product.In order to improve ethanol yield,glycerol synthesis gene CgGPD was knocked out,which termed as C.glycerinogenes UG21.In this mutant strain,ethanol yield increased by 16.2%.Deletion of CgGPD had no effect on cell growth under high osmotic pressure or high concentration of acetic acid.C.glycerinogenes UG21 also exhibited excellent ethanol fermentation performance under low pH and high temperature conditions.The ethanol yield reached a maximum of 76.3 g·L-1 at 40°C.Through analysis of different aeration rates,we found that the maximal ethanol yield was achieved at 0.3 vvm aeration,and the productivity of ethanol was 3.0 g·L-1·h-1.?2?Anti-growth inhibitory factor experiments showed that C.glycerinogenes had significant furfural tolerance and furfural degradation ability compared with Saccharomyces cerevisiae.qRT-PCR results showed that furfural induced up-regulation of alcohol dehydrogenase gene CgADH1 to accelerate furfural degradation and reduced the inhibition of furfural on cells.In addition,overexpression of CgADH1 in S.cerevisiae showed better growth compared to S.cerevisiae overexpressing ScADH1 under furfural stress,indicating that the high expression activity of CgADH1 caused better furfural tolerance.In addition to CgAdh1,other oxidoreductases including CgNuo1 and CgAdh7 were also involved in furfural degradation.Overexpression of sulfite reductase gene CgSIA1 in C.glycerinogenes improved cell furfural tolerance but no significant effect on the degradation of furfural,indicating that C.glycerinogenes had a pressure response mechanism in addition to furfural degradation.?3?To explore other furfural tolerance mechanism of C.glycerinogenes,RNA-Seq was performed to study the whole transcriptome of C.glycerinogene exposed to 4 g·L-1 furfural stress.A total of 170 genes were up-regulated and 245 genes were down-regulated.Among them,it was found that some transporter genes including CgSNQ2,CgSTE6 and some ubiquitin-proteasome system genes including CgUFD2,CgECM29 were significantly up-regulated,indicating that furfural induced cell transport and damage protein ubiquitination degradation in response to furfural damage.In contrast to S.cerevisiae,a large number of ribosomal function-related genes were up-regulated in C.glycerinogenes,indicating that up-regulation of ribosome function-related gene stress under furfural stress might be a unique mechanisms to resist furfural stress in C.glycerinogene.The results of RNA-Seq and qRT-PCR indicated that the furfural-induced the expression of the cysteine synthesis genes CgCYS1,CgSIR1 and CgSIR2.Overexpression of CgCYS1 in C.glycerinogenes improved cell growth under furfural stress.These results indicated that the ubiquitin-proteasome system accelerates the degradation of damage proteins,ribosome function-related genes and cysteine synthesis gene stress expression was the main mechanism to resist furfural stress in C.glycerinogenes.?4?RNA-Seq and qRT-PCR results revealed that transcription factors,such as Pdr1,Rpn4,NirA and Bas1p,were up-regulated under furfural stress.The biomass of C.glycerinogenes decreased by 27.1%as PDR1 expression was inhibited by antisense RNA technique.qRT-PCR revealed that the transporter genes CgSNQ2 and CgSTE6 were not induced by furfural,indicating that the furfural-induced transcription factor Pdr1 regulated the expression of ABC transporter Snq2 and Ste6.The biomass of C.glycerinogenes decreased by 33.2%as RPN4 expression was inhibited by antisense RNA technique.qRT-PCR analysis revealed that the ubiquitin-proteasome system involved genes CgUFD2 and CgECM29 were not induced by furfural,indicating that furfural regulates the expression of the ubiquitin-proteasome system by transcription factor Rpn4.The overexpressed genes BAS1 and NIRA promoted the metabolism under furfural pressure,which enhanced the ethanol fermentation performance of C.glycerinogenes.The yield of ethanol increased by 35.2%and 31.4%,respectively.Antisense RNA inhibited the expression of BAS1 and NIRA,and the cell biomass decreased by 22.3%and 18.9%respectively.The above results indicated that the transcription factors Pdr1 and Rpn4 in C.glycerinogenes regulated cell transport and ubiquitination damage protein degradation to resist furfural pressure.In addition,transcription factors NirA and Bas1p were also involved in resisting furfural stress in C.glycerinogenes.?5?During ethanol fermentation under high concentrations of furfural and acetic acid,C.glycerinogenes UG21 showed an excellent cell growth and ethanol production compared with industrial ethanol product yeast S.cerevisiae ZWA46 and commercial Angel yeast.Using undetoxified bagasse hydrolysate as substrate,the productivity of ethanol reached 1.24 g·L-1·h-1 in C.glycerinogenes UG21,while S.cerevisiae ZWA46 and Angel Yeast could not grow under this condition.In addition,the treated low concentration of furfural acetate bagasse hydrolysate as the substrate for ethanol fermentation,the ethanol yield of C.glycerinogenes UG21 represented an 2.42-fold and 1.58-fold increased compared with S.cerevisiae ZWA46and Angel Yeast,respectively.Combined with D-1,2,4-butanetriol-producing strain,the important national defense chemicals D-1,2,4-butanetriol and ethanol were simultaneously achieved using two-stage method,the final yield was 4.73 g·L-1 and 43.2 g·L-1,respectively. |
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