| A large number of toxic inhibitors are produced during the pretreatment of the second generation bio-ethanol industry,which seriously affect the growth and fermentation process of Saccharomyces cerevisiae.Furfural is the most important inhibitor.So overcoming the inhibitory effect of toxic inhibitors such as furfural is the key factor to realize the second generation bio-ethanol production.Inhibitors such as furfural can be removed by physical,chemical and biological methods,but these methods will increase the production cost of bio-ethanol and the removal effect is limited.Based on the ability of Saccharomyces cerevisiae to tolerate and toxify furfural,researchers have been carrying out many scientific studies on the mechanism of furfural detoxification as well as tolerance in Saccharomyces cerevisiae,and found many transcriptional regulatory genes related to furfural tolerance.In this study,engineered Saccharomyces cerevisiae strains were constructed by overexpression of PDR1,YAP1 and RPN4 transcriptional regulatory genes.Phenotypes of engineered strains and original strain under furfural stress were determined and compared,and transcriptome sequencing data were analyzed.The following experimental results are obtained:1.Using YB-A-6-1 industrial Saccharomyces cerevisiae diploid strain as the original strain,we selected TEF1 p constitutive strong promoter and PDR1,YAP1,RPN4 key transcriptional regulatory genes,and constructed P strain overexpressing PDR1 gene by homologous recombination in yeast.PY strain overexpressing PDR1 and YAP1 genes and PYR strain overexpressing PDR1,YAP1 and RPN4 genes were constructed based on P strain respectively.By measuring the liquid growth curve of strains under the condition of furfural stress,it is concluded that the lag phase of three engineered strains is obviously shorter than that of the original strain.With the increase of the concentration of furfural,the difference of the lag phase between them becomes more and more obvious.In the maximum concentration of 45 m M furfural,the lag phase of PYR engineered strain is shorter than that of PY,and the lag phase of PY is shorter than that of P.Without any inhibitors,there is no lag phase between the three engineered strains and the original strain,showing almost similar growth trends.2.The specific activity of aldehyde reductase was determined by crude enzyme extraction and using two coenzymes NADH and NADPH,respectively.Under the pressure of furfural stress,the specific activity of aldehyde reductase dependent on NADH of three engineered strains increased gradually compared with that of the original strain,while the specific activity of aldehyde reductase dependent on NADPH of three engineered strains is significantly lower than that of the original strain.The determination of cell wall resistance of yeast strains under furfural stress demonstrates the cell wall resistance of three engineered strains is significantly stronger than that of the original strain.In addition,there was no significant difference in cell wall resistance between engineered strains.The growth lag phase of three engineered strain in 85 m M 5-hydroxymethyl furfural,5.2 g/L acetic acid,10.5% ethanol and 13% corn straw hydrolysate was shorter than that of the original strain,and there was no significant difference between engineered strains.The growth lag phase of P engineered strain was the shortest under 1.8 g/L formic acid stress,and there was no significant difference between PY,PYR engineered strains and the original strain.There was no growth lag phase under phenol inhibition,but only the difference of growth rate.3.Significant up-regulation and down-regulation genes related to furfural tolerance in engineered strains were preliminarily obtained by transcriptome sequencing data.The up-regulation genes of membrane efflux protein were mainly concentrated in the PY and the up-regulation genes of cell membrane component synthesis were mainly concentrated in the strain PYR.CHS2 participates in the synthesis of chitin,an important component of cell wall,which is up-regulated significantly in all three engineered strains.And the down-regulation of damage protein modification and degradation genes was all found in three engineered strains.The genes related to pentose phosphate pathway were mainly concentrated in PY and PYR,specifically reflected in the up-regulation of the cascade enzyme gene to synthesize the metabolite PRPP(5-ribose phosphate-1-pyrophosphate).4.Through the analysis of transcriptional regulation relationship,it was found that Yap1 p has a significant indirect up-regulation effect on downstream genes HXT8,YJL163 C,PDR10,PDR15,ICT1,URA7,YKL071 W.Yap1p is the dominant regulator involved in gene regulation involved in fufural tolerance.The co-overexpression of transcriptional regulation genes was not a simple,overlapping and isolated regulation of each transcriptional regulation gene,which involved transcriptional regulation factors,which involves the interaction between transcription regulators and the indirect regulation of downstream functional genes by transcription regulators,etc.In this study,PDR1 related to multidrug resistance,YAP1 related to oxidative stress response,RPN4 related to damage protein degradation,constitutive strong promoter TEF1 p were selected to successfully construct engineered strain with co-overexpression of transcriptional regulatory genes PDR1,YAP1,RPN4,engineered strain with co-overexpression of PDR1,YAP1 and engineered strain with overexpression of PDR1.The tolerance mechanism and transcriptional regulation of engineered strains were studied at the phenotypic and molecular levels.The results of this study have enabled us to have a deeper understanding of the mechanism of furfural tolerance and the relationship between transcriptional regulation,which lays a foundation for further co-overexpression of transcriptional regulation genes with different functions in the future,and has important scientific and practical significance for industrial production of lignocellulose bio-ethanol. |
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