Study On Candida Glycerinogenes Furfural Tolerance Transcription Factors

Cellulose is one of the main raw materials for biotransformation to solve energy problems.Furfural,which seriously affects the growth of strains,exists in its hydrolysate and needs to be detoxified before it can be applied in fermentation.In this paper,the transcription factors related to furfural tolerance of Candida glycerinogenes were used to screen for transcription factors that can improve furfural tolerance and applied to the ethanol fermentation of undetoxified cellulose hydrolysate.The details of the research in this paper were as follows:Based on the transcriptome data of C.glycerinogenes under furfural stress,we pre-screened furfural stress-related genes Cg SEF1,Cg STB5,Cg ETP1 and Cg CAS5,by combining real-time fluorescence quantitative PCR.The C.glycerinogenes genome was used as a template to clone the transcription factors,and the C.glycerinogenes overexpression strains and S.cerevisiae recombinant strains were constructed.Cg SEF1,Cg STB5,Cg ETP1 and Cg CAS5transcription factors were analyzed bioinformatically.The furfural tolerance of the constructed transcription factor overexpression strains and recombinant strains was examined.The recombinant strains S.cerevisiae expressing Cg STB5,Cg ETP1 and Cg SEF1 was significantly improved by more than 25%in the spot plate tolerance test,and the growth retardation period was shortened under 1.5 g·L^-1 and 2 g·L^-1 furfural stress.The results of q RT-PCR indicated that the tolerance of S.cerevisiae to cellulose-hydrolyzed furfural involved the combined effect of multiple genes regulation.C.glycerinogenes overexpression strains under different glucose concentration medium conditions with the addition of 3 g·L^-1 furfural showed that C.glycerinogenes ETP1 and C.glycerinogenes STB5showed better tolerance to furfural.C.glycerinogenes ETP1 showed significantly elevated furfural tolerance under high glucose(250 g·L^-1),which favored fermentation with concentrated undetoxified cellulose hydrolysate.The recombinant strains S.cerevisiae expressing Cg STB5,Cg ETP1 and Cg SEF1 had some growth advantages over the control strain,with 12.5%higher ethanol production efficiency for the strain expressing Cg STB5 and 15.7%higher ethanol production for the strain expressing Cg ETP1.Applying the engineered strains with significantly elevated furfural tolerance to the ethanol fermentation of undetoxified cellulose,showed that the recombinant strains had obvious advantages in the ethanol fermentation of undetoxified cellulose hydrolysate.On the 5L fermenter,showed a significant increase in furfural tolerance and ethanol production,and the ethanol yield of C.glycerinogenes ETP1 was 2.79 g·L^-1·h^-1,which was 34%higher than the wild strain.Through q RT-PCR,it was found that Cg STB5 firstly resisted stress by lowering metabolism while decomposing furfural,and when furfural was reduced to a certain level,the strain resumed rapid growth and eventually had a certain advantage in biomass,while Cg ETP1was directly up-regulated furfural degradation-related genes to rapidly degrade high concentrations of furfural so that the strain growth did not produce significant inhibition.C.glycerinogenes ETP1 fed batch fermentation significantly showed higher ethanol production capacity than wild strains and faster consumption of glucose for ethanol production,with better cellulosic ethanol production efficiency.This result provided a reference for the modification of undetoxified cellulose hydrolysate fermentation strains.