Construction Of High-Yield Xylanase Saccharomyces Cerevisiae Engineering Bacteria And Optimization Of Fermentation Conditions

Xylanase is considered as an important industrial enzyme preparation and has been maturely used in industrial production such as pulp and papermaking,feed processing,and brewing,etc.β-1,4-endo-xylanase is a key enzyme in xylan complete hydrolase system.Its structure includes catalytic domain,binding domain,thermal stability domain and unknown functional domain,etc.It catalyzes the use of endo-hydrolysis and specifically acts on the β-1,4-glycosidic linkage in the backbone molecule.In order to successfully achieve efficient secretion of xylanaseB by Saccharomyces cerevisiae,In this study,the constitutive promoter PGK,exogenous protein secretion α-factor,xylanase B(XynB)and terminator CYC1 were constructed into PαXC expression cassettes by overlap extension PCR method.The recombinant expression vector pYES2-PαXC-rDNA was integrated into the Saccharomyces cerevisiae genome using rDNA integration method.The positive transformants were obtained by uracil auxotrophy screening,The copy number of the xylanaseB gene of the recombinant Saccharomyces cerevisiae strain was identified based on a digital polymerase chain reaction(ddPCR)method.Using 3,5-dinitrosalicylic acid(DNS)method to detect the enzyme activity of different copy number xylanaseB gene recombinant strains,and the relationship between the different copy number of xylanaseB gene and its enzyme activity was investigated.The genetic stability of the recombinant strain with the highest enzyme activity was studied.The fermentation conditions of xylanase produced by recombinant strains were optimized by single factor analysis and response surface methodology.The results were as follows:1.The PαXC expression cassette was successfully constructed by the overlap extension PCR method.The constitutive expression vector pYES2-PαXC-rDNA was integrated into the Saccharomyces cerevisiae genome by rDNA integration method.A large number of positive transformants were selected from the pyrimidine auxotrophs.2.The ddPCR method was used to identify the xylanaseB gene copy number of the recombinant strain obtained by the above screening.A total of 10 strains of recombinant bacteria with 1,2,3,6,7,8,10,14,17 and 21 xylanaseB gene copy numbers were obtained.The activity of xylanaseB produced by 10 strains of recombinant bacteria was determined by DNS method,indicating that there was no positive correlation between gene copy number and enzyme activity.When the xylanaseB gene copy number was 7,the activity of xylanaseB produced by the recombinant strain(LX7)was the highest,and the enzyme activity was up to 355.03 U/mL,which was 6.4 times that of the single-copy xylanase production activity.When the enzyme gene exceeds 7 copies,the enzyme activity decreases instead.3.Compared with the activity of xylanaseB produced by recombinant Saccharomyces cerevisiae strains preserved in our laboratory,the xylanaseB production activity of the recombinant strain constructed in this experiment increased by 47 U/mL,which was 1.15 times higher than of its xylanase activity.The genetic stability of the LX7 strain showed that the recombinant strain obtained by the rDNA-integrated xylanaseB gene method had a good stability.After 6 passages,the enzyme activity changes little,and the enzyme activity was 349.5U/mL at the 6th passage.4.The single factor analysis of the fermentation conditions of high yield xylanaseB recombinant Saccharomyces cerevisiae was performed.The results of variance analysis showed that the inoculation amount,liquid loading,and glucose addition had no significant effect on the xylanaseB production conditions of recombinant strains.The initial pH,fermentation temperature,and stirring speed had significant effects on the fermentation conditions of the xylanaseB produced by the recombinant strain.Response surface method was used to optimize the fermentation conditions of LX7 strain.The results showed that the optimal conditions for the fermentation of xylanaseB from the recombinant strain were as follows: the initial pH of the medium was 5.2,the fermentation temperature was 29 ℃,the stirring speed was 180 rpm,and the xylanaseB activity of the LX7 strain produced was up to 401.3 U/mL,which was 46 U/mL higher than that before optimization.