Effects Of Candida Boydin’s XylI Gene On Xylitol Production By Escherichia Coli Fermentation

Xylitol(C₅H₁₂O5)is widely used in chemical,pharmaceutical,food and other fields.The use of engineered bacteria to synthesize xylitol is becoming a research hotspot at home and abroad.This thesis intends to study the effect of xyl I gene（encoding aldose reductase）from Candida Boydin on xylitol fermentation.With the help of constructing a vector capable of expressing the xyl I gene without repressor protein,the addition of chemical inducer is realized when Escherichia coli produces xylitol.At the same time,the effect of co-expression of xyl I gene and soluble pyridine nucleotide transhydrogenase gene（UdhA）gene on xylitol production by Escherichia coli was explored.The main research contents and results are as follows:（1）The enzymatic properties of the aldose reductase（encoded by the xyl I gene）from Candida boidin were studied.In this work,the expression plasmid p ET28a（+）-xyl I was constructed,and the expression of the recombinant plasmid in Escherichia coli BL21（DE3）was analyzed.The results showed that the molecular weight of aldose reductase was 39 KD,and the enzyme activity was 3.3 U/m L.The optimal catalytic temperature is 25℃,and the optimal catalytic p H is 7.The enzyme is relatively stable when stored below 25℃,and can maintain more than 60%of the enzyme activity within 12 hours.When stored at a p H of about 7,it can maintain more than 50%of the enzyme activity within 12 hours.（2）In this work,we successfully constructed a plasmid（p WYZ-2）capable of expressing the xyl I gene without repressor protein,and realized that no chemical inducer was added when Escherichia coli produces xylitol.The plasmid p WYZ-1,which used lac P as the promoter,had 1.8 times higher xylitol production than the plasmid p AG102that used the anaerobic promoter pfl B-p6（shaking speed is 200 r/min）.The xyl I gene was expressed in the plasmid p WYZ-2（based on the mid-copy plasmid p BR322,without lac I fragment）,which produced more xylitol than expressed in the plasmid p WYZ-1（based on the high-copy plasmid p UC19）.The addition of the inducer IPTG had little effect on the xyl I gene expression of the plasmid p WYZ-2 to produce xylitol.Combining the knockout of xylose isomerase gene（xyl A）and xylulose kinase gene（xyl B）gene,the yield of E.coli xylitol could reach 48.7 g/L without adding inducer.（3）The UdhA gene was further cloned into the p WYZ-2 plasmid to achieve co-expression with xyl I,and the plasmid p WYZ-4 was constructed.When the substrate D-xylose concentration was 20 g/L,the shake flask fermentation results showed that E.coli was fermented for 36 h.The xylitol yield of E.coli AI07/p WYZ-4 strain was 19.91g·L^-1,which was 34%higher than that of E.coli AI07/p WYZ-2 strain.When the concentration of substrate D-xylose was increased to 110 g·L^-1,the final yield of xylitol could reach 49.76 g/L.