Construction Of Recombinant Candida Tropicalis To Produce D-arabitol

As a functional sugar alcohol,D-arabitol has been widely used in food,chemical,medical and other fields.Microbial production of D-arabitol has great potential and application prospects,and how to improve the production of D-arabitol strains has also been studied.In this study,Candida tropicalis CU-208 was used as the starting strain to explore the metabolic pathway of D-arabitol.CRISPR Cas9 system was used for gene editing and marker gene reuse for metabolic transformation.It constructed strains use xylose or glucose as a substrate to produce D-arabitol.Yhe main results are as follows:1.Through the whole genome sequencing of Candida tropicalis ATCC 20336 and the alignment of the D-arabitol dehydrogenase（ARD）gene on NCBI,the CDS sequence of the endogenous ard gene was found,using CRISPR Cas9,The strains ARDD and ARDO with knockout and overexpression of ard gene were constructed,and fermentation experiments were carried out on ARDD and ARDO to determine the change of D-arabitol production.The results showed that ARDD accumulated more D-arabitol than wild type,while ARDO had lower D-arabitol accumulation than both ARDD and wild type,indicating that ard gene was involved in the catabolism of D-arabitol.2.constructing a strain that used xylose as a substrate and glucose as an auxiliary carbon source to produce D-arabitol.The metabolic transformation is as follows.The xylukinase（XKS）gene was knocked out,and the upstream gene xylitol dehydrogenation was overexpressed at the same time.Enzyme（xylitol dehydrogenase XDH）gene to construct strain FYM01;I knocked out endogenous D-arabitol dehydrogenase gene to construct strain FYM02;overexpress heterologous D-arabitol dehydrogenase（D-arabitol dehydrogenase ADH）,Construct strain FYM04;overexpressing endogenous glucose-6-phosphate dehydrogenase（Glucose-6-phosphate dehydrogenase,ZWF）to construct strain FYM05.The results showed that FYM01 could not grow with xylose as the sole carbon source due to blocking the entry of xylose into the pentose phosphate pathway;FYM04 could use glucose as an auxiliary carbon source to produce D-arabitol from xylose,but due to the utilization efficiency of xylose.lower,the D-arabitol yield was 4.72 g·L^-1;FYM05 accumulated more coenzyme NADPH and was able to utilize more xylose.Fermentation experiments were performed on FYM05,and FYM05accumulated 11.35 g·L^-1 of D-arabitol.The single factor optimization of FYM05 was carried out,and it was found that the optimum amount of xylose for the production of D-arabitol was80 g·L^-1,the optimum temperature was 35^oC,and the optimum p H was 5,Attempt to ferment with xylose mother liquor as substrate,and accumulated 3.28 g·L^-1 D-arabitol..3.constructeing a strain that used glucose as a substrate to produce D-arabitol,and the metabolic transformation is as follows.Taking ARDD as the starting strain,overexpressing glucose 6-phosphate dehydrogenase,constructing strain FYM06,single-copy knockout endogenous ribulose-5-phosphate isomerase（Ribulose-5 phosphate isomerase,RPE）.The results showed that compared with the starting strain,FYM06 and FYM07 accumulated more D-arabitol,and the yields were 8.36 g·L^-1 and 10.37 g·L^-1,respectively.The yield of FYM07was higher than that of FYM06,and the results indicated that RPE The knockout of D-arabitol favored the accumulation of D-arabitol.And through the single factor experiment on FYM07,and on this basis,the orthogonal experiment was carried out.The optimum p H was 7,the optimum substrate concentration was 150 g·L^-1 glucose,and the optimum temperature was35^oC;Under these conditions,13.92 g·L^-1 of D-arabitol was obtained by shaking flask fermentation,and.26.65 g·L^-1 D-arabitol was obtained by 5L fermenter fermentation.