Construction Of Recombinant Yeast For Efficient Synthesis Of Ursodeoxycholic Acid

Ursodeoxycholic acid（UDCA）is mainly used for treating hepatobiliary diseases and it is more effective than chenodeoxycholic acid（CDCA）.Currently,UDCA production is largely by chemical synthesis and biosynthesis.The chemical synthetic route is complex,costly and polluting,while the biosynthesis is mainly by the two-step enzymatic reactions based on7α-hydroxysteroid dehydrogenase（7αHSDH）and 7β-hydroxysteroid dehydrogenase（7βHSDH）,which limits its application in industrial production due to the need to add expensive cofactors.This study aimed at constructing a recombinant Saccharomyces cerevisiae whole-cell catalyst by expressing the7αHSDH and 7βHSDH in yeast cells simultaneously to explore a new process for converting CDCA to UDCA.The main research contents were as follows:（1）The E.coli 7αHSDH gene and the Ruminococcus torques 7βHSDH gene were used to construct a recombinant yeast plasmid expressing both 7αHSDH and 7βHSDH,which was introduced into yeast cells to obtain recombinant yeast SE7αRt7β.The 12 h fermentation results showed that at 2 g/L of substrate CDCA,the conversion was 93.50%.To improve the enzymatic activity of7βHSDH,recombinant yeast SE7αRt7β189207 expressing 7αHSDH and7βHSDH mutant T189V/V207M was constructed,and at 2 g/L of CDCA and 12h fermentation,the conversion was 90.91%.（2）The fermentation was optimized for 2 g/L of CDCA by recombinant yeast SE7αRt7β:inducer galactose 2 g/m L,the OD₆₀₀ 0.4 for inoculation,0.06%（v/v）of co-solvent Tween 80,and ethanol as the substrate solvent.（3）Using recombinant yeast SE7αRt7βas the catalyst,UDCA was synthesized by fermentation using batch feeding of substrate:1)fermentation in 50 m L centrifuge tubes（2.5 m L nutrient medium）showed that with a total loading of 4g/L CDCA,the conversion reached 81.66%when 2 g/L CDCA was added first and 2 g/L CDCA was supplemented after 24 h fermentation and the process continued further 24 h;2)the fermentation in 250 m L shake flasks（50 m L medium）showed that with a total loading of 8 g/L CDCA,the conversion was72.36%when 2 g/L CDCA was loaded first and 2 g/L CDCA was supplemented every 24 h.（4）Using whole-cell for UDCA synthesis by resting cultures of recombinant yeast SE7αRt7β:cultures concentrated 10 times,1)a single feeding of 4 g/L of CDCA,the conversion reached 87.36%at 24 h fermentation;2)separate feeding of CDCA,with the total feeding of 8 g/L,4 g/L was added first and after 24 h,4g/L CDCA was supplemented,and the fermentation continued further 24 h.The conversion reached 85.22%.（5）At the 5L fermenter level,the conversion was 82.50%for UDCA synthesis by recombinant yeast SE7αRt7β,with a total loading of 6 g/L CDCA and 120 h fermentation.The above results show that the construction of recombinant yeast SE7αRt7βwhole cell catalyst and the optimization of substrate transformation conditions have provided a solid foundation for the research and development of efficient and green processes for UDCA production.