长寿型工程酵母构建及其高效催化合成熊去氧胆酸的研究OA

Ursodeoxycholic acid(UDCA)is a first-line drug for the clinical treatment of hepatobiliary diseases.The bioconversion of chenodeoxycholic acid(CDCA)based on whole-cell catalysis provides an important alternative approach for its green production.However,during fermentation,cellular aging and CDCA-induced stress lead to decreased stability of the catalytic system,which severely restricts its industrial application.This study took the extension of cellular chronological lifespan as the entry point,and systematically evaluated the effects of overexpressing the chronological lifespan regulatory factor heme activator protein 4 gene(HAP4)on whole-cell catalytic performance.The results showed that,under CDCA stress conditions after 8 days of fermentation,the survival rate of cells overexpressing HAP4 was 4.5-fold higher than that of the wild type,indicating that HAP4 overexpression significantly prolonged the CLS of Saccharomyces cerevisiae.Furthermore,by screening efficient genetic elements,an efficient catalytic module was obtained,consisting of 7α-hydroxysteroid dehydrogenase(7α-HSDH)from Escherichia coli and RT-7β-hydroxysteroid dehydrogenase(RT-7β-HSDH)from Ruminococcus torques.These enzymes were assembled in S.cerevisiae BY4742 to construct an efficient CDCA whole-cell catalytic strain,BY-RT,which achieved a UDCA conversion rate of 96.0%.On this basis,HAP4 was further overexpressed to obtain a long-lived engineered strain,BY-RT-HAP4.After 20 days of shake-flask fermentation,the catalytic efficiency of BY-RT-HAP4 remained at 44.4%,representing a 14.8-fold increase compared with BY-RT.Finally,industrial fermentation conditions were simulated in a 5 L bioreactor to systematically evaluate the effect of enhanced HAP4 expression on UDCA whole-cell catalytic performance.Under conditions of greater environmental stress and higher substrate concentration stress,the catalytic efficiency of BY-RT-HAP4 at the end of fermentation was still 13.4%higher than that of BY-RT.These results demonstrate that enhanced HAP4 expression can effectively maintain cellular activity and metabolic homeostasis,significantly improve the whole-cell conversion efficiency of UDCA,and provide a new metabolic engineering strategy and theoretical basis for the green and efficient biosynthesis of UDCA.