Machine-Learning-Guided Engineering of an NADH-Dependent 7β-Hydroxysteroid Dehydrogenase for Economic Synthesis of Ursodeoxycholic Acid

• Published in: Journal of agricultural and food chemistry 2023-12, Vol.71 (49), p.19672-19681
• Main Authors: Wang, Mu-Qiang, You, Zhi-Neng, Yang, Bing-Yi, Xia, Zi-Wei, Chen, Qi, Pan, Jiang, Li, Chun-Xiu, Xu, Jian-He
• Format: Article
• Language: English
• Subjects: Bayes Theorem; Biotechnology and Biological Transformations; Hydroxysteroid Dehydrogenases - genetics; Hydroxysteroid Dehydrogenases - metabolism; NAD; NADP - metabolism; Ursodeoxycholic Acid - metabolism
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.3c06339

Enzymatic synthesis of ursodeoxycholic acid (UDCA) catalyzed by an NADH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH) is more economic compared with an NADPH-dependent 7β-HSDH when considering the much higher cost of NADP+/NADPH than that of NAD+/NADH. However, the poor catalytic performance of NADH-dependent 7β-HSDH significantly limits its practical applications. Herein, machine-learning-guided protein engineering was performed on an NADH-dependent Rt7β-HSDHM0 from Ruminococcus torques. We combined random forest, Gaussian Naïve Bayes classifier, and Gaussian process regression with limited experimental data, resulting in the best variant Rt7β-HSDHM3 (R40I/R41K/F94Y/S196A/Y253F) with improvements in specific activity and half-life (40 °C) by 4.1-fold and 8.3-fold, respectively. The preparative biotransformation using a “two stage in one pot” sequential process coupled with Rt7β-HSDHM3 exhibited a space–time yield (STY) of 192 g L–1 d–1, which is so far the highest productivity for the biosynthesis of UDCA from chenodeoxycholic acid (CDCA) with NAD+ as a cofactor. More importantly, the cost of raw materials for the enzymatic production of UDCA employing Rt7β-HSDHM3 decreased by 22% in contrast to that of Rt7β-HSDHM0, indicating the tremendous potential of the variant Rt7β-HSDHM3 for more efficient and economic production of UDCA.