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Updated: Jan 18, 2026
Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
Published on: February 5, 2019
Structure-guided single-point mutagenesis and carboxyl-terminal truncation improved the substrate selectivity of
Lina Jin1, Min Chen1, Li Yang1
1State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, The SATCM Key Laboratory for New Resources & Quality Evaluation of Chinese Medicine, The MOE Key Laboratory for Standardization of Chinese Medicines and Shanghai Key Laboratory of Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, PR China.
Abstract:
Directional biotransformation using whole microbial cell factory coexpressing 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) with readily available chicken bile powder (CBP) as substrate holds potential for producing rare bear bile alternatives. However, the low specificity of wild-type 7α-HSDH toward taurochenodeoxycholic acid (TCDCA) results in the formation of unwanted by-products. In this study, guided by structure analysis, 7α-HSDH was engineered through active site-directed saturation mutagenesis and carboxyl-terminal (C-terminal) truncation. High-throughput screening, stepwise activity assays, and enzyme kinetic analyses identified T94 as key active site residue and confirmed C-terminal's importance for substrate specificity and catalytic activity. The T94FΔC1 mutant, combining C-terminal truncation and T94F mutation, exhibited high selectivity and activity toward TCDCA. Coupled with 7β-HSDH and applied in biotransformation using CBP as substrate, T94FΔC1-Rg7β-HSDH (β2) eliminated by-products tauroursocholic acid (TUCA) and 3,12-dihydroxy-7-oxocholanoyltaurine (3,12-HOCT) and produced the desired ratio of tauroursodeoxycholic acid (TUDCA):TCDCA in M9-GY medium. This study highlights the crucial role of T94 in determining the substrate specificity and activity of 7α-HSDH, and provides strategies for rational designing and engineering enzymes to enhance their substrate specificity and catalytic activity in practical applications.
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