Mutants of 3-ketosteroid-9alpha-hydroxylase ksha and uses thereof

CN122168549APending Publication Date: 2026-06-09TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
Filing Date
2024-12-08
Publication Date
2026-06-09

AI Technical Summary

Technical Problem

In the existing technology, the soluble expression of 3-sterone-9α-hydroxylase KshA in Escherichia coli is poor and its efficiency in catalyzing the conversion of 4-androstenedione substrates is low, which makes it difficult to meet the requirements for efficient preparation of 9-hydroxy-androstenedione.

Method used

By using genetic engineering techniques to perform site-directed mutations on KshA, particularly introducing specific mutations at positions 182, 210, and 362 of the amino acid sequence, a KshA mutant with high soluble expression and high catalytic performance was constructed. This mutant was then co-expressed with the ferroreductase KshB, and the reaction conditions were optimized to improve catalytic efficiency.

Benefits of technology

The efficient synthesis of 9-OH-4-AD under mild reaction conditions was achieved, which significantly improved the catalytic activity of KshA and enhanced the preparation efficiency of 9-hydroxy-androstenedione.

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Abstract

The application belongs to the field of molecular biology and enzyme engineering, and relates to 3-steroid ketone-9 alpha-hydroxylase oxidase KshA mutants and application thereof in synthesis of 9-hydroxy-androstenedione, in particular to 3-steroid ketone-9 alpha-hydroxylase oxidase KshA mutants, recombinant host cells, cell cultures, and an enzyme method for synthesizing 9-hydroxy-androstenedione. The application solves the problem of low concentration of substrate 4-androstenedione in the existing industrial synthesis route, which leads to high production cost of 9-hydroxy-androstenedione, and has a broad market prospect.
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