Multilevel Optimization of 3-Ketosteroid-9α-Hydroxylase for Enhanced 9α-Hydroxy-4-androstene-3,17-dione Production.

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-10-01 DOI:10.1021/acssynbio.5c00476

Jing Tao, Ting Zhang, Kun Jiang, Xiaohui Cheng, Yuting Zhang, Mengfei Long, Guojian Liao

{"title":"Multilevel Optimization of 3-Ketosteroid-9α-Hydroxylase for Enhanced 9α-Hydroxy-4-androstene-3,17-dione Production.","authors":"Jing Tao, Ting Zhang, Kun Jiang, Xiaohui Cheng, Yuting Zhang, Mengfei Long, Guojian Liao","doi":"10.1021/acssynbio.5c00476","DOIUrl":null,"url":null,"abstract":"9α-Hydroxylation represents a critical modification step in steroid pharmaceutical synthesis, where 9α-hydroxy-4-androstene-3,17-dione (9α-OH-AD) serves as an important intermediate for producing high-potency steroids, such as dexamethasone. The biosynthesis of 9α-OH-AD is catalyzed by a 3-ketosteroid-9α-hydroxylase (KSH) system comprising Rieske oxygenase (KshA) and ferredoxin reductase (KshB). In this study, we cloned the kshA and kshB genes from Mycobacterium smegmatis mc2155 and optimized their heterologous expression in E. coli, enhancing 9α-OH-AD yield by 4.9-fold. We implemented a 17β-carbonyl reductase (17β-CR)-mediated NADH regeneration system to ensure sufficient cofactor supply, which increased 9α-OH-AD production by 5.8-fold, while computer-aided design yielded the optimal KshA mutant A83V-G186L-A249W with a remarkable 13.8-fold improvement in catalytic efficiency compared to the wild-type. Through biotransformation optimization, the engineered microbial cell factory demonstrated a 17.2 times enhancement over the starting strain at the flask level, with a fed-batch strategy. This enzymatic conversion strategy establishes an eco-efficient platform for the sustainable synthesis of steroid therapeutics.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9000,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Synthetic Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1021/acssynbio.5c00476","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}

引用次数: 0

Abstract

9α-Hydroxylation represents a critical modification step in steroid pharmaceutical synthesis, where 9α-hydroxy-4-androstene-3,17-dione (9α-OH-AD) serves as an important intermediate for producing high-potency steroids, such as dexamethasone. The biosynthesis of 9α-OH-AD is catalyzed by a 3-ketosteroid-9α-hydroxylase (KSH) system comprising Rieske oxygenase (KshA) and ferredoxin reductase (KshB). In this study, we cloned the kshA and kshB genes from Mycobacterium smegmatis mc²155 and optimized their heterologous expression in E. coli, enhancing 9α-OH-AD yield by 4.9-fold. We implemented a 17β-carbonyl reductase (17β-CR)-mediated NADH regeneration system to ensure sufficient cofactor supply, which increased 9α-OH-AD production by 5.8-fold, while computer-aided design yielded the optimal KshA mutant A83V-G186L-A249W with a remarkable 13.8-fold improvement in catalytic efficiency compared to the wild-type. Through biotransformation optimization, the engineered microbial cell factory demonstrated a 17.2 times enhancement over the starting strain at the flask level, with a fed-batch strategy. This enzymatic conversion strategy establishes an eco-efficient platform for the sustainable synthesis of steroid therapeutics.

查看原文本刊更多论文

3-酮甾-9α-羟化酶促进9α-羟基-4-雄烯-3,17-二酮合成的多级优化

9α-羟基化是类固醇药物合成的关键修饰步骤，其中9α-羟基-4-雄烯-3,17-二酮（9α-OH-AD）是生产高效类固醇（如地塞米松）的重要中间体。9α-OH-AD的生物合成是由Rieske加氧酶（KshA）和铁氧还蛋白还原酶（KshB）组成的3-酮类固醇-9α-羟化酶（KSH）系统催化的。本研究从耻垢分枝杆菌mc2155中克隆了kshA和kshB基因，并对其在大肠杆菌中的异种表达进行了优化，使9α-OH-AD的产量提高了4.9倍。为了保证足够的辅助因子供应，我们建立了17β-羰基还原酶（17β-CR）介导的NADH再生系统，使9α-OH-AD的产量提高了5.8倍，而计算机辅助设计产生了最佳的KshA突变体A83V-G186L-A249W，其催化效率比野生型提高了13.8倍。通过生物转化优化，采用分批补料策略，工程微生物细胞工厂在烧瓶水平上比初始菌株提高17.2倍。这种酶转化策略建立了一个可持续合成类固醇治疗的生态高效平台。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.