福氏分枝杆菌ATCC 6842植物甾醇通过多基因修饰高效生产9α-羟基类固醇

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal Pub Date : 2025-02-18 DOI:10.1016/j.bej.2025.109689

Ruijie Zhang , Wen Sun , Suwan Han , Xiaoxuan Sun , Xinghui Zhai , Beiru He , Xianfeng Zhu , Baoguo Zhang

{"title":"福氏分枝杆菌ATCC 6842植物甾醇通过多基因修饰高效生产9α-羟基类固醇","authors":"Ruijie Zhang , Wen Sun , Suwan Han , Xiaoxuan Sun , Xinghui Zhai , Beiru He , Xianfeng Zhu , Baoguo Zhang","doi":"10.1016/j.bej.2025.109689","DOIUrl":null,"url":null,"abstract":"<div><div>The steroid drug industry is increasingly utilizing microbial biotransformation, employing genetically modified mycobacteria to convert phytosterols into steroid intermediates, with an emphasis on improving yield and purity. This study enhances the production of 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), a vital C19 steroid intermediate for glucocorticoid synthesis, by genetically modifying <em>Mycobacterium fortuitum</em> ATCC 6842. The study involved the targeted disruption of five 3-ketosteroid-Δ<sup>1</sup>-dehydrogenase (<em>kstD</em>) genes to prevent Δ<sup>1</sup>-dehydrogenation. The purity of 9-OH-AD is initially low at 81.85 % due to two main by-products: 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one (9-OH-HP) and 9,24-dihydroxychol-4-en-3-one (9,24-DHC). To address this, the steroid aldolase (<em>sal</em>) gene was deleted to block the C22 metabolic pathway, which completely eliminated 9-OH-HP and increased the purity of 9-OH-AD to 88.19 %. To further reduce 9,24-DHC levels, acyl-CoA dehydrogenases ChsE1 and ChsE2 were overexpressed. The resulting strain, MFKS_<em>chsE1</em>-<em>chsE2</em>, achieved a high purity of 9-OH-AD at 94.96 %, with a molar yield of 87.17 % from 10 g/L phytosterols, and converted up to 30 g/L of phytosterols into 15.91 g/L of 9-OH-AD. This method effectively enhances both the production and purity of this important steroid intermediate.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"218 ","pages":"Article 109689"},"PeriodicalIF":3.7000,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Efficient production of 9α-hydroxy-steroid from phytosterols in Mycobacterium fortuitum ATCC 6842 by modifying multiple genes\",\"authors\":\"Ruijie Zhang , Wen Sun , Suwan Han , Xiaoxuan Sun , Xinghui Zhai , Beiru He , Xianfeng Zhu , Baoguo Zhang\",\"doi\":\"10.1016/j.bej.2025.109689\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>The steroid drug industry is increasingly utilizing microbial biotransformation, employing genetically modified mycobacteria to convert phytosterols into steroid intermediates, with an emphasis on improving yield and purity. This study enhances the production of 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), a vital C19 steroid intermediate for glucocorticoid synthesis, by genetically modifying <em>Mycobacterium fortuitum</em> ATCC 6842. The study involved the targeted disruption of five 3-ketosteroid-Δ<sup>1</sup>-dehydrogenase (<em>kstD</em>) genes to prevent Δ<sup>1</sup>-dehydrogenation. The purity of 9-OH-AD is initially low at 81.85 % due to two main by-products: 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one (9-OH-HP) and 9,24-dihydroxychol-4-en-3-one (9,24-DHC). To address this, the steroid aldolase (<em>sal</em>) gene was deleted to block the C22 metabolic pathway, which completely eliminated 9-OH-HP and increased the purity of 9-OH-AD to 88.19 %. To further reduce 9,24-DHC levels, acyl-CoA dehydrogenases ChsE1 and ChsE2 were overexpressed. The resulting strain, MFKS_<em>chsE1</em>-<em>chsE2</em>, achieved a high purity of 9-OH-AD at 94.96 %, with a molar yield of 87.17 % from 10 g/L phytosterols, and converted up to 30 g/L of phytosterols into 15.91 g/L of 9-OH-AD. This method effectively enhances both the production and purity of this important steroid intermediate.</div></div>\",\"PeriodicalId\":8766,\"journal\":{\"name\":\"Biochemical Engineering Journal\",\"volume\":\"218 \",\"pages\":\"Article 109689\"},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2025-02-18\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochemical Engineering Journal\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1369703X25000622\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemical Engineering Journal","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1369703X25000622","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}

引用次数: 0

摘要

类固醇药物行业越来越多地利用微生物生物转化，利用转基因分枝杆菌将植物甾醇转化为类固醇中间体，重点是提高产量和纯度。本研究通过基因修饰fortuitum分枝杆菌ATCC 6842，提高了9α-羟基-4-雄烯-3,17-二酮（9-OH-AD）的产量，9α-羟基-4-雄烯-3,17-二酮是糖皮质激素合成的重要C19类固醇中间体。该研究涉及靶向破坏5个3-酮类固醇-Δ1-dehydrogenase （kstD）基因以预防Δ1-dehydrogenation。由于两个主要副产物：9,22-二羟基-23,24-双去甲胆-4-烯-3-one （9- oh - hp）和9,24-二羟基胆-4-烯-3-one (9,24- dhc)， 9- oh - ad的纯度最初较低，为81.85 %。为了解决这个问题，我们删除了类固醇醛缩酶（sal）基因来阻断C22代谢途径，完全消除了9-OH-HP，将9-OH-AD的纯度提高到88.19 %。为了进一步降低9,24- dhc水平，酰基辅酶a脱氢酶ChsE1和ChsE2过表达。得到的菌株MFKS_chsE1-chsE2纯度为94.96 %，10 g/L植物甾醇的摩尔产率为87.17 %，最高可将30 g/L植物甾醇转化为15.91 g/L 9-OH-AD。该方法有效地提高了这一重要类固醇中间体的产量和纯度。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文本刊更多论文

Efficient production of 9α-hydroxy-steroid from phytosterols in Mycobacterium fortuitum ATCC 6842 by modifying multiple genes

The steroid drug industry is increasingly utilizing microbial biotransformation, employing genetically modified mycobacteria to convert phytosterols into steroid intermediates, with an emphasis on improving yield and purity. This study enhances the production of 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), a vital C19 steroid intermediate for glucocorticoid synthesis, by genetically modifying Mycobacterium fortuitum ATCC 6842. The study involved the targeted disruption of five 3-ketosteroid-Δ¹-dehydrogenase (kstD) genes to prevent Δ¹-dehydrogenation. The purity of 9-OH-AD is initially low at 81.85 % due to two main by-products: 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one (9-OH-HP) and 9,24-dihydroxychol-4-en-3-one (9,24-DHC). To address this, the steroid aldolase (sal) gene was deleted to block the C22 metabolic pathway, which completely eliminated 9-OH-HP and increased the purity of 9-OH-AD to 88.19 %. To further reduce 9,24-DHC levels, acyl-CoA dehydrogenases ChsE1 and ChsE2 were overexpressed. The resulting strain, MFKS_chsE1-chsE2, achieved a high purity of 9-OH-AD at 94.96 %, with a molar yield of 87.17 % from 10 g/L phytosterols, and converted up to 30 g/L of phytosterols into 15.91 g/L of 9-OH-AD. This method effectively enhances both the production and purity of this important steroid intermediate.

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

来源期刊

Biochemical Engineering Journal 工程技术-工程：化工

CiteScore

7.10

自引率

5.10%

发文量

380

审稿时长

34 days

期刊介绍： The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology. The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields: Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics Biosensors and Biodevices including biofabrication and novel fuel cell development Bioseparations including scale-up and protein refolding/renaturation Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells Bioreactor Systems including characterization, optimization and scale-up Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis Protein Engineering including enzyme engineering and directed evolution.