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

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

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引用次数: 0

Abstract

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.

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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.