Abstract
C22 steroid drug intermediates are suitable for corticosteroids synthesis, and the production of C22 steroids is unsatisfactory due to the intricate steroid metabolism. Among the C22 steroids, 21-hydroxy-20-methyl-pregna-1,4-dien-3-one (1,4-HP) could be used for Δ1-steroid drug synthesis, such as prednisolone. Nevertheless, the production of 1,4-HP remains unsatisfactory. In this study, an ideal 1,4-HP producing strain was constructed. By the knockout of 3-ketosteroid-9-hydroxylase (KshA) genes and 17β-hydroxysteroid dehydrogenase (Hsd4A) gene, the steroid nucleus degradation and the accumulation of C19 steroids in Mycolicibacterium neoaurum were blocked. The mutant strain could transform phytosterols into 1,4-HP as the main product and 21-hydroxy-20-methyl-pregna-4-ene-3-one as a by-product. Subsequently, the purity of 1,4-HP improved to 95.2% by the enhancement of 3-ketosteroid-Δ1-dehydrogenase (KSTD) activity, and the production of 1,4-HP was improved by overexpressing NADH oxidase (NOX) and catalase (KATE) genes. Consequently, the yield of 1,4-HP achieved 10.5 g/L. The molar yield and the purity of 1,4-HP were optimal so far, and the production of 1,4-HP provides a new intermediate for the pharmaceutical steroid industry.
Key points
• A third 3-ketosteroid-9-hydroxylase was identified in Mycolicibacterium neoaurum.
• An 1,4-HP producer was constructed by KshA and Hsd4A deficiency.
• The production of 1,4-HP was improved by KSTD, NOX, and KATE overexpression.
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Acknowledgements
We are grateful to Dr. Yu Jiang (CAS Center for Excellence in Molecular Plant Sciences Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China) and Dr. Yichen Sun (Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China) for helping us set up the Cprispr-Cpf1 system.
Funding
This work is supported by the National Key R&D Program of China (No.2017YFE0112700) and Shi Jiping Expert Workstation project of Yunnan Province in 2021.
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BGZ, CYY, and ZGM designed and conceived the study. CYY, ZGM, and SWH performed the research. CYY, JXZ, and XCL analyzed the data. CYY and YXL wrote the paper. YXL, BGZ, and GLD revised the paper. JSS, JPS, and GLD contributed analytical tools and supervised the project. All authors read and approved the manuscript.
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Yuan, C., Ma, Z., Li, Y. et al. Production of 21-hydroxy-20-methyl-pregna-1,4-dien-3-one by modifying multiple genes in Mycolicibacterium. Appl Microbiol Biotechnol 107, 1563–1574 (2023). https://doi.org/10.1007/s00253-023-12399-2
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DOI: https://doi.org/10.1007/s00253-023-12399-2