Production of 14α-hydroxysteroids by a recombinant Saccharomyces cerevisiae biocatalyst expressing of a fungal steroid 14α-hydroxylation system
Abstract
The 14α-hydroxysteroids have specific anti-gonadotropic and carcinolytic biological activities and can be produced by microbial biotransformation. The steroid 11β-/14α-hydroxylase P-450 lun from Cochliobolus lunatus is the only fungal cytochrome P450 enzyme identified to date with steroid C14 hydroxylation ability. Previous work has mainly revealed the 11β-hydroxylation activity of the P-450 lun towards cortexolone (RSS) substrate; however, the potential steroid 14α-hydroxylation activity of this enzyme, especially for androstenedione (AD) substrate, has not yet conducted in-depth testing. In this work, we further tested the steroid 14α-hydroxylation activity of the P-450 lun towards RSS and AD in the Saccharomyces cerevisiae system. We demonstrated that P-450 lun functions as the specific 14α-hydroxylase towards the AD substrate (regiospecificity > 99%); however, it showed a poor C14-hydroxylation regiospecificity (around 40%) for the RSS substrate. In addition, through transcriptome analysis combined with gene functional characterizations, we also identified and cloned the gene for the P-450 lun-associated redox partner CPR lun. Finally, through codon optimization, knockout of genes for the side reactions related enzymes GCY1 and YPR1, and increasing copies of the P-450 lun and CPR lun, we developed a recombinant S. cerevisiae biocatalyst based on the C. lunatus steroid 14α-hydroxylation system to produce 14α-hydroxysteroids. Initial production of 14α-OH-AD (150 mg/L day productivity, 99% regioisomeric purity, and 60% w/w yield) and 14α-OH-RSS (64 mg/L day productivity, 40% regioisomeric purity, and 26% w/w yield) were separately achieved in shake flasks; these results represent the highest level of 14α-hydroxysteroid production in the current yeast system.
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Most enzymes of ecdysteroid biosynthesis, except 14α-hydroxylase and 22-hydroxylase, have been identified only in insects/arthropods.
“Moreover, most of the enzymes, except 14α-hydroxylase [179] and 22-hydroxylase [36], have been identified only in insects/arthropods.”
From Ecdysteroids (2021) · cites this paper as [179]
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- Authors
- Chen, J., Tang, J., Xi, Y., Dai, Z., Bi, C., Chen, X., Fan, F., & Zhang, X.
- Affiliations
- Chinese Academy of Sciences (CN)
- Corresponding author
- Fan, Feiyu
- Zhang, Xueli
- Venue
- Applied Microbiology and Biotechnology
- Published
- 2019-08-14
- Volume
- 103
- Issue
- 20
- Pages
- 8363-8374
- ISSN
- 0175-7598 (ISSN-L)
- 1432-0614
- OpenAlex
- W2969055489
- Cited by
- 35
- References
- 44
- Citations by year
- 2026: 3 · 2025: 4 · 2024: 5 · 2023: 7 · 2022: 9 · 2021: 4 · 2020: 3
- FWCI
- 1.33
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Applied Microbiology and Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Key Research Program of the Chinese Academy of Sciences (KFZD-SW-215)
- National Natural Science Foundation of China (31861143019, 31700066)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Steroid Chemistry and Biochemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Keywords
- Curvularia lunata, cytochrome P450, recombinant protein expression, regiospecificity, androstenedione, cortexolone, redox partners, codon optimization, microbial transformation
- MeSH terms
- Mixed Function Oxygenases, Hydroxylation, Hydroxysteroids, Recombinant Proteins, Saccharomyces cerevisiae, Metabolic Engineering
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