Cited by 35Steroid Chemistry and BiochemistryEnzyme Catalysis and ImmobilizationFungal and yeast genetics research

Production of 14α-hydroxysteroids by a recombinant Saccharomyces cerevisiae biocatalyst expressing of a fungal steroid 14α-hydroxylation system

Chen, Jing, Tang, Jinlei, Xi, Yongyan, Dai, Zhubo, Bi, Changhao, Chen, Xi, Fan, Feiyu, Zhang, Xueli·2019·Applied Microbiology and Biotechnology

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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Crossref · 10.1007/s00253-019-10076-x

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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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