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

  • Jing Chen
  • Jinlei Tang
  • Yongyan Xi
  • Zhubo Dai
  • Changhao Bi
  • Xi Chen
  • Feiyu FanEmail author
  • Xueli ZhangEmail author
Biotechnological products and process engineering

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-450lun 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-450lun 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-450lun towards RSS and AD in the Saccharomyces cerevisiae system. We demonstrated that P-450lun 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-450lun-associated redox partner CPRlun. Finally, through codon optimization, knockout of genes for the side reactions related enzymes GCY1 and YPR1, and increasing copies of the P-450lun and CPRlun, 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.

Keywords

Steroid 14α-hydroxylation 14α-Hydroxysteroids Cytochrome P450 Cochliobolus lunatus Saccharomyces cerevisiae 

Notes

Acknowledgments

We sincerely thank our colleague Mr. Cai Yi for his help with the NMR experiments.

Funding information

This research was supported by the Key Research Program of the Chinese Academy of Sciences (KFZD-SW-215) and the National Natural Science Foundation of China (31861143019, 31700066).

Compliance with ethical standards

Competing interests

The authors declare that they have no conflict of interest.

Ethical statements

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_10076_MOESM1_ESM.pdf (666 kb)
ESM 1 (PDF 666 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jing Chen
    • 1
    • 2
    • 3
  • Jinlei Tang
    • 1
    • 3
  • Yongyan Xi
    • 1
    • 2
    • 3
  • Zhubo Dai
    • 1
    • 3
  • Changhao Bi
    • 1
    • 3
  • Xi Chen
    • 1
    • 3
  • Feiyu Fan
    • 1
    • 3
    Email author
  • Xueli Zhang
    • 1
    • 3
    Email author
  1. 1.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.Key Laboratory of Systems Microbial BiotechnologyChinese Academy of SciencesTianjinChina

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