Title:
STEROID 5β REDUCTASE VARIANT AND APPLICATION THEREFOR
Document Type and Number:
WIPO Patent Application WO/2021/098506
Kind Code:
A1
Abstract:
A steroid 5β reductase variant and an application therefor. The steroid 5β reductase variant has steroid 5β reductase activity, and the amino acid sequence of the variant includes R63K and R64H mutations. The variant can use NADH as a coenzyme, and has an amino acid sequence selected from the group consisting of: 1) an amino acid sequence as indicated in SEQ ID NO: 1, 3 or 5; 2) an amino acid sequence produced by an amino acid sequence as indicated in SEQ ID NO: 1, 3 or 5 going through the substitution, deletion or addition of one or a plurality of amino acids; or 3) an amino acid sequence that has 69% or more of its sequence in common with SEQ ID NO: 1, 3 or 5. The transformation product of the steroid 5β reductase variant has a single conformation and only has a β-type reduction product that occurs, the reductase has spatial hydrogenation specificity, and the product does not contain an α-type structure. The variant is efficient and stable.
Inventors:
QI FUPING (CN)
LI YUANYUAN (CN)
LI YUANYUAN (CN)
Application Number:
PCT/CN2020/126374
Publication Date:
May 27, 2021
Filing Date:
November 04, 2020
Export Citation:
Assignee:
SHENYANG BOTAI PHARMACEUTICAL CO LTD (CN)
International Classes:
C12N9/02; C12N15/53; C12P33/00
Foreign References:
| CN106434705A | 2017-02-22 | |||
| CN101565709A | 2009-10-28 | |||
| US20070298456A1 | 2007-12-27 |
Other References:
LI, Y. ET AL.: "Identification of key sites determining the cofactor specificity and improvement of catalytic activity of a steroid 5β-reductase from Capsella rubella", ENZYME AND MICROBIAL TECHNOLOGY, vol. 134, 30 November 2019 (2019-11-30), XP086025157, DOI: 10.1016/j.enzmictec.2019.109483
RIECK CHRISTOPH, GEIGER DANIEL, MUNKERT JENNIFER, MESSERSCHMIDT KATRIN, PETERSEN JAN, STRASSER JULIANE, MEITINGER NADINE, KREIS WO: "Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae", MICROBIOLOGYOPEN, vol. 8, no. 12, 1 December 2019 (2019-12-01), XP055814002, ISSN: 2045-8827, DOI: 10.1002/mbo3.925
YAN QIAO-SHUN, YUE WANG, JUN HE, QING-HUI LUO, LI CUN-FU, XU JIAN-CHU, YUNNAN KUNMING, CHINAI P R, CHINAL R: "Cloning and Sequence Analysis of 5β-reductase of Progesterone f Cgp5Bra of Calotropis gigantea Ailaoshan Station for Subtropical Forest Ecosystem Studies Chinese Academy of Sciences Jingdong Yunnan 676200 P", JOURNAL OF WEST CHINA FORESTRY SCIENCE, vol. 48, no. 3, 1 June 2019 (2019-06-01), pages 53 - 60, XP055814102, DOI: 10.16473/j.cnki.xblykx1972.2019.03.009
HUANG YAO-YAO, JIN-FEN WEN, DENG MING-HUA, MING GONG, HAO-WEI CHEN: "Cloning and Expression Analysis of Jc5β-StR Gene in Jatropha curcas", BIOTECHNOLOGY BULLETIN, vol. 33, no. 7, 1 July 2017 (2017-07-01), pages 89 - 95, XP055814099, DOI: 10.13560/j.cnki.biotech.bull.1985.2016-1114
HERL, V. ; FISCHER, G. ; REVA, V.A. ; STIEBRITZ, M. ; MULLER, Y.A. ; MULLER-URI, F. ; KREIS, W.: "The VEP1 gene (At4g24220) encodes a short-chain dehydrogenase/reductase with 3-oxo-@D^4^,^5-steroid 5@b-reductase activity in Arabidopsis thaliana L.", BIOCHIMIE, MASSON, PARIS, FR, vol. 91, no. 4, 1 April 2009 (2009-04-01), FR, pages 517 - 525, XP026032155, ISSN: 0300-9084, DOI: 10.1016/j.biochi.2008.12.005
CHEN MO, DRURY JASON E., CHRISTIANSON DAVID W., PENNING TREVOR M.: "Conversion of Human Steroid 5β-Reductase (AKR1D1) into 3β-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 287, no. 20, 10 March 2011 (2011-03-10), US, pages 16609 - 16622, XP055813998, ISSN: 0021-9258, DOI: 10.1074/jbc.M111.338780
RIECK CHRISTOPH, GEIGER DANIEL, MUNKERT JENNIFER, MESSERSCHMIDT KATRIN, PETERSEN JAN, STRASSER JULIANE, MEITINGER NADINE, KREIS WO: "Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae", MICROBIOLOGYOPEN, vol. 8, no. 12, 1 December 2019 (2019-12-01), XP055814002, ISSN: 2045-8827, DOI: 10.1002/mbo3.925
YAN QIAO-SHUN, YUE WANG, JUN HE, QING-HUI LUO, LI CUN-FU, XU JIAN-CHU, YUNNAN KUNMING, CHINAI P R, CHINAL R: "Cloning and Sequence Analysis of 5β-reductase of Progesterone f Cgp5Bra of Calotropis gigantea Ailaoshan Station for Subtropical Forest Ecosystem Studies Chinese Academy of Sciences Jingdong Yunnan 676200 P", JOURNAL OF WEST CHINA FORESTRY SCIENCE, vol. 48, no. 3, 1 June 2019 (2019-06-01), pages 53 - 60, XP055814102, DOI: 10.16473/j.cnki.xblykx1972.2019.03.009
HUANG YAO-YAO, JIN-FEN WEN, DENG MING-HUA, MING GONG, HAO-WEI CHEN: "Cloning and Expression Analysis of Jc5β-StR Gene in Jatropha curcas", BIOTECHNOLOGY BULLETIN, vol. 33, no. 7, 1 July 2017 (2017-07-01), pages 89 - 95, XP055814099, DOI: 10.13560/j.cnki.biotech.bull.1985.2016-1114
HERL, V. ; FISCHER, G. ; REVA, V.A. ; STIEBRITZ, M. ; MULLER, Y.A. ; MULLER-URI, F. ; KREIS, W.: "The VEP1 gene (At4g24220) encodes a short-chain dehydrogenase/reductase with 3-oxo-@D^4^,^5-steroid 5@b-reductase activity in Arabidopsis thaliana L.", BIOCHIMIE, MASSON, PARIS, FR, vol. 91, no. 4, 1 April 2009 (2009-04-01), FR, pages 517 - 525, XP026032155, ISSN: 0300-9084, DOI: 10.1016/j.biochi.2008.12.005
CHEN MO, DRURY JASON E., CHRISTIANSON DAVID W., PENNING TREVOR M.: "Conversion of Human Steroid 5β-Reductase (AKR1D1) into 3β-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 287, no. 20, 10 March 2011 (2011-03-10), US, pages 16609 - 16622, XP055813998, ISSN: 0021-9258, DOI: 10.1074/jbc.M111.338780
Attorney, Agent or Firm:
PANAWELL & PARTNERS, LLC (CN)
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