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Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants.

ABSTRACT: Diosgenin is a spiroketal steroidal natural product extracted from plants and used as the single most important precursor for the world steroid hormone industry. The sporadic occurrences of diosgenin in distantly related plants imply possible independent biosynthetic origins. The characteristic 5,6-spiroketal moiety in diosgenin is reminiscent of the spiroketal moiety present in anthelmintic avermectins isolated from actinomycete bacteria. How plants gained the ability to biosynthesize spiroketal natural products is unknown. Here, we report the diosgenin-biosynthetic pathways in himalayan paris (Paris polyphylla), a monocot medicinal plant with hemostatic and antibacterial properties, and fenugreek (Trigonella foenum-graecum), an eudicot culinary herb plant commonly used as a galactagogue. Both plants have independently recruited pairs of cytochromes P450 that catalyze oxidative 5,6-spiroketalization of cholesterol to produce diosgenin, with evolutionary progenitors traced to conserved phytohormone metabolism. This study paves the way for engineering the production of diosgenin and derived analogs in heterologous hosts.

SUBMITTER: Christ B

PROVIDER: S-EPMC6642093 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants.

Christ Bastien B Xu Chengchao C Xu Menglong M Li Fu-Shuang FS Wada Naoki N Mitchell Andrew J AJ Han Xiu-Lin XL Wen Meng-Liang ML Fujita Makoto M Weng Jing-Ke JK

Nature communications 20190719 1

Diosgenin is a spiroketal steroidal natural product extracted from plants and used as the single most important precursor for the world steroid hormone industry. The sporadic occurrences of diosgenin in distantly related plants imply possible independent biosynthetic origins. The characteristic 5,6-spiroketal moiety in diosgenin is reminiscent of the spiroketal moiety present in anthelmintic avermectins isolated from actinomycete bacteria. How plants gained the ability to biosynthesize spiroketa ...[more]

PMID: 31324795

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Project description:BackgroundCytochrome P450 monooxygenases--able to regio- and stereoselectively hydroxylate non-activated carbon atoms--are important enzymes for the synthesis of valuable intermediates in the production of steroid hormones in the pharmaceutical industry. However, up to now only a few bacterial enzymes able to hydroxylate steroids have been reported. CYP154C5 from Nocardia farcinica IFM 10152, a bacterial P450 monooxygenase, was previously shown to convert testosterone to 16?-hydroxytestosterone. Since the hydroxylation at 16?-position is of special interest for the pharmaceutical industry, we have studied this enzyme in more detail to investigate its activity and selectivity in bioconversions of further steroids.ResultsCYP154C5 was coexpressed in Escherichia coli together with putidaredoxin and putidaredoxin reductase from Pseudomonas putida as redox partners for electron transfer and applied in bioconversions of various pregnanes and androstanes [pregnenolone (1), dehydroepiandrosterone (2), progesterone (3), androstenedione (4), testosterone (5) and nandrolone (6)]. Structure elucidation of the formed products revealed an exclusive regio- and stereoselectivity of CYP154C5, always yielding the corresponding 16?-hydroxylated steroids. Application of whole cells expressing the three components, P450, Pdx and PdR, in steroid biotransformations resulted in significantly higher conversions and total turnover numbers (TTN) compared to reactions using cell-free extracts. Additionally, considerably higher substrate loads (up to 15 mM) were tolerated by the whole-cell system. Furthermore, turnover numbers (TON) were determined for the six different steroids using whole cells. Thus, testosterone was found to be the worst substrate with a TON of only 0.8 ?mol substrate consumed min-1 ?mol(-1) CYP154C5, while progesterone and pregnenolone were converted the fastest resulting in TON of 3.3 ?mol substrate consumed min(-1) ?mol(-1) CYP154C5.ConclusionCYP154C5 from N. farcinica constitutes a promising catalyst due to its high regio- and stereoselectivity in the hydroxylation of different steroids as well as its efficient expression in E. coli at high yields. Using this enzyme, 16?-hydroxylated steroids, which are important precursors for the synthesis of high value steroidal drugs in the pharmaceutical industry, can be selectively produced on preparative scale with TTN (?mol substrate consumed ?mol(-1) CYP154C5) exceeding 2000.

Dataset Information

Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants.

Publications

Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants.

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