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Generate a bioactive natural product library by mining bacterial cytochrome P450 patterns.

ABSTRACT: The increased number of annotated bacterial genomes provides a vast resource for genome mining. Several bacterial natural products with epoxide groups have been identified as pre-mRNA spliceosome inhibitors and antitumor compounds through genome mining. These epoxide-containing natural products feature a common biosynthetic characteristic that cytochrome P450s (CYPs) and its patterns such as epoxidases are employed in the tailoring reactions. The tailoring enzyme patterns are essential to both biological activities and structural diversity of natural products, and can be used for enzyme pattern-based genome mining. Recent development of direct cloning, heterologous expression, manipulation of the biosynthetic pathways and the CRISPR-CAS9 system have provided molecular biology tools to turn on or pull out nascent biosynthetic gene clusters to generate a microbial natural product library. This review focuses on a library of epoxide-containing natural products and their associated CYPs, with the intention to provide strategies on diversifying the structures of CYP-catalyzed bioactive natural products. It is conceivable that a library of diversified bioactive natural products will be created by pattern-based genome mining, direct cloning and heterologous expression as well as the genomic manipulation.

SUBMITTER: Liu X

PROVIDER: S-EPMC5640691 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Generate a bioactive natural product library by mining bacterial cytochrome P450 patterns.

Liu Xiangyang X

Synthetic and systems biotechnology 20160226 2

The increased number of annotated bacterial genomes provides a vast resource for genome mining. Several bacterial natural products with epoxide groups have been identified as pre-mRNA spliceosome inhibitors and antitumor compounds through genome mining. These epoxide-containing natural products feature a common biosynthetic characteristic that cytochrome P450s (CYPs) and its patterns such as epoxidases are employed in the tailoring reactions. The tailoring enzyme patterns are essential to both b ...[more]

PMID: 29062932

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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

Generate a bioactive natural product library by mining bacterial cytochrome P450 patterns.

Publications

Generate a bioactive natural product library by mining bacterial cytochrome P450 patterns.

Similar Datasets

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