Characterization of P450 FcpC, the Enzyme Responsible for Bioconversion of Diosgenone to Isonuatigenone in Streptomyces virginiae IBL-14

Abstract

A new cytochrome P450 monooxygenase, FcpC, from Streptomyces virginiae IBL-14 has been identified. This enzyme is found to be responsible for the bioconversion of a pyrano-spiro steroid (diosgenone) to a rare nuatigenin-type spiro steroid (isonuatigenone), which is a novel C-25-hydroxylated diosgenone derivative. A whole-cell P450 system was developed for the production of isonuatigenone via the expression of the complete three-component electron transfer chain in an Escherichia coli strain.Nuatigenin-type steroids, such as nuatigenin and isonuatigenin (9, 13, 22), are rare natural steroidal sapogenins that are important pharmacological compounds. They are found in several healthy foods and traditional medicinal herbs. These compounds have been shown to have potential anticancer effects, antagonistic effects on rheumatoid arthritis, beneficial cardiovascular activities, and antimalarial activities. Examples include ophiofurospiside in Ophiopogon japonicus (28), nuatigenosido in Solanum sisymbriifolium (13), avenacoside in oat (20), and glycosides in Paris polyphylla SM (7). Since the majority of these nuatigenin-type steroids are very rare, strategies for their isolation can lead to very high production costs. As a result, with a more economical production process in mind, it would be worthwhile to search for a suitable reagent capable of converting the abundant amounts of pyrano-spirostanol sapogenins found in nature, such as diosgenone, to rare nuatigenintype steroids. At this time, we plan to focus on microbial transformation systems.

Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function

“…31–35 One advantage of bacterial P450s, and those from Streptomyces in particular, is the inherent flexibility of the P450s to accept electrons from heterologous redox partners. While each P450 behaves differently, streptomycete P450s have been shown to accept redox proteins from other Streptomyces species, 36–38 other bacterial genera including putidaredoxin reductase and putidaredoxin from the P450 cam system in Pseudomonas putida 39,40 and flavodoxin reductase and flavodoxin from Escherichia coli , 41,42 and eukaryotes such as the commercially available spinach ferredoxin reductase and ferredoxin. 43,44 Inspired by the one-component systems, Streptomyces P450s have also been engineered into redox self-sufficient enzymes by the fusion of the P450 protein to the reductase domains of P450 RhF 45–47 or CYP102D1.…”

CYP105-diverse structures, functions and roles in an intriguing family of enzymes inStreptomyces

“…) (Wang et al . , ). CYP105D7, involved in pentalenic acid biosynthesis in S. avermitilis , has also been shown to catalyse regiospecific hydroxylation of isoflavones such as daidzein both in vitro and in vivo (Pandey et al .…”

Identification and functional analysis of cytochrome P450 complement in Streptomyces virginiaeIBL14

“…The cytochrome P450 Svh01 (responsible for the C25-hydroxylation of diosgenin) [32] belongs to the class I (prokaryotic/mitochondrial) P450 system based on a taxonomic split, in which electrons are transferred from NADPH or NADH to ferredoxin reductase and ferredoxin. Sequence analysis revealed the complete sequence of svh 01 with ATG as the start codon has 70% G + C content.…”

“…From the SDS-PAGE, we can find that the two distinctly additional protein bands should be Svh01 with an about MW of 44 kDa and Svf09 with an about MW of 8.0 kDa, respectively. The further functional identification of the Svh01/FcpC of S. virginiae IBL14, hydroxylating the C25-tertiary carbon of diosgenin to form isonuatigenone, was experimentally confirmed [32]. …”