Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brev
- PDF / 3,150,295 Bytes
- 13 Pages / 595.276 x 790.866 pts Page_size
- 34 Downloads / 145 Views
Microbial Cell Factories Open Access
RESEARCH
Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea Junheng Liang1†, Huimin Wang1†, Xiaoying Bian2, Youming Zhang2, Guoping Zhao1,3 and Xiaoming Ding1*
Abstract Background: Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the market, having been approved by the FDA in 2007. The terminal step in epothilone B biosynthesis is catalysed by the cytochrome P450 enzyme EpoK (CYP167A1), which catalyses the epoxidation of the C12–C13 double bond (in epothilone C and D) to form epothilone A and B, respectively. Although redox partners from different sources support the catalytic activity of EpoK in vitro, the conversion rates are low, and these redox partners are not applied to produce epothilone B in heterologous hosts. Results: Schlegelella brevitalea DSM 7029 contains electron transport partners that efficiently support the catalytic activity of EpoK. We screened and identified one ferredoxin, Fdx_0135, by overexpressing putative ferredoxin genes in vivo and identified two ferredoxin reductases, FdR_0130 and FdR_7100, by whole-cell biotransformation of epothilone C to effectively support the catalytic activity of EpoK. In addition, we obtained strain H7029-3, with a high epothilone B yield and found that the proportion of epothilone A + B produced by this strain was 90.93%. Moreover, the whole-cell bioconversion strain 7029-10 was obtained; this strain exhibited an epothilone C conversion rate of 100% in 12 h. Further RT-qPCR experiments were performed to analyse the overexpression levels of the target genes. Gene knock-out experiments showed that the selected ferredoxin (Fdx_0135) and its reductases (FdR_0130 and FdR_7100) might participate in critical physiological processes in DSM 7029. Conclusion: Gene overexpression and whole-cell biotransformation were effective methods for identifying the electron transport partners of the P450 enzyme EpoK. In addition, we obtained an epothilone B high-yield strain and developed a robust whole-cell biotransformation system. This strain and system hold promise for the industrial production of epothilone B and its derivatives. Keywords: Epothilone B, EpoK (CYP167A1), Electron transport partner, Gene overexpression, Whole-cell biotransformation
*Correspondence: [email protected] † Junheng Liang and Huimin Wang contributed equally to this work 1 Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, Shanghai 200438, People’s Republic of China Full list of author information is available at the end of the article
Introduction Epothilones are microtubule-stabilizing antitumour compounds with a mechanism similar to t
Data Loading...
