Chloroplastic metabolic engineering coupled with isoprenoid pool enhancement for committed taxanes biosynthesis in Nicotiana benthamiana

Jianhua Li,Kaibo Wang,Lei Yang,Ishmael Mutanda,Yong Wang,Jiawei Wang

doi:10.1038/s41467-019-12879-y

Abstract

Production of the anticancer drug Taxol and its precursors in heterologous hosts is more sustainable than extraction from tissues of yew trees or chemical synthesis. Although attempts to engineer the Taxol pathway in microbes have made significant progress, challenges such as functional expression of plant P450 enzymes remain to be addressed. Here, we introduce taxadiene synthase, taxadiene-5α-hydroxylase, and cytochrome P450 reductase in a high biomass plant Nicotiana benthamiana. Using a chloroplastic compartmentalized metabolic engineering strategy, combined with enhancement of isoprenoid precursors, we show that the engineered plants can produce taxadiene and taxadiene-5α-ol, the committed taxol intermediates, at 56.6 μg g−1 FW and 1.3 μg g−1 FW, respectively. In addition to the tools and strategies reported here, this study highlights the potential of Nicotiana spp. as an alternative platform for Taxol production.

Highlights

Production of the anticancer drug Taxol and its precursors in heterologous hosts is more sustainable than extraction from tissues of yew trees or chemical synthesis
Full-length encoding sequences of taxadiene synthase (TS), T5αH, and CPR were amplified from the needles of Taxus chinensis and fused with FLAG, HA and c-Myc tags at their C-terminals, respectively
TS was detectable at 2 dpi and accumulated to its highest levels at 4 dpi followed by a slow decrease afterwards

Summary

Introduction

Production of the anticancer drug Taxol and its precursors in heterologous hosts is more sustainable than extraction from tissues of yew trees or chemical synthesis. An alternative approach is production by semi-synthesis from two key intermediates of Taxol: baccatin III and 10-deacetylbaccatin III, both still extracted from renewable needles of Taxus trees or cell cultures[11,12]. It is against this backdrop that great efforts have been directed toward synthetic production of committed Taxol intermediates through optimization of microbial hosts[13,14,15,16,17]. Unlike in aforementioned microbial systems where at least three oxygenated taxoids including 5(13)-oxa-3(11)-cyclotaxane (iso-OCT), 5(12)-oxa-3 (11)-cyclotaxane (OCT), and taxadiene-5α-ol have been produced in E. coli, co-expression of T5αH with TS in tobacco trichomes produced an unexpected OCT rather than taxadience-5α-ol[43]

Methods

Results

Conclusion