Construction of an Escherichia coli cell factory to synthesize taxadien-5α-ol, the key precursor of anti-cancer drug paclitaxel

Abstract

Paclitaxel (Taxol™), an alkaloid of diterpenoid family, is one of the most widely used anti-cancer drugs due to its effectiveness against a variety of tumors. Rather than directly extraction and chemical synthesis of paclitaxel or its intermediates from yew plants, construction of a microbial cell factory for paclitaxel biosynthesis will be more efficient and sustainable. The challenge for biosynthesis of paclitaxel lies on the insufficient precursor, such as taxadien-5α-ol. In this study, we report a recombinant Escherichia coli strain constructed with a heterologous mevalonate pathway, a taxadiene synthase from yew, and a cytochrome P450-mediated oxygenation system for the de novo production of taxadien-5α-ol, the first product of the multi-step taxadiene oxygenation metabolism. The key enzymes including taxadiene synthases and cytochrome P450 reductases were screened, and the linker for fusing taxadiene-5α-hydroxylase with its reductase partner cytochrome P450 reductase was optimized. By reducing the metabolic burden and optimizing the fermentation conditions, the final production of total oxygenated taxanes was raised up to 27 mg L⁻¹ in a 50-mL flask cultivation, of which the yield of taxadien-5α-ol was 7.0 mg L⁻¹, representing approximately a 12-fold and 23-fold improvements, respectively, as compared with the initial titers. The engineered MVA pathway for the overproduction of terpenoid precursors can serve as an efficient platform for the production of other valuable terpenoids.

Highlights

We report a recombinant Escherichia coli BL21(DE3) strain for de novo production of taxadien-5α-ol, the key precursor of paclitaxel.

Through screening of key enzymes and the fermentation condition optimization, the final production of total oxygenated taxanes was raised up to 27 mg L⁻¹ in 50-mL flask cultivation, of which the yield of taxadien-5α-ol was 7.0 mg L⁻¹, representing approximately a 12-fold and 23-fold improvements, respectively.

It is believed that the strategy used in this study will guide in the synthesis of terpenoids.