Enhanced production of taxadiene in Saccharomyces cerevisiae

Behnaz Nowrouzi,Jose R Morones-Ramirez,Leonardo Rios-Solis,Laura E Walls,Leo D’Espaux,Albert I Lerma-Escalera,Jay D Keasling,Lungang Liang,Nestor Jonguitud-Borrego,Koray Malcı,Rachel A Li

doi:10.1186/s12934-020-01458-2

Abstract

BackgroundCost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck.ResultsMulti-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at microscale, compared to expressing a single episomal copy of TASY. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively at 20 and 30 °C. Maximum taxadiene titres of 129 ± 15 mg/L and 127 mg/L were achieved through shake flask and bioreactor cultivations, respectively, of the optimal strain at a reduced temperature of 20 °C.ConclusionsThe results of this study highlight the benefit of employing a combination of molecular biology and bioprocess tools during synthetic pathway development, with which TASY activity was successfully improved by 6.5-fold compared to the highest literature titre in S. cerevisiae cell factories.

Highlights

Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands
This study reports the successful optimisation of taxadiene biosynthesis in a Saccharomyces cerevisiae microbial chassis
Low expression and poor solubility of taxadiene synthase (TASY) were identified as critical bottlenecks. This was alleviated through multi-copy chromosomal integration of TASY with a combination of fusion protein tags, improving taxadiene titres 22-fold to 57 ± 3 mg/L at 30 °C

Summary

Introduction

Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. Nowrouzi et al Microb Cell Fact (2020) 19:200 The reconstitution of this enzymatic step has been successfully achieved in both E. coli [6, 7] and S. cerevisiae [8, 9] (Table 1). When the authors expressed the subsequent enzyme, taxadien-5α-hydroxylase, which is a membrane-bound cytochrome P450, a tenfold reduction in total taxane titre was observed. Membrane-bound cytochrome P450s like taxadien-5α-hydroxylase are estimated to comprise around half of the 19 enzymatic steps in the paclitaxel biosynthetic pathway [10]. As the overexpression of such membrane-bound enzymes is greatly

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Conclusion