Enhanced production of taxadiene in Saccharomyces cerevisiae

Abstract

Background 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. 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. Results Multi-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. Conclusions The 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.

Developing a Yeast Platform Strain for an Enhanced Taxadiene Biosynthesis by CRISPR/Cas9

“…Most of the previous studies on improving taxadiene production in yeast did not use BTS1 overexpression since BTS1 , like human GGPPS , is active only when FPP is present and may compete with squalene synthase for the common substrate, FPP [ 24 , 25 , 26 , 27 ]. However, we had overexpressed BTS1 to enhance diterpenes production in yeast and a significant increase in diterpene production was observed [ 35 ].…”

“…Production of taxadiene in S . cerevisiae has been previously attempted [ 23 , 24 , 25 , 26 ]. The first attempt was performed by the Croteau lab and successfully yielded 1 mg/L taxadiene.…”

“…The first attempt was performed by the Croteau lab and successfully yielded 1 mg/L taxadiene. Currently, the highest reported titer of taxadiene production is ~57 mg/L, which was achieved by optimizing TS expression and providing multi-copies of TS in the genome [ 26 ]. Since S .…”

“…cerevisiae does not synthesize diterpenoid-derived metabolites, one essential modification is enhancing the production of GGPP [ 27 ]. Previous studies utilized characterized geranylgeranyl diphosphate synthase ( GGPPS ) from Taxus spp., archaea, and carotenoid-producing bacteria ( Escherichia vulneris ) [ 24 , 25 , 26 ]. However, different GGPPSs have not been comparatively examined to enhance taxadiene production in yeast.…”

Sustainable Production of Microbial Isoprenoid Derived Advanced Biojet Fuels Using Different Generation Feedstocks: A Review

“…They have a vast range of applications in the pharmaceutical, fragrance and fuel industries and have enriched the everyday lives of humans for millennia (Peralta-Yahya et al, 2011;George et al, 2015). Isoprenoid research predominantly focused on medicinal applications such as the construction of microbial pathways for the ground-breaking antimalarial (Paddon and Keasling, 2014) and anticancer drugs (Wong et al, 2018;Nowrouzi et al, 2020;Walls et al, 2020). However, the aforementioned introduction of stringent greenhouse gas emission reduction targets in recent years has increased interest in their potential as advanced biofuels (Peralta-Yahya et al, 2012;George et al, 2015;Wong et al, 2017).…”