Dataset Information

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.

SUBMITTER: Nowrouzi B

PROVIDER: S-EPMC7607689 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Enhanced production of taxadiene in Saccharomyces cerevisiae.

Nowrouzi Behnaz B Li Rachel A RA Walls Laura E LE d'Espaux Leo L Malcı Koray K Liang Lungang L Jonguitud-Borrego Nestor N Lerma-Escalera Albert I AI Morones-Ramirez Jose R JR Keasling Jay D JD Rios-Solis Leonardo L

Microbial cell factories 20201102 1

<h4>Background</h4>Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol<sup>®</sup>), 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.<h4>Results</h4>Multi-copy chromosomal integration of ...[more]

PMID: 33138820

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Project description:Replacement of petrochemical-based materials with microbially produced biodegradable alternatives calls for industrially attractive fermentation processes. Lignocellulosic materials offer non-edible alternatives for cultivated sugars, but require often use of expensive sugar releasing enzymes, such as β-glucosidases. These cellulose treatment costs could be reduced if microbial production hosts could use short cellodextrins such as cellobiose directly as their substrates. In this study, we demonstrate production of poly(hydroxybutyrate) (PHB) in yeast Saccharomyces cerevisiae using cellobiose as a sole carbon source. Yeast strains expressing PHB pathway genes from Cupriavidus necator and cellodextrin transporter gene CDT-1 from Neurospora crassa were complemented either with β-glucosidase gene GH1-1 from N. crassa or with cellobiose phosphorylase gene cbp from Ruminococcus flavefaciens. These cellobiose utilization routes either with Gh1-1 or Cbp enzymes differ in energetics and dynamics. However, both routes enabled higher PHB production per consumed sugar and higher PHB accumulation % of cell dry weight (CDW) than use of glucose as a carbon source. As expected, the strains with Gh1-1 consumed cellobiose faster than the strains with Cbp, both in flask and bioreactor batch cultures. In shake flasks, higher final PHB accumulation % of CDW was reached with Cbp route (10.0 ± 0.3%) than with Gh1-1 route (8.1 ± 0.2%). However, a higher PHB accumulation was achieved in better aerated and pH-controlled bioreactors, in comparison to shake flasks, and the relative performance of strains switched. In bioreactors, notable PHB accumulation levels per CDW of 13.4 ± 0.9% and 18.5 ± 3.9% were achieved with Cbp and Gh1-1 routes, respectively. The average molecular weights of accumulated PHB were similar using both routes; approximately 500 kDa and 450 kDa for strains expressing either cbp or GH1-1 genes, respectively. The formation of PHB with high molecular weights, combined with efficient cellobiose conversion, demonstrates a highly potential solution for improving attractiveness of sustainable polymer production using microbial cells.

Dataset Information

Enhanced production of taxadiene in Saccharomyces cerevisiae.

Publications

Enhanced production of taxadiene in Saccharomyces cerevisiae.

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