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.

Keywords: Minibioreactor; Paclitaxel; Saccharomyces cerevisiae; Taxadiene synthase; Taxol™; Yeast metabolic engineering.

Fig. 1

Engineered taxadiene biosynthetic pathway in S. cerevisiae. Genes highlighted in red represent native genes which have been overexpressed through the integration of one additional copy. Genes highlighted in blue are exogenous genes which were heterologously expressed. Hydroxymethylglutaryl-CoA synthase (mvaS) and Acetyl-CoA acetyltransferase (mvaE) from Enterococcus faecalis. Geranylgeranyl diphosphate synthase (crtE) from Xanthophyllomyces dendrorhous and Taxadiene synthase (TASY) from Taxus cuspidata

Fig. 2

Optimisation of taxadiene titres using episomal TASY expression. a Effect of promoter on taxadiene concentration produced by cells incubated at 30 °C. b Effect of cultivation temperature on taxadiene titre. c Effect of cofactor (Mg²⁺) availability at 20 and 30 °C. d Effect of gene truncation length for taxadiene production at 20 °C. Values shown are mean ± standard deviation for 96-h triplicate cultivations

Fig. 3

Chromosomally integrated TASY genetic constructs. From top to bottom are TASY cassettes integrated at loci: ARS1114a, ARS1014a, and ARS1622b, respectively. The genetic designs were visualized by SBOLDesigner 2 [41]

Fig. 4

Summary of taxadiene titers microscale optimisation study. LRS1 indicates titers resulting from episomal TASY expression using the high copy 2-micron plasmid (LRS1) at 30 and 20 °C, respectively. The other titers are from strains with chromosomally integrated TASY variants: LRS2 (TDH3p-TASY-ADH1t), LRS3 (TASY-GFP), LRS4 (TASY-GFP; MBP-TASY-ERG20*) and LRS5 (TASY-GFP; MBP-TASY-ERG20*; MBP-TASY-ERG20*), respectively, grown at 30 °C

Fig. 5

Effect of temperature on LRS5 performance in shake flasks. LRS5 was cultivated in 250 mL shake flasks in YPG media at 20 or 30 °C. Taxane accumulation (a) and yeast growth (b) were evaluated after 72 h of cultivation. Error bars represent ± standard deviation for triplicate cultivations

Fig. 6

LRS5 gas chromatogram. Results show products produced by LRS5 during the 30 °C shake flask cultivation

Fig. 7

Bioreactor studies results and respective taxane concentration kinetics at 20 °C (a, b) and at 30 °C (c, d). LRS5 was cultivated in an Applikon MiniBio 500 bioreactor in yeast extract (1%), peptone (2%), galactose (2%). The pH and dissolved oxygen were monitored online and controlled to set points of 6 and 30%, respectively online

Fig. 8

Kinetics of intracellular and total taxadiene titres in high-yielding 20 °C bioreactor cultivation