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bioRxiv · 10.1101/2020.06.08.139600

Enhanced production of taxadiene in Saccharomyces cerevisiae

Nowrouzi, B.·Li, R.·Walls, L. E.·d'Espaux, L.·Malci, K.·Liang, L.·Jonguitud Borrego, N.·Lerma Escalera, A. I.·Morones-Ramirez, J. R.·Keasling, J. D.·Rios Solis, L.
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Abstract

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. 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 shake flask scale. 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. Maximum taxadiene titres of 129 ± 15 mg/L and 119 mg/L were achieved through shake flask and bioreactor cultivation, respectively, of the optimal strain at a reduced temperature of 20 °C. The results highlight the positive effect of coupling molecular biology tools with bioprocess variable optimisation on synthetic pathway development.HighlightsMaximum taxadiene titre of 129 ± 15 mg/L in Saccharomyces cerevisiae at 20 °CIntegrating fusion protein tagged-taxadiene synthase improved taxadiene titre.Consistent taxadiene titres were achieved at the micro-and mini-bioreactor scales.Competing Interest StatementJ.D.K. has financial interests in Amyris, Lygos, Demetrix, Napigen, Maple Bio, Apertor Labs, Ansa Biotechnologies, and Berkeley Brewing Sciences.AbbreviationsBTS1Geranylgeranyl diphosphate synthasecrtEGeranylgeranyl diphosphate synthaseDMAPPDimethylallyl pyrophosphateERG8Phosphomevalonate kinaseERG9Farnesyl-diphosphate farnesyl transferase (squalene synthase)ERG103-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthaseERG12Mevalonate kinaseERG133-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthaseERG19Mevalonate pyrophosphate decarboxylaseERG20Farnesyl pyrophosphate synthetaseFPPFarnesyl diphosphateGGOH(E,E,E)-geranylgeraniolGGPP(E,E,E)-Geranylgeranyl diphosphateGPPGeranyl diphosphateHMG13-hydroxy-3-methylglutaryl-coenzyme A reductase 1HMG23-hydroxy-3-methylglutaryl-coenzyme A reductase 2IDIIsopentenyl-diphosphate delta-isomeraseIPPIsopentenyl pyrophosphateMBPMaltose binding proteinmvaEAcetyl-CoA acetyltransferasemvaSHydroxymethylglutaryl-CoA synthaseMVA pathwayMevalonate pathwayTASYTaxadiene synthase.View Full Text

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Nowrouzi, B., Li, R., Walls, L. E., d'Espaux, L., Malci, K., Liang, L., Jonguitud Borrego, N., Lerma Escalera, A. I., Morones-Ramirez, J. R., Keasling, J. D., Rios Solis, L.. 2020-06-08. Enhanced production of taxadiene in Saccharomyces cerevisiae. https://doi.org/10.1101/2020.06.08.139600

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