A modular engineering strategy for high-level production of protopanaxadiol from ethanol by Saccharomyces cerevisiae |
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Authors: | Fanglong Zhao Peng Bai Weihua Nan Dashuai Li Chuanbo Zhang Chunzhe Lu Haishan Qi Wenyu Lu |
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Affiliation: | Dept. of Biological Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, P.R. China |
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Abstract: | Ethanol is a more reduced substrate than sugars. Here, 13C-metabolic flux analysis (MFA) revealed that ethanol catabolism could supply sufficient acetyl-CoA and reducing equivalent for PPD biosynthesis. Then, we described modular engineering strategy to optimize a multigene pathway for protopanaxadiol (PPD) production from ethanol in Saccharomyces cerevisiae. PPD biosynthesis was divided into four modules: mevalonate (MVA) pathway module, triterpene biosynthesis module, sterol biosynthesis module, and acetyl-CoA formation module. Combinatorially overexpressing every gene in MVA pathway and optimizing metabolic balance in triterpene biosynthesis module led to significantly enhanced PPD production (42.34 mg/L/OD600). In sterol biosynthesis module, fine-tuning lanosterol synthase gene (ERG7) expression using TetR–TetO gene regulation system enabled further production improvement (51.26 mg/L/OD600). Furthermore, increasing cytoplasmic acetyl-CoA supply by overexpressing a Salmonella ACS (acetyl-CoA synthetase gene) mutant ACSseL641P improved PPD production to 66.55 mg/L/OD600. In 5 L bioreactor, PPD production of the best-performing strain WLT-MVA5 reached 8.09 g/L, which has been the highest titer of plant triterpene produced in yeast. © 2018 American Institute of Chemical Engineers AIChE J, 65: 866–874, 2019 |
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Keywords: | protopanaxadiol Saccharomyces cerevisiae 13C-metabolic flux analysis metabolic engineering synthetic biology |
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