Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH |
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| Institution: | 1. State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China;2. School of Life Science, Linyi University, Linyi 276000, China;3. School of Energy and Environment, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong SAR;1. School of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China;2. College of Life and Environmental Sciences, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, China;3. Shanghai Laiyi Center for Biopharmaceutical R&D, 800 Dongchuan Road, Shanghai 200240, China;4. George Stevens Academy, 23 Union St, Blue Hill, ME 04614, USA;5. Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Road, Shanghai 200040, China;6. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China;7. Shanghai Research and Development Center of Industrial Biotechnology, 528 Ruiqing Road, Shanghai 201201, China;8. Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), 200 North Zhongshan Road, Nanjing 211816, China;1. Department of Biotechnology and Food Microbiology, Wroc?aw University of Environmental and Life Sciences, Che?mońskiego 37, Wroc?aw 51-630, Poland;2. Institute of Environmental Engineering, Wroc?aw University of Environmental and Life Sciences, Grunwaldzki Sq 24, Wroc?aw 50-363, Poland |
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| Abstract: | Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7 g/L succinic acid with a yield of 0.53 g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects. |
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| Keywords: | Succinic acid production Acetic acid overflow Acetate: succinate CoA-transferase |
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