Metabolic engineering of Corynebacterium glutamicum for shikimate overproduction by growth-arrested cell reaction |
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| Institution: | 1. Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan;2. Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan;1. Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany;2. Technology Platform Genomics, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany;1. Department of Agricultural and Life Sciences, Faculty of Agriculture, Shinshu University, Nagano 399-4598, Japan;2. Bioprocess Development Center, Kyowa Hakko Bio Co., Ltd., Tsukuba, Ibaraki 305-0841, Japan;1. Laboratory of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China;2. Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China;1. Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Environmental Microbial Technology Center of Hubei Province, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China;2. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, PR China;3. Department of Chemistry, The State University of New York College of Environmental Science and Forestry (SUNY-ESF), Syracuse, NY 13210, USA |
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| Abstract: | Corynebacterium glutamicum with the ability to simultaneously utilize glucose/pentose mixed sugars was metabolically engineered to overproduce shikimate, a valuable hydroaromatic compound used as a starting material for the synthesis of the anti-influenza drug oseltamivir. To achieve this, the shikimate kinase and other potential metabolic activities for the consumption of shikimate and its precursor dehydroshikimate were inactivated. Carbon flux toward shikimate synthesis was enhanced by overexpression of genes for the shikimate pathway and the non-oxidative pentose phosphate pathway. Subsequently, to improve the availability of the key aromatics precursor phosphoenolpyruvate (PEP) toward shikimate synthesis, the PEP: sugar phosphotransferase system (PTS) was inactivated and an endogenous myo-inositol transporter IolT1 and glucokinases were overexpressed. Unexpectedly, the resultant non-PTS strain accumulated 1,3-dihydroxyacetone (DHA) and glycerol as major byproducts. This observation and metabolome analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-catalyzed reaction as a limiting step in glycolysis. Consistently, overexpression of GAPDH significantly stimulated both glucose consumption and shikimate production. Blockage of the DHA synthesis further improved shikimate yield. We applied an aerobic, growth-arrested and high-density cell reaction to the shikimate production by the resulting strain and notably achieved the highest shikimate titer (141 g/l) and a yield (51% (mol/mol)) from glucose reported to date after 48 h in minimal medium lacking nutrients required for cell growth. Moreover, comparable shikimate productivity could be attained through simultaneous utilization of glucose, xylose, and arabinose, enabling efficient shikimate production from lignocellulosic feedstocks. These findings demonstrate that C. glutamicum has significant potential for the production of shikimate and derived aromatic compounds. |
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| Keywords: | Shikimic acid Shikimate pathway Metabolic engineering Oseltamivir Phosphoenolpyruvate |
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