Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields |
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| Institution: | 1. Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072, PR China;2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China;3. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China;1. Beijing Bioprocess Key Laboratory, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, PR China;2. Amoy - BUCT Industrial Bio-technovation Institute, Amoy 361022, PR China;1. TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, PR China;2. Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China;3. Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin 300457, PR China;4. Key Laboratory of System Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, PR China;5. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China;6. University of Chinese Academy of Sciences, Beijing 100039, PR China;1. School of Agriculture, Yunnan University, Kunming 650500, China;2. Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, Yunnan Minzu University, Kunming 650031, China;3. School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China;4. Dehong Tropical Agriculture Research Institute, Dehong 678600, China;1. Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China;2. Center for Systems Biology, University of Iceland, Reykjavik, Iceland |
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| Abstract: | As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production. |
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| Keywords: | Site-specific integration Constitutive expression Shikimic acid |
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