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Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline
Institution:1. Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin University of Science & Technology, Tianjin, 300457, PR China;2. College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, PR China;3. College of Food Science and Engineering, Tianjin University of Science & Technology, Tianjin, 300457, PR China;4. Ningxia Eppen Biotech Co, Ltd, Ningxia, 750000, PR China;1. National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, China;2. Key Laboratory of Industrial Microbiology of the Ministry of Education, Tianjin 300457, China;3. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China;1. Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, Jülich D-52425, Germany;2. Center for Structural Studies (CSS), Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225, Düsseldorf, Germany;3. Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225, Düsseldorf, Germany;4. Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225, Düsseldorf, Germany;5. Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, Jülich D-52425, Germany;1. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China;2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China;3. Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China;4. Biotechnology Institute, Laiwu College for Vocational Technology, Laiwu, Shandong 271100, China;1. Institute of Synthetic Biology, Biomedical Center, Guangdong Province Key Laboratory of Improved Variety Reproduction in Aquatic Economic Animals, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China;2. School of Food Science and Engineering, South China University of Technology, Guangzhou, 510641, China;1. State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China;2. School of Chemical, Materials and Biomedical Engineering, College of Engineering, University of Georgia, Athens, GA 30602, USA
Abstract:L-citrulline is a high-value amino acid with promising application in medicinal and food industries. Construction of highly efficient microbial cell factories for L-citrulline production is still an open issue due to complex metabolic flux distribution and L-arginine auxotrophy. In this study, we constructed a nonauxotrophic cell factory in Escherichia coli for high-titer L-citrulline production by coupling modular engineering strategies with dynamic pathway regulation. First, the biosynthetic pathway of L-citrulline was enhanced after blockage of the degradation pathway and introduction of heterologous biosynthetic genes from Corynebacterium glutamicum. Specifically, a superior recycling biosynthetic pathway was designed to replace the native linear pathway by deleting native acetylornithine deacetylase. Next, the carbamoyl phosphate and L-glutamate biosynthetic modules, the NADPH generation module, and the efflux module were modified to increase L-citrulline titer further. Finally, a toggle switch that responded to cell density was designed to dynamically control the expression of the argG gene and reconstruct a nonauxotrophic pathway. Without extra supplement of L-arginine during fermentation, the final CIT24 strain produced 82.1 g/L L-citrulline in a 5-L bioreactor with a yield of 0.34 g/g glucose and a productivity of 1.71 g/(L ? h), which were the highest values reported by microbial fermentation. Our study not only demonstrated the successful design of cell factory for high-level L-citrulline production but also provided references of coupling the rational module engineering strategies and dynamic regulation strategies to produce high-value intermediate metabolites.
Keywords:L-citrulline  Recycling biosynthetic pathway  Dynamic regulation  Quorum sensing
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