Selection of an endogenous 2,3-butanediol pathway in Escherichia coli by fermentative redox balance |
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| Affiliation: | 1. Key Laboratory of Environment Correlative Dietology (Ministry of Education), College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China;2. Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Sciences, Hubei University, Wuhan 430062, China;3. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China;4. Department of Food Science and Human Nutrition, Iowa State University, 50011 Ames, IA, USA;1. State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People''s Republic of China;2. Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao Tong University, Shanghai 200240, People''s Republic of China;3. Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, People''s Republic of China;1. Synthetic Biology and Biofuels Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India;2. DBT-ICGEB Centre for Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology, New Delhi, India;1. Key Laboratory of Systems Bioengineering, Ministry of Education (Tianjin University), Tianjin 300072, People’s Republic of China;2. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China;3. Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, People’s Republic of China |
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| Abstract: | Fermentative redox balance has long been utilized as a metabolic evolution platform to improve efficiency of NADH-dependent pathways. However, such system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. In this study, endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. In this case, feeding of the 2,3-BD precursor acetoin effectively restored anaerobic growth of the mixed-acid fermentation mutant that remained otherwise inhibited even in the presence of a functional 2,3-BD pathway. Using established 2,3-BD dehydrogenases as model enzyme, we verified that the redox-based selection system is responsive to NADPH-dependent reactions but with lower sensitivity. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol/1,2-propanediol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity (kcat/Km≈0.4 mM−1 s−1) observed in E. coli. Significant shift of 2,3-BD configuration upon withdrawal of the heterologous acetolactate decarboxylase revealed that the endogenous synthesis of acetoin occurs via diacetyl. Among the predicted diacetyl reductase in E. coli, Ec-UcpA displayed the most significant activity towards diacetyl reduction into acetoin (Vmax≈6 U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3 g/L without introduction of foreign genes. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme bioprospecting and metabolic evolution. |
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| Keywords: | Fermentative growth selection Redox balance Metabolic engineering 2,3-butanediol |
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