Dynamic control of the mevalonate pathway expression for improved zeaxanthin production in Escherichia coli and comparative proteome analysis |
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| Institution: | 1. Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China;2. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;1. Joint BioEnergy Institute, 5885 Hollis St., Emeryville, CA 94608, USA;2. Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA;3. Department of Bioengineering and Biotechnology, Sandia National Laboratory, Livermore, CA, USA;4. Department of Bioengineering, University of California, Berkeley, CA, USA;5. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, USA;6. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Alle, DK2970 Hørsholm, Denmark;1. Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China;2. Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University, Hangzhou 310027, PR China;1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China;2. Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, Hubei Collaborative Innovation Center of Industrial Fermentation, Hubei University of Technology, Wuhan 430068, PR China;3. Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai 200237, PR China;1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, PR China;2. J1 Biotech Co., Ltd., Wuhan 430075, PR China;3. Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, PR China;4. Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan 430068, PR China |
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| Abstract: | Engineered heterologous multi-gene metabolic pathways often suffer from flux imbalance and toxic metabolites, as the production host typically lacks the regulatory mechanisms for the heterologous pathway. Here, we first coordinated the expression of all genes of the mevalonate (MEV) pathway from Saccharomyces cerevisiae using the tunable intergenic regions (TIGRs), and then dynamically regulated the TIGR-mediated MEV pathway to prevent the accumulation of toxic metabolites by using IPP/FPP-responsive promoter. After introduction of the dynamically controlled TIGR-mediated MEV pathway into Escherichia coli, the content and concentration of zeaxanthin in shaker flask cultures were 2.0- and 2.1-fold higher, respectively, than those of the strain harboring the statically controlled non-TIGR-mediated MEV pathway. The content and concentration of zeaxanthin in E. coli ZEAX (pZSPgadE-MevTTIGR-MevBTIGRIS-2) reached 722.46 mg/L and 23.16 mg/g dry cell weight (DCW), respectively, in 5.0 L fed-batch fermentation. We also comparatively analyzed the proteomes between E. coli ZEAX and E. coli ZEAX (pZSPgadE-MevTTIGR-MevBTIGRIS-2) to understand the mechanism of zeaxanthin biosynthesis. The results of the comparative proteomes demonstrate that zeaxanthin overproduction may be associated with increased precursor availability, increased NADPH availability, increased ATP availability, oxidative stress response, and increased membrane storage capacity for zeaxanthin due to changes in both cellular shape and membrane composition. |
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| Keywords: | Zeaxanthin TIGR Dynamic regulation Proteome |
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