Pathway construction and metabolic engineering for fermentative production of ectoine in Escherichia coli |
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| Affiliation: | 1. National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, PR China;2. Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science and Technology, Tianjin 300457, PR China;3. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, 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 Microbial Engineering of China Light Industry, Tianjin University of Science and Technology, Tianjin 300457, China;3. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China;1. Graduate School of Biotechnology and Bioengineering, Yuan Ze University, No. 135 Yuan-Tung Rd., Chung-Li Dist., Taoyuan City 32003, Taiwan, ROC;2. Department of Applied Chemistry and Materials Science, Fooyin University, No. 151 Jinxue Rd., Daliao Dist., Kaohsiung City 83102, Taiwan, ROC;3. Department of Chemical Engineering and Materials Science, Yuan Ze University, No. 135 Yuan-Tung Rd., Chung-Li Dist., Taoyuan City 32003, Taiwan, ROC;4. Department & Graduate Institute of Chemical Engineering, Ming Chi University of Technology, No. 84 Gungjuan Rd., Taishan Dist., New Taipei City 24301, Taiwan, ROC;1. Biorefinery and Bioprocess Engineering Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan 32003, Taiwan;2. Faculty of Applied Sciences, UCSI University, UCSI Heights, Cheras, Kuala Lumpur 56000, Malaysia;3. Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan 32003, Taiwan;1. State Key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China;2. Huzhou University, College of Life Science, Huzhou, China;3. School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, Singapore |
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| Abstract: | Ectoine is a protective agent and stabilizer whose synthesis pathway exclusively exists in select moderate halophiles. A novel established process called “bacterial milking” efficiently synthesized ectoine in moderate halophiles, however, this method places high demands on equipment and is cost prohibitive. In this study, we constructed an ectoine producing strain by introducing the ectoine synthesis pathway into Escherichia coli and improved its production capacity. Firstly, the ectABC gene cluster from Halomonas elongata was introduced into E. coli W3110 and the resultant strain synthesized 4.9 g/L ectoine without high osmolarity. Subsequently, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was deleted to weaken the competitive l-threonine branch, resulting in an increase of ectoine titer by 109%. Furthermore, a feedback resistant lysC from Corynebacterium glutamicum encoding the aspartate kinase was introduced to complement the enzymatic activity deficiency caused by thrA deletion and a 9% increase of ectoine titer was obtained. Finally, the promoter of ppc that encodes phosphoenolpyruvate carboxylase was replaced by a trc promoter, and iclR, a glyoxylate shunt transcriptional repressor gene, was deleted. The oxaloacetate pool, was thus reinforced and ectoine titer increased by 21%. The final engineered strain ECT05 (pTrcECT, pSTVLysC-CG) produced 25.1 g/L ectoine by fed-batch fermentation in low salt concentration with glucose as a carbon source. The specific ectoine production and productivity was 0.8 g/g DCW and 0.84 g L−1 h−1 respectively. The overall ectoine yield was 0.11 g/g of glucose. |
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| Keywords: | Ectoine Aspartokinase Phosphoenolpyruvate carboxylase Glyoxylate shunt |
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