Ning Yike, Wu Xuejiao, Zhang Chenglin, Xu Qingyang, Chen Ning, Xie Xixian
National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, PR China; Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science and Technology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, PR China; Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science and Technology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
Metab Eng. 2016 Jul;36:10-18. doi: 10.1016/j.ymben.2016.02.013. Epub 2016 Mar 9.
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 4.9g/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 25.1g/L ectoine by fed-batch fermentation in low salt concentration with glucose as a carbon source. The specific ectoine production and productivity was 0.8g/g DCW and 0.84gL(-)(1)h(-)(1) respectively. The overall ectoine yield was 0.11g/g of glucose.
依克多因是一种保护剂和稳定剂，其合成途径仅存在于特定的中度嗜盐菌中。一种名为“细菌挤奶”的新型成熟工艺可在中度嗜盐菌中高效合成依克多因，然而，该方法对设备要求很高且成本高昂。在本研究中，我们通过将依克多因合成途径引入大肠杆菌构建了一株产依克多因的菌株，并提高了其生产能力。首先，将来自伸长盐单胞菌的ectABC基因簇引入大肠杆菌W3110，所得菌株在无高渗透压的情况下合成了4.9g/L依克多因。随后，删除编码双功能天冬氨酸激酶/高丝氨酸脱氢酶的thrA以削弱竞争性L-苏氨酸分支，导致依克多因产量提高了109%。此外，引入来自谷氨酸棒杆菌的反馈抗性lysC编码天冬氨酸激酶，以弥补因thrA缺失导致的酶活性不足，依克多因产量提高了9%。最后，将编码磷酸烯醇丙酮酸羧化酶的ppc启动子替换为trc启动子，并删除乙醛酸分流转录阻遏基因iclR。由此增强了草酰乙酸库，依克多因产量提高了21%。最终的工程菌株ECT05（pTrcECT，pSTVLysC-CG）以葡萄糖为碳源，在低盐浓度下通过分批补料发酵产生了25.1g/L依克多因。依克多因的比产量和生产率分别为0.8g/g干细胞重量和0.84gL⁻¹h⁻¹。依克多因的总产率为0.11g/g葡萄糖。
