Biorefinery Laboratory, Shanghai Advanced Research Institute, Chinese Academy of Sciences, 99 Haike Road, Shanghai, 201210, China.
University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing, 100049, China.
Microb Cell Fact. 2020 Mar 14;19(1):66. doi: 10.1186/s12934-020-01323-2.
Microbial de novo production of L-serine, which is widely used in a range of cosmetic and pharmaceutical products, has attracted increasing attention due to its environmentally friendly characteristics. Previous pioneering work mainly focused on L-serine anabolism; however, in this study, it was found that L-serine could be reimported through the L-serine uptake system, thus hampering L-serine production.
To address this challenge, engineering via deletion of four genes, namely, sdaC, cycA, sstT and tdcC, which have been reported to be involved in L-serine uptake in Escherichia coli, was first carried out in the L-serine producer E. coli ES. Additionally, the effects of these genes on L-serine uptake activity and L-serine production were investigated. The data revealed an abnormal phenomenon regarding serine uptake activity. The serine uptake activity of the ΔsdaC mutant was 0.798 nmol min (mg dry weight) after 30 min, decreasing by 23.34% compared to that of the control strain. However, the serine uptake activity of the single sstT, cycA and tdcC mutants increased by 34.29%, 78.29% and 48.03%, respectively, compared to that of the control strain. This finding may be the result of the increased level of sdaC expression in these mutants. In addition, multigene-deletion strains were constructed based on an sdaC knockout mutant. The ΔsdaCΔsstTΔtdcC mutant strain exhibited 0.253 nmol min (mg dry weight) L-serine uptake activity and the highest production titer of 445 mg/L in shake flask fermentation, which was more than three-fold the 129 mg/L production observed for the parent. Furthermore, the ΔsdaCΔsstTΔtdcC mutant accumulated 34.8 g/L L-serine with a yield of 32% from glucose in a 5-L fermenter after 36 h.
The results indicated that reuptake of L-serine impairs its production and that an engineered cell with reduced uptake can address this problem and improve the production of L-serine in E. coli.
微生物从头合成 L-丝氨酸，由于其环保特性，广泛应用于各种化妆品和制药产品，越来越受到关注。以前的开创性工作主要集中在 L-丝氨酸的合成代谢上；然而，在这项研究中，发现 L-丝氨酸可以通过 L-丝氨酸摄取系统再输入，从而阻碍 L-丝氨酸的生产。
为了解决这一挑战，首先对 L-丝氨酸产生菌大肠杆菌 ES 中的四个基因（sdaC、cycA、sstT 和 tdcC）进行了缺失工程改造，这些基因已被报道参与大肠杆菌中的 L-丝氨酸摄取。此外，还研究了这些基因对 L-丝氨酸摄取活性和 L-丝氨酸生产的影响。数据显示丝氨酸摄取活性出现异常现象。ΔsdaC 突变体的丝氨酸摄取活性在 30 分钟后为 0.798 nmol min（mg 干重），比对照菌株下降 23.34%。然而，sstT、cycA 和 tdcC 突变体的丝氨酸摄取活性分别比对照菌株增加了 34.29%、78.29%和 48.03%。这一发现可能是由于这些突变体中 sdaC 表达水平的增加所致。此外，还基于 sdaC 敲除突变体构建了多基因缺失菌株。ΔsdaCΔsstTΔtdcC 突变株在摇瓶发酵中的 L-丝氨酸摄取活性为 0.253 nmol min（mg 干重），最高产量为 445mg/L，比亲本菌株的 129mg/L 产量高出三倍以上。此外，在 5L 发酵罐中，ΔsdaCΔsstTΔtdcC 突变株在 36h 内从葡萄糖中积累了 34.8g/L 的 L-丝氨酸，产率为 32%。
结果表明，L-丝氨酸的再摄取会损害其生产，而减少摄取的工程细胞可以解决这一问题，提高大肠杆菌中 L-丝氨酸的生产。
