Wang Jia, Jian Xingjin, Xing Xin-Hui, Zhang Chong, Fei Qiang
School of Chemical Engineering and Technology, Xi'an Jiaotong University, Xi'an, China.
Department of Chemical Engineering, Tsinghua University, Beijing, China.
Front Bioeng Biotechnol. 2020 Jul 9;8:570. doi: 10.3389/fbioe.2020.00570. eCollection 2020.
Recently, a methanol-essential was constructed; this strain is highly dependent on a supply of gluconate as a co-substrate for growth. Adaptive laboratory evolution is commonly applied to obtain mutants with specific phenotypes under certain selected pressure. However, conventional adaptive evolution approaches are not only laborious and time consuming, but they also come with lower throughput and inefficiency. In order to empower the aforementioned with reduced gluconate usage and enhanced growth rate, an irrational strategy based on a microbial microdroplet culture (MMC) platform was developed in this study. Given the automatic high-throughput selection of the MMC, a three-stage regime of an adaptive evolution experiment via gradually decreasing the availability of gluconate during the cultivation was performed for 50 days continuously in order to obtain the mutations. Finally, a candidate mutant was obtained with a 3-fold faster growth rate, a 43% shorter lag phase, and 40% less gluconate usage compared with the starting strain. Moreover, the gene mutations of , and were identified by analyzing the whole-genome sequencing of mutants, which are strongly associated with the efficiency of gluconate uptake and cell growth. In conclusion, we have successfully demonstrated the feasibility of using MMC platform to empower the target strain with specific requirements in a manner of labor, time efficiency, and directed evolution.
最近，构建了一种甲醇必需菌株；该菌株高度依赖葡萄糖酸盐作为共底物来供应以实现生长。适应性实验室进化通常用于在特定选择压力下获得具有特定表型的突变体。然而，传统的适应性进化方法不仅费力且耗时，而且通量较低且效率不高。为了使上述菌株减少葡萄糖酸盐的使用并提高生长速率，本研究开发了一种基于微生物微滴培养（MMC）平台的非理性策略。鉴于MMC的自动高通量筛选，在培养过程中通过逐渐降低葡萄糖酸盐的可用性进行了为期50天的三阶段适应性进化实验，以获得突变。最后，获得了一个候选突变体，与起始菌株相比，其生长速率快3倍，延迟期短43%，葡萄糖酸盐使用量减少40%。此外，通过分析突变体的全基因组测序鉴定了、和的基因突变，这些突变与葡萄糖酸盐摄取效率和细胞生长密切相关。总之，我们成功证明了使用MMC平台以省力、时间高效和定向进化的方式赋予目标菌株特定要求的可行性。
