Empowering a Methanol-Dependent Escherichia coli via Adaptive Evolution Using a High-Throughput Microbial Microdroplet Culture System

Recently, a methanol-essential Escherichia coli 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 E. coli 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 gntU, idnT, edd, and pckA 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.