Abstract
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.
Highlights
Due to the steady increase in both the global population and the demand for food (Ganesh, 2014), it is urgent to explore more available substrates with which to replace limited editable feedstocks used as carbon sources for biomanufacturing (Wen-Liang et al, 2016; Hu et al, 2017; Wang et al, 2017; Fei et al, 2018, 2020; Cui et al, 2018a)
A methanol-dependent E. coli was developed by heterotrophically expressing ribulose monophosphate cycle (RuMP) with mdh, hps (3-hexulose6-phosphate synthase), and phi (6-phospho-3-hexuloisomerase) genes, which enabled E. coli to utilize methanol directly as a carbon source (Meyer et al, 2018)
The starting samples of all experiments were from the same medium with 2% methanol, which were individually transferred to 96-well plates, shake flasks, and microbial microdroplet culture (MMC)
Summary
Due to the steady increase in both the global population and the demand for food (Ganesh, 2014), it is urgent to explore more available substrates with which to replace limited editable feedstocks used as carbon sources for biomanufacturing (Wen-Liang et al, 2016; Hu et al, 2017; Wang et al, 2017; Fei et al, 2018, 2020; Cui et al, 2018a). Genetic engineering of Escherichia coli capable of assimilating methanol has attracted considerable interest in terms of biosynthesis and fermentation applications (Cocks et al, 1974; Müller et al, 2015; Whitaker et al, 2015; Bennett et al, 2018; Gonzalez et al, 2018). It is worth noting that the growth of the aforementioned E. coli strongly relies on the supply of gluconate as a co-substrate for growth, which drastically limits the versatile of this strain in commercialization. This reduces the usage of substrate during the culture of this methanol-dependent E. coli, and this is one of the key puzzles leading to its applications in the future
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