UC San Diego

Adaptive laboratory evolution (ALE) has become an increasingly common method in recent years, but only a few experiments have adapted Escherichia coli to low-oxygen conditions (Finn et al., 2017; Puentes-Téllez et al., 2013; Wang et al., 2019). This is surprising, given its nature as a facultative anaerobe and the dramatic shift from respiration to mixed-acid fermentation that is observed (Clark, 1989). Here, a preexisting automated platform is used to adapt E. coli K-12 MG1655 to oxygen limitation, and the resulting strains are compared to those previously evolved aerobically. Additionally, an automated workflow is developed to measure the uptake and secretion rates of common E. coli substrates and products. Although mixed acid fermentation was observed during ALE, the resulting strains had similar genotypes to those adapted aerobically. Mutations in both cases focused on mitigating strain defects, increasing flux through central metabolism, or reducing stress response. Differences in growth, carbon uptake, and fermentation product secretion rates were the largest under aerobic growth between the strains having an hns/tdk intergenic mutation and those without, regardless of the oxygen level during adaptation or the presence and absence of other mutations. Similar differences were observed for growth under oxygen limited conditions, but the magnitude of the changes was much smaller and not statistically significant. Although the oxygen-limited adapted strains generally lacked this intergenic mutation, two mutations not found in the aerobic ALEs may function in a similar capacity. Overall, it appears that the strains were adapted primarily to the media conditions, regardless of oxygen levels.