•Parallel ALEs of E. coli were performed to obtain IPA tolerant strains.•Genome re-sequencing and transcriptome analyses of IPA tolerant strains were performed.•Mutation of relA, marC, proQ, yfgO, and rraA provided IPA tolerance.•Expression change of genes related to several amino acid biosynthesis, iron ion, and central metabolism was observed.•Results from these experiments provide clues to understanding the mechanism of IPA tolerance.Isopropanol (IPA) is the secondary alcohol that can be dehydrated to yield propylene. To produce IPA using microorganisms, a significant issue is that the toxicity of IPA causes retardation or inhibition of cell growth, decreasing the yield. One possible strategy to overcome this problem is to improve IPA tolerance of production organisms. For the understanding of tolerance to IPA, we performed parallel adaptive laboratory evolution (ALE) of Escherichia coli under IPA stress. To identify the genotypic change during ALE, we performed genome re-sequencing analyses of obtained tolerant strains. To verify which mutations were contributed to IPA tolerance, we constructed the mutant strains and quantify the IPA tolerance of the constructed mutants. From these analyses, we found that five mutations (relA, marC, proQ, yfgO, and rraA) provided the increase of IPA tolerance. To understand the phenotypic change during ALE, we performed transcriptome analysis of tolerant strains. From transcriptome analysis, we found that expression levels of genes related to biosynthetic pathways of amino acids, iron ion homeostasis, and energy metabolisms were changed in the tolerant strains. Results from these experiments provide fundamental bases for designing IPA tolerant strains for industrial purposes.

Background: the acquisition of antibiotic resistance in bacterial cells is often accompanied with a reduction of fitness in the absence of antibiotics, known as the “fitness cost”. The magnitude of this fitness cost is an important biological parameter that influences the degree to which antibiotic resistant strains become widespread. However, the relationship between the fitness cost and comprehensive phenotypic and genotypic changes remains unclear. Here, we quantified the fitness cost of resistant strains obtained by experimental evolution in the presence of various antibiotics, and analyzed how the cost correlated to phenotypic and genotypic changes in the resistant strains. Results: we measured the specific growth rate of the resistant strains in the presence of various concentrations of drugs or in their absence. In the absence of drugs, the resistant strains showed reductions of approximately 20% to 50% in growth rate compared with the parent strain, which corresponded to the fitness cost. We found that the decrease of the specific growth rate was correlated with overall expression changes between the parent and resistant strains, measured by the Euclid distance between expression profiles. We also found that there are a number of genes whose changes in expression levels were significantly correlated with the growth rate, which may account for the observed correlation between the fitness cost and overall expression changes. Conclusions: our analysis provides a basis for quantitative understanding of the mechanism of the fitness cost. This understanding may provide clues on how to influence the fitness cost that accompanies resistance acquisition and consequently how to limit the spread of antibiotic resistant strains.

We analyzed the effect of combinatorial use of antibiotics with a trade-off relationship of resistance, i.e., resistance acquisition to one drug causes susceptibility to the other drug, and vice versa, on the evolution of antibiotic resistance. We demonstrated that this combinatorial use of antibiotics significantly suppressed the acquisition of resistance.

Saccharomyces cerevisiae shows a Crabtree effect that produces ethanol in a high glucose concentration even under fully aerobic condition. For efficient production of cake yeast or compressed yeast for baking, ethanol by-production is not desired since glucose limited chemostat or fed-batch cultivations are performed to suppress the Crabtree effect. In this study, the 13C-based metabolic flux analysis (13C-MFA) was performed for the S288C derived S. cerevisiae strain to characterize a metabolic state under the reduced Crabtree effect. S. cerevisiae cells were cultured at a low dilution rate (0.1 h−1) under the glucose-limited chemostat condition. The estimated metabolic flux distribution showed that the acetyl-CoA in mitochondria was mainly produced from pyruvate by pyruvate dehydrogenase (PDH) reaction and that the level of the metabolic flux through the pentose phosphate pathway was much higher than that of the Embden–Meyerhof–Parnas pathway, which contributes to high biomass yield at low dilution rate by supplying NADPH required for cell growth.