Evolution, genomic analysis, and reconstruction of isobutanol tolerance in Escherichia coli

Abstract

Escherichia coli has been engineered to produce isobutanol, with titers reaching greater than the toxicity level. However, the specific effects of isobutanol on the cell have never been fully understood. Here, we aim to identify genotype-phenotype relationships in isobutanol response. An isobutanol-tolerant mutant was isolated with serial transfers. Using whole-genome sequencing followed by gene repair and knockout, we identified five mutations (acrA, gatY, tnaA, yhbJ, and marCRAB) that were primarily responsible for the increased isobutanol tolerance. We successfully reconstructed the tolerance phenotype by combining deletions of these five loci, and identified glucosamine-6-phosphate as an important metabolite for isobutanol tolerance, which presumably enhanced membrane synthesis. The isobutanol-tolerant mutants also show increased tolerance to n-butanol and 2-methyl-1-butanol, but showed no improvement in ethanol tolerance and higher sensitivity to hexane and chloramphenicol than the parental strain. These results suggest that C4, C5 alcohol stress impacts the cell differently compared with the general solvent or antibiotic stresses. Interestingly, improved isobutanol tolerance did not increase the final titer of isobutanol production.

Figure 1

Comparison of growth with isobutanol stress. Cells were incubated in LB at 37°C. (A, B) Time courses for the growth of E. coli strain JCL260 (A) and SA481 (B) in the absence of isobutanol (open triangles) or in the presence of 6 g/l (closed triangles), 8 g/l (closed circles), 10 g/l (closed diamond), and 15 g/l (closed square) isobutanol (C). The ratio of viable cells at 0 and 24 h in the presence of 6 g/l (open bar) and 8 g/l (closed bar) isobutanol. All data were performed in triplicate.

Figure 2

Summary of specific mutations on SA481 genome. Genes without annotation contain an IS insertion. Brackets represent an IS insertion in non-coding region or deletion (specified) between the two genes in the bracket.

Figure 3

Effect of mutation repairs on isobutanol tolerance. (A) Single repair of mutations on SA481 and (B) multiple repairs. Each of the mutations on SA481, including SNP (polA), deletions (hipA–flxA), IS mutations (the remaining), was repaired. Brackets represent IS insertions in intergenic regions. Cell was treated with 8 g/l isobutanol in LB for 24 h. The y axis indicates the ratio of OD₆₀₀ at 0 and 24 h. Gene names below the axis indicate repaired genes in SA481. The numbers below the gene names indicate the OD₆₀₀ values after 24 h isobutanol treatment. All data were performed in triplicate. The closed bars represent genes that were selected for multiple repairs.

Figure 4

Reconstruction of tolerance phenotype in the parental strain (JCL260) by gene deletion. Significant mutations identified in Figure 3 were reconstructed in JCL260 to test isobutanol tolerance. Growth test of the E. coli strains in LB at 37°C without isobutanol (A, C, E) and with 6 g/l isobutanol (B, D, F). All data were performed in triplicate.

Figure 5

Analysis of ΔyhbJ effect. Cells were incubated in LB at 37°C. The y axis indicates the ratio of OD₆₀₀ at 0 and 24 h. (A) Open and closed bars represent E. coli strains containing the control plasmid (pSA40) and the glmZ overexpression plasmid (pHW29), respectively, with 6 g/l isobutanol. (B, C) Open and closed bars represent without and with 10 g/l GlcNAc supplement, respectively, in the absence (B) of and the presence (C) of 6 g/l isobutanol. All data were performed in triplicate.

Figure 6

Effects of other solvents and antibiotics. Cells were incubated in LB at 37°C. The y axis indicates the ratio of OD₆₀₀ at 0 and 4 h (A) and at 0 and 24 h (B) of JCL260 (white bar), SA481 (black bar), and TW306 (gray bar) under various types of solvent and antibiotics stress: hexane 3.3 g/l, chloramphenicol 5 mg/l, ethanol 32 g/l, n-butanol 6 g/l, 2-methyl-1-butanol 3 g/l. All data were performed in triplicate.

Figure 7

Isobutanol production from the engineered E. coli. (A–C) Isobutanol production in M9 medium containing 63 g/l glucose and 10 g/l yeast extract in a shake flask at 30°C (A), time profiles of cell growth (B), glucose concentration (C). At 48 h, 30 g/l glucose was added to the culture. (D–F) Isobutanol production with 8 g/l isobutanol supplement (D), time profiles of cell growth (E), glucose concentration (F) All data were performed in triplicate.