Identification and manipulation of a novel locus to improve cell tolerance to short-chain alcohols in Escherichia coli
Escherichia coli KO11 is a popular ethanologenic strain, but is more sensitive to ethanol than other producers. Here, an ethanol-tolerant mutant EM was isolated from ultraviolet mutagenesis library of KO11. Comparative genomic analysis added by piecewise knockout strategy and complementation assay revealed EKO11_3023 (espA) within the 36.6-kb deletion from KO11 was the only locus responsible for ethanol sensitivity. Interestingly, when espA was deleted in strain W (the parent strain of KO11), ethanol tolerance was dramatically elevated to the level of espA-free hosts [e.g., MG1655 and BL21(DE3)]. And overexpression of espA in strains MG1655 and BL21(DE3) led to significantly enhanced ethanol sensitivity. In addition to ethanol, deletion of espA also improved cell tolerance to other short-chain (C2–C4) alcohols, including methanol, isopropanol, n-butanol, isobutanol and 2-butanol. Therefore, espA was responsible for short-chain alcohol sensitivity of W-strains compared to other cells, which provides a potential engineering target for alcohols production.
KeywordsSynthetic biology Cell tolerance Short-chain alcohols Escherichia coli espA
This work was financially supported by the Ministry of Science and Technology of China (“973” Program, 2014CB745100), the National Natural Science Foundation of China (21621004, 21390203, 21622605 and 21576198) and Innovative Talents and Platform Program of Tianjin (16PTSYJC00050 and 16PTGCCX00140).
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- 7.Chevreux B, Pfisterer T, Drescher B, Driesel AJ, Muller WE, Wetter T, Suhai S (2004) Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs. Genome Res 14:1147–1159. https://doi.org/10.1101/gr.1917404 CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Gonzalez R, Tao H, Purvis JE, York SW, Shanmugam KT, Ingram LO (2003) Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant). Biotechnol Progr 19:612–623. https://doi.org/10.1021/bp025658q CrossRefGoogle Scholar
- 16.Jh M (1992) A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, PlainviewGoogle Scholar
- 23.Ohta K, Beall DS, Mejia JP, Shanmugam KT, Ingram LO (1991) Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II. Appl Environ Micro 57:893–900Google Scholar
- 26.Studier FW, Daegelen P, Lenski RE, Maslov S, Kim JF (2009) Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 genomes. J Mol Biol 394:653–680. https://doi.org/10.1016/j.jmb.2009.09.021 CrossRefPubMedGoogle Scholar