Submitted to: World Journal of Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/23/2020
Publication Date: 11/6/2020
Citation: Liu, S., Skory, C.D., Qureshi, N. 2020. Ethanol tolerance assessment in recombinant E. coli of ethanol responsive genes from Lactobacillus buchneri NRRL B-30929. World Journal of Microbiology and Biotechnology. 36. Article 179. https://doi.org/10.1007/s11274-020-02953-9.
DOI: https://doi.org/10.1007/s11274-020-02953-9

Interpretive Summary: Bacteria are sometimes used to produce certain types of alcohol as alternative liquid fuels, but growth inhibition from the alcohol often limits productivity. Lactic acid bacteria (LAB), which are used in food applications, are found to tolerate high concentrations of alcohol. The reasons behind this trait are not well understood, so uncovering the genetic mechanisms associated with alcohol tolerance may allow us to engineer this trait into other production strains. In this study, we isolated 14 genes from an ethanol tolerant LAB that were found to be highly expressed in the presence of ethanol. We then introduced these individual genes into another bacterial host that cannot grow in high alcohol content. We showed that six of these genes conferred ethanol tolerance to the new host. This is an important functional assessment to demonstrate that these unique genes can be used to improve resistance to alcohols in other bacteria. The potential application of this work will lead to more efficient production strains that will benefit fuel ethanol producers.

Technical Abstract: We previously identified numerous proteins associated with ethanol stress response in a Lactobacillus buchneri strain capable of growing in 10% ethanol and have shown that several of these are capable of conferring ethanol tolerance in other bacteria. . In the current study, the functional roles of additional ethanol responsive genes are examined to determine if they can increase ethanol tolerance in E.coli host cells. The recombinant strains carrying ethanol responsive genes were subjected to growth analyses in media with and without 4% ethanol. Among the tested, six genes Lbuc_0522 (NADPH-dependent methylglyoxal reductase), Lbuc_0354 (succinate semialdehyde dehydrogenase), Lbuc_1211(threonyl_tRNA synthetase), Lbuc_2051 (nitroreductase), Lbuc_0707 (branched chain amino acid aminotransferase) and Lbuc_1852 (proline-specific peptidase) conferred host cells tolerance to 4% ethanol. Two genes Lbuc_1523 (phage major capsid protein, HK 97 family) and Lbuc_1319 (phosphoglycerate kinase) conferred host cells slight tolerance to 4% ethanol. Four genes Lbuc_0787 encoding fumarylacetoacetate hydrolase, Lbuc_1219 encoding UDP-N-acetylmuramate-L-alanine ligase, Lbuc_0466 encoding ornithine carbamoyltransferase and Lbuc_0858 encoding glycine hydroxymethyltransferase showed no impact on growth in media with 4% ethanol with IPTG induction when compared with E.coli carrying control pET28b plasmid. The expression of two genes Lbuc_1557 (S-layer glycoprotein) and Lbuc_2157 (6-phosphogluconate dehydrogenase) resulted ethanol sensitivity phenotype.