He figure also illustrated a longer exponential growth phase to a higher OD600 value (0.19) than the control (0.10). It was noted that the deletion of crp (JW5702 Dkan+blank plasmid pKSC) did not improve the ethanol tolerance of E. coli.Chromosomal IntegrationSince E2 had demonstrated the best ethanol resistance among all three mutants, its crp operon was integrated into the chromosome of E. coli JW5702 Dkan, the native crp operon location, to create strain iE2. Chromosomal integration was performed to avoid the disadvantages of using plasmid-based system, such as genetic instability arising from segregation or horizontal gene transfer, metabolic burden, and the need for supplementing antibiotics [44,45]. DNA sequencing results confirmed the same amino acid substitution in the crp operon of iE2 as E2. The growth of iE2 under ethanol stress was investigated with its parent strain BW25113 and JW5702. In the absence of ethanol, iE2 shared similar growth pattern with BW25113, with a growth rate 25331948 around 0.45 h21, faster than that of JW5702 (Figure 2A). When all strains were facing ethanol challenge (62 g/l ethanol), iE2 (0.07 h21) not only outgrew BW25113 (0.055 h21) but also reached a higher OD600 value (0.13) than BW25113 (0.09) after 12 h (Figure 2B). The crp knock-out strain JW5702 exhibited the worst ethanol tolerance among all three strains. It was also noted that when ethanol concentration was low, iE2 might result in worse growth than the parent strain (data not shown). To further prove the ethanol tolerance of iE2, both iE2 and BW25113 were exposed to 150 g/l ethanol and 
their survival was recorded over time (Figure 3). iE2 exhibited significantly better survival than BW25113 over the 1-h period examined. For instance, after 15-min exposure to 150 g/l ethanol, iE2 displayed more than 10 survival whereas BW25113 only had less than 0.01 . Even after 1-h exposure, iE2 still demonstrated 15900046 over 10,000-fold survival than BW25113.Cross Resistance to other AlcoholsCell culture was prepared by diluting overnight seed into fresh LB medium containing different alcohols. Cell growth was recorded by OD600 readings with the incubation at 37uC, 200 rpm. The alcohol concentrations used are presented in CUDC-907 biological activity volume ratio: 3.1 1-propanol, 1.3 1-butanol, and 0.45 1pentanol.Results Isolation of Ethanol-tolerant CRP MutantsError-prone PCR was carried out to introduce 2? nucleotide substitutions per crp by varying the amount of DNA MedChemExpress CTX-0294885 template. Recombinant plasmids with mutated crp inserts were transformed into competent E. coli JW5702 Dkan (crp knock-out strain) and the total error-prone library size was greater than 106. The mutagenesis libraries were then enriched through repeated subcultures containing 40?5 g/l ethanol to separate “winners” with enhanced ethanol tolerance. The mutated crp inserts of these “winners” were digested, re-ligated to freshly prepared plasmid pKSC, and the resulting recombinant plasmids were re-transformed into E. coli JW5702 Dkan background to eliminate false positives or chromosomal mutations. Three ethanol-tolerant mutants (E1 3) with improved growth under ethanol stress were selected and their amino acid substitutions are summarized in Table 2.Resistance towards other AlcoholsThe tolerance ability of iE2 towards other alcohols, namely 1propanol, 1-butanol, and 1-pentanol, was also studied to demonstrate its alcohol tolerance in general (Figure 4). iE2 showed much better growth than BW25113 and was able to achieve a higher.He figure also illustrated a longer exponential growth phase to a higher OD600 value (0.19) than the control (0.10). It was noted that the deletion of crp (JW5702 Dkan+blank plasmid pKSC) did not improve the ethanol tolerance of E. coli.Chromosomal IntegrationSince E2 had demonstrated the best ethanol resistance among all three mutants, its crp operon was integrated into the chromosome of E. coli JW5702 Dkan, the native crp operon location, to create strain iE2. Chromosomal integration was performed to avoid the disadvantages of using plasmid-based system, such as genetic instability arising from segregation or horizontal gene transfer, metabolic burden, and the need for supplementing antibiotics [44,45]. DNA sequencing results confirmed the same amino acid substitution in the crp operon of iE2 as E2. The growth of iE2 under ethanol stress was investigated with its parent strain BW25113 and JW5702. In the absence of ethanol, iE2 shared similar growth pattern with BW25113, with a growth rate 25331948 around 0.45 h21, faster than that of JW5702 (Figure 2A). When all strains were facing ethanol challenge (62 g/l ethanol), iE2 (0.07 h21) not only outgrew BW25113 (0.055 h21) but also reached a higher OD600 value (0.13) than BW25113 (0.09) after 12 h (Figure 2B). The crp knock-out strain JW5702 exhibited the worst ethanol tolerance among all three strains. It was also noted that when ethanol concentration was low, iE2 might result in worse growth than the parent strain (data not shown). To further prove the ethanol tolerance of iE2, both iE2 and BW25113 were exposed to 150 g/l ethanol and their survival was recorded over time (Figure 3). iE2 exhibited significantly better survival than BW25113 over the 1-h period examined. For instance, after 15-min exposure to 150 g/l ethanol, iE2 displayed more than 10 survival whereas BW25113 only had less than 0.01 . Even after 1-h exposure, iE2 still demonstrated 15900046 over 10,000-fold survival than BW25113.Cross Resistance to other AlcoholsCell culture was prepared by diluting overnight seed into fresh LB medium containing different alcohols. Cell growth was recorded by OD600 readings with the incubation at 37uC, 200 rpm. The alcohol concentrations used are presented in volume ratio: 3.1 1-propanol, 1.3 1-butanol, and 0.45 1pentanol.Results Isolation of Ethanol-tolerant CRP MutantsError-prone PCR was carried out to introduce 2? nucleotide substitutions per crp by varying the amount of DNA template. Recombinant plasmids with mutated crp inserts were transformed into competent E. coli JW5702 Dkan (crp knock-out strain) and the total error-prone library size was greater than 106. The mutagenesis libraries were then enriched through repeated subcultures containing 40?5 g/l ethanol to separate “winners” with enhanced ethanol tolerance. The mutated crp inserts of these “winners” were digested, re-ligated to freshly prepared plasmid pKSC, and the resulting recombinant plasmids were re-transformed into E. coli JW5702 Dkan background to eliminate false positives or chromosomal mutations. Three ethanol-tolerant mutants 
(E1 3) with improved growth under ethanol stress were selected and their amino acid substitutions are summarized in Table 2.Resistance towards other AlcoholsThe tolerance ability of iE2 towards other alcohols, namely 1propanol, 1-butanol, and 1-pentanol, was also studied to demonstrate its alcohol tolerance in general (Figure 4). iE2 showed much better growth than BW25113 and was able to achieve a higher.
