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Improving ethanol tolerance of Escherichia coli by rewiring its global regulator cAMP receptor protein (CRP).

Chong H, Huang L, Yeow J, Wang I, Zhang H, Song H, Jiang R - PLoS ONE (2013)

Bottom Line: These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA), iron ion transport (entH, entD, fecA, fecB), and general stress response (osmY, rpoS).Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol.Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore.

ABSTRACT
A major challenge in bioethanol fermentation is the low tolerance of the microbial host towards the end product bioethanol. Here we report to improve the ethanol tolerance of E. coli from the transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP), which is known to regulate over 400 genes in E. coli. Three ethanol tolerant CRP mutants (E1- E3) were identified from error-prone PCR libraries. The best ethanol-tolerant strain E2 (M59T) had the growth rate of 0.08 h(-1) in 62 g/L ethanol, higher than that of the control at 0.06 h(-1). The M59T mutation was then integrated into the genome to create variant iE2. When exposed to 150 g/l ethanol, the survival of iE2 after 15 min was about 12%, while that of BW25113 was <0.01%. Quantitative real-time reverse transcription PCR analysis (RT-PCR) on 444 CRP-regulated genes using OpenArray® technology revealed that 203 genes were differentially expressed in iE2 in the absence of ethanol, whereas 92 displayed differential expression when facing ethanol stress. These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA), iron ion transport (entH, entD, fecA, fecB), and general stress response (osmY, rpoS). Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol. Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol.

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Growth of iE2, parent strain BW25113, and JW5702 in LB medium.Cells were grown in (A) no ethanol (B) 62 g/L ethanol at 37°C.
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pone-0057628-g002: Growth of iE2, parent strain BW25113, and JW5702 in LB medium.Cells were grown in (A) no ethanol (B) 62 g/L ethanol at 37°C.

Mentions: Since E2 had demonstrated the best ethanol resistance among all three mutants, its crp operon was integrated into the chromosome of E. coli JW5702 Δkan, 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 around 0.45 h−1, faster than that of JW5702 (Figure 2A). When all strains were facing ethanol challenge (62 g/l ethanol), iE2 (0.07 h−1) not only outgrew BW25113 (0.055 h−1) 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).


Improving ethanol tolerance of Escherichia coli by rewiring its global regulator cAMP receptor protein (CRP).

Chong H, Huang L, Yeow J, Wang I, Zhang H, Song H, Jiang R - PLoS ONE (2013)

Growth of iE2, parent strain BW25113, and JW5702 in LB medium.Cells were grown in (A) no ethanol (B) 62 g/L ethanol at 37°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585226&req=5

pone-0057628-g002: Growth of iE2, parent strain BW25113, and JW5702 in LB medium.Cells were grown in (A) no ethanol (B) 62 g/L ethanol at 37°C.
Mentions: Since E2 had demonstrated the best ethanol resistance among all three mutants, its crp operon was integrated into the chromosome of E. coli JW5702 Δkan, 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 around 0.45 h−1, faster than that of JW5702 (Figure 2A). When all strains were facing ethanol challenge (62 g/l ethanol), iE2 (0.07 h−1) not only outgrew BW25113 (0.055 h−1) 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).

Bottom Line: These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA), iron ion transport (entH, entD, fecA, fecB), and general stress response (osmY, rpoS).Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol.Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore.

ABSTRACT
A major challenge in bioethanol fermentation is the low tolerance of the microbial host towards the end product bioethanol. Here we report to improve the ethanol tolerance of E. coli from the transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP), which is known to regulate over 400 genes in E. coli. Three ethanol tolerant CRP mutants (E1- E3) were identified from error-prone PCR libraries. The best ethanol-tolerant strain E2 (M59T) had the growth rate of 0.08 h(-1) in 62 g/L ethanol, higher than that of the control at 0.06 h(-1). The M59T mutation was then integrated into the genome to create variant iE2. When exposed to 150 g/l ethanol, the survival of iE2 after 15 min was about 12%, while that of BW25113 was <0.01%. Quantitative real-time reverse transcription PCR analysis (RT-PCR) on 444 CRP-regulated genes using OpenArray® technology revealed that 203 genes were differentially expressed in iE2 in the absence of ethanol, whereas 92 displayed differential expression when facing ethanol stress. These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA), iron ion transport (entH, entD, fecA, fecB), and general stress response (osmY, rpoS). Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol. Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol.

Show MeSH
Related in: MedlinePlus