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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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iE2 tolerance towards other alcohols as compared to parent strain BW25113.Cells were cultivated at 37°C in LB medium containing different alcohols: (A) 3.1% (v/v) 1-propanol, (B) 1.3% (v/v) 1-butanol, (C) 0.45% (v/v) 1-pentanol.
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pone-0057628-g004: iE2 tolerance towards other alcohols as compared to parent strain BW25113.Cells were cultivated at 37°C in LB medium containing different alcohols: (A) 3.1% (v/v) 1-propanol, (B) 1.3% (v/v) 1-butanol, (C) 0.45% (v/v) 1-pentanol.

Mentions: The tolerance ability of iE2 towards other alcohols, namely 1-propanol, 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 OD600 value at stationery phase with all alcohols tested. For instance, the presence of 3.1% (v/v) 1-propanol led to 0.129 h−1 growth rate in iE2, higher than that of BW25113 at 0.087 h−1. Although iE2 and BW25113 shared similar growth rate (0.068 h−1) when cultivated in 1.3% (v/v) 1-butanol, iE2 was able to reach a higher OD600 value.


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)

iE2 tolerance towards other alcohols as compared to parent strain BW25113.Cells were cultivated at 37°C in LB medium containing different alcohols: (A) 3.1% (v/v) 1-propanol, (B) 1.3% (v/v) 1-butanol, (C) 0.45% (v/v) 1-pentanol.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057628-g004: iE2 tolerance towards other alcohols as compared to parent strain BW25113.Cells were cultivated at 37°C in LB medium containing different alcohols: (A) 3.1% (v/v) 1-propanol, (B) 1.3% (v/v) 1-butanol, (C) 0.45% (v/v) 1-pentanol.
Mentions: The tolerance ability of iE2 towards other alcohols, namely 1-propanol, 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 OD600 value at stationery phase with all alcohols tested. For instance, the presence of 3.1% (v/v) 1-propanol led to 0.129 h−1 growth rate in iE2, higher than that of BW25113 at 0.087 h−1. Although iE2 and BW25113 shared similar growth rate (0.068 h−1) when cultivated in 1.3% (v/v) 1-butanol, iE2 was able to reach a higher OD600 value.

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