Improving ethanol tolerance of Escherichia coli by rewiring Its global regulator cAMP receptor protein (CRP)
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...
| Main Authors: | , , , , , , |
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| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
| Published: |
2013
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| Subjects: | |
| Online Access: | https://hdl.handle.net/10356/96553 http://hdl.handle.net/10220/9887 |
| _version_ | 1826123013927469056 |
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| author | Chong, Huiqing Huang, Lei Yeow, Jianwei Wang, Ivy Zhang, Hongfang Song, Hao Jiang, Rongrong |
| author2 | School of Chemical and Biomedical Engineering |
| author_facet | School of Chemical and Biomedical Engineering Chong, Huiqing Huang, Lei Yeow, Jianwei Wang, Ivy Zhang, Hongfang Song, Hao Jiang, Rongrong |
| author_sort | Chong, Huiqing |
| collection | NTU |
| description | 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 OpenArrayH 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. |
| first_indexed | 2024-10-01T05:57:41Z |
| format | Journal Article |
| id | ntu-10356/96553 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T05:57:41Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | ntu-10356/965532023-12-29T06:50:04Z Improving ethanol tolerance of Escherichia coli by rewiring Its global regulator cAMP receptor protein (CRP) Chong, Huiqing Huang, Lei Yeow, Jianwei Wang, Ivy Zhang, Hongfang Song, Hao Jiang, Rongrong School of Chemical and Biomedical Engineering DRNTU::Science::Biological sciences::Genetics 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 OpenArrayH 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. Published version 2013-05-06T08:49:28Z 2019-12-06T19:32:27Z 2013-05-06T08:49:28Z 2019-12-06T19:32:27Z 2013 2013 Journal Article Chong, H., Huang, L., Yeow, J., Wang, I., Zhang, H., Song, H., & Jiang, R. (2013). Improving Ethanol Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP). PLoS ONE, 8(2). 1932-6203 https://hdl.handle.net/10356/96553 http://hdl.handle.net/10220/9887 10.1371/journal.pone.0057628 23469036 en PLoS ONE © 2013 The Author(s). application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences::Genetics Chong, Huiqing Huang, Lei Yeow, Jianwei Wang, Ivy Zhang, Hongfang Song, Hao Jiang, Rongrong Improving ethanol tolerance of Escherichia coli by rewiring Its global regulator cAMP receptor protein (CRP) |
| title | Improving ethanol tolerance of Escherichia coli by rewiring Its global regulator cAMP receptor protein (CRP) |
| title_full | Improving ethanol tolerance of Escherichia coli by rewiring Its global regulator cAMP receptor protein (CRP) |
| title_fullStr | Improving ethanol tolerance of Escherichia coli by rewiring Its global regulator cAMP receptor protein (CRP) |
| title_full_unstemmed | Improving ethanol tolerance of Escherichia coli by rewiring Its global regulator cAMP receptor protein (CRP) |
| title_short | Improving ethanol tolerance of Escherichia coli by rewiring Its global regulator cAMP receptor protein (CRP) |
| title_sort | improving ethanol tolerance of escherichia coli by rewiring its global regulator camp receptor protein crp |
| topic | DRNTU::Science::Biological sciences::Genetics |
| url | https://hdl.handle.net/10356/96553 http://hdl.handle.net/10220/9887 |
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