Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance
The naturally existing microbial hosts can rarely satisfy industrial requirements, thus there has always been an intense effort in strain engineering to meet the needs of these bioprocesses. Here, in this work, we want to prove the concept that engineering global transcription factor cAMP receptor p...
| Main Authors: | , , , |
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| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
| Published: |
2013
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| Online Access: | https://hdl.handle.net/10356/98901 http://hdl.handle.net/10220/12722 |
| _version_ | 1826124504415338496 |
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| author | Zhang, Hongfang Chong, Huiqing Ching, Chi Bun Jiang, Rongrong |
| author2 | School of Chemical and Biomedical Engineering |
| author_facet | School of Chemical and Biomedical Engineering Zhang, Hongfang Chong, Huiqing Ching, Chi Bun Jiang, Rongrong |
| author_sort | Zhang, Hongfang |
| collection | NTU |
| description | The naturally existing microbial hosts can rarely satisfy industrial requirements, thus there has always been an intense effort in strain engineering to meet the needs of these bioprocesses. Here, in this work, we want to prove the concept that engineering global transcription factor cAMP receptor protein (CRP) of Escherichia coli can improve cell phenotypes. CRP is one of the global regulatory proteins that can regulate the transcription of over 400 genes in E. coli. The target phenotype in this study is strain osmotolerance. Amino acid mutations were introduced to CRP by either error-prone PCR or DNA shuffling, and the random mutagenesis libraries were subjected to enrichment selection under NaCl stress. Five CRP mutants (MT1–MT5) were selected from the error-prone PCR libraries with enhanced osmotolerance. DNA shuffling technique was employed to generate mutant MT6 with MT1–MT5 as templates. All of these variants showed much better growth in the presence of NaCl compared to the wild type, and MT6 presented the best tolerance towards NaCl. In the presence of 0.9 M NaCl, the growth rate of MT6 is 0.113 h−1, while that of WT is 0.077 h−1. MT6 also exhibited resistance to other osmotic stressors, such as KCl, glucose, and sucrose. DNA microarray analysis showed that genes involved in colanic acid biosynthesis are up-regulated in the absence of salt stress, whereas carbohydrate metabolic genes are differentially expressed under NaCl stress when comparing MT6 to WT. Scanning electron microscopy images confirmed the elongation of both WT and MT6 when exposed to NaCl but the cell surface of MT6 was relatively smooth. |
| first_indexed | 2024-10-01T06:21:43Z |
| format | Journal Article |
| id | ntu-10356/98901 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T06:21:43Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | ntu-10356/989012020-03-07T11:35:38Z Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance Zhang, Hongfang Chong, Huiqing Ching, Chi Bun Jiang, Rongrong School of Chemical and Biomedical Engineering The naturally existing microbial hosts can rarely satisfy industrial requirements, thus there has always been an intense effort in strain engineering to meet the needs of these bioprocesses. Here, in this work, we want to prove the concept that engineering global transcription factor cAMP receptor protein (CRP) of Escherichia coli can improve cell phenotypes. CRP is one of the global regulatory proteins that can regulate the transcription of over 400 genes in E. coli. The target phenotype in this study is strain osmotolerance. Amino acid mutations were introduced to CRP by either error-prone PCR or DNA shuffling, and the random mutagenesis libraries were subjected to enrichment selection under NaCl stress. Five CRP mutants (MT1–MT5) were selected from the error-prone PCR libraries with enhanced osmotolerance. DNA shuffling technique was employed to generate mutant MT6 with MT1–MT5 as templates. All of these variants showed much better growth in the presence of NaCl compared to the wild type, and MT6 presented the best tolerance towards NaCl. In the presence of 0.9 M NaCl, the growth rate of MT6 is 0.113 h−1, while that of WT is 0.077 h−1. MT6 also exhibited resistance to other osmotic stressors, such as KCl, glucose, and sucrose. DNA microarray analysis showed that genes involved in colanic acid biosynthesis are up-regulated in the absence of salt stress, whereas carbohydrate metabolic genes are differentially expressed under NaCl stress when comparing MT6 to WT. Scanning electron microscopy images confirmed the elongation of both WT and MT6 when exposed to NaCl but the cell surface of MT6 was relatively smooth. 2013-08-01T02:49:11Z 2019-12-06T20:00:56Z 2013-08-01T02:49:11Z 2019-12-06T20:00:56Z 2011 2011 Journal Article Zhang, H., Chong, H., Ching, C. B.,& Jiang, R. (2012). Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance. Biotechnology and Bioengineering, 109(5), 1165-1172. 0006-3592 https://hdl.handle.net/10356/98901 http://hdl.handle.net/10220/12722 10.1002/bit.24411 en Biotechnology and bioengineering |
| spellingShingle | Zhang, Hongfang Chong, Huiqing Ching, Chi Bun Jiang, Rongrong Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance |
| title | Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance |
| title_full | Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance |
| title_fullStr | Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance |
| title_full_unstemmed | Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance |
| title_short | Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance |
| title_sort | random mutagenesis of global transcription factor camp receptor protein for improved osmotolerance |
| url | https://hdl.handle.net/10356/98901 http://hdl.handle.net/10220/12722 |
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