title: Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance.
abstract: 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.
authors.affiliation: School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore.
authors.forename: Hongfang
authors.initials: H
authors.lastname: Zhang
authors.forename: Huiqing
authors.initials: H
authors.lastname: Chong
authors.forename: Chi Bun
authors.initials: CB
authors.lastname: Ching
authors.forename: Rongrong
authors.initials: R
authors.lastname: Jiang
mesh_terms: D002373:Cyclic AMP Receptor Protein / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D032621:DNA Shuffling; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D020869:Gene Expression Profiling; D046228:Microarray Analysis; D008855:Microscopy, Electron, Scanning; D016296:Mutagenesis; D009997:Osmotic Pressure; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D012641:Selection, Genetic; D012965:Sodium Chloride / Q000378:metabolism; D013312:Stress, Physiological; D014157:Transcription Factors
chemical_list: D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C481662:crp protein, E coli; D012965:Sodium Chloride