title: Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance.
abstract: One major challenge in biofuel production, including biobutanol production, is the low tolerance of the microbial host towards increasing biofuel concentration during fermentation. Here, we have demonstrated that Escherichia coli 1-butanol tolerance can be greatly enhanced through random mutagenesis of global transcription factor cyclic AMP receptor protein (CRP). Four mutants (MT1-MT4) with elevated 1-butanol tolerance were isolated from error-prone PCR libraries through an enrichment screening. A DNA shuffling library was then constructed using MT1-MT4 as templates and one mutant (MT5) that exhibited the best tolerance ability among all variants was selected. In the presence of 0.8 % (v/v, 6.5 g/l) 1-butanol, the growth rate of MT5 was found to be 0.28 h(-1) while that of wild type was 0.20 h(-1). When 1-butanol concentration increased to 1.2 % (9.7 g/l), the growth rate of MT5 (0.18 h(-1)) became twice that of the wild type (0.09 h(-1)). Microbial adhesion to hydrocarbon test showed that cell surface of MT5 was less hydrophobic and its cell length became significantly longer in the presence of 1-butanol, as observed by scanning electron microscopy. Quantitative real-time reverse transcription PCR analysis revealed that several CRP regulated, 1-butanol stress response related genes (rpoH, ompF, sodA, manX, male, and marA) demonstrated differential expression in MT5 in the presence or absence of 1-butanol. In conclusion, direct manipulation of the transcript profile through engineering global transcription factor CRP can provide a useful tool in strain engineering.
authors.affiliation: School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, 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: Hao
authors.initials: H
authors.lastname: Song
authors.forename: Rongrong
authors.initials: R
authors.lastname: Jiang
mesh_terms: D020001:1-Butanol / Q000633:toxicity*; D001422:Bacterial Adhesion / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D020869:Gene Expression Profiling; D060847:Metabolic Engineering; D008855:Microscopy, Electron, Scanning; D016296:Mutagenesis; D060888:Real-Time Polymerase Chain Reaction; D011953:Receptors, Cyclic AMP / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003470:Culture Media; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011953:Receptors, Cyclic AMP; C047947:cyclic AMP receptor protein, E coli; D020001:1-Butanol