| Escherichia coli is the most widely used prokaryotic system for the synthesis of heterologous proteins because it is such a well-characterized system. A primary goal of fermentation research is the costeffective production of desired products. However, E. coli generates undesirable by-product that has several negative effects on cell growth. A global regulon is defined as a network of operons under the control of a common regulatory protein in a global regulatory network. FNR is the major global regulator and it controls its own synthesis. FNR may regulate transcription at more than 100 promoters. Here in this work, we apply directed evolution method to engineer FNR to improve strain ethanol and butanol tolerance.Firstly, the fnr gene was amplified from the E. coli genome and cloned into plasmid pUC18 to construct the pUC18-fnr operon. To mimic the genome level fnr expression in E. coli, a low cope plasmid pSK was used to construct the pSK-fnr operon.In order to improve transformation efficiency, the preparation of E. coli competent cells and electrotransformation conditions were optimized, including different volume of medium, growth phase of E. coli,electric field strength and time of posttransformation culture. Under the optimized conditions, the transformation efficiency could reach 1010 cfu/μg DNA with plasmid and 105 colonies with ligation transformation.The conditions of error-prone PCR reaction were optimized. A random mutagenesis library of fnr mutants was prepared by error-prone PCR using the constructed plasmid"pUC18-fnr operon"as the template.The error-prone PCR product was ligated into low copy pSK-fnr operon. The mutants were then transformed into E. coli DH5α. The mutant library was screened out for improved ethanol and butanol tolerence. Finally, we have demonstrated that the ethanol and butanol tolerance of E. coli can be improved through introducing mutations to the global transcription factor FNR. |
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