Engineering Of Escherichia Coli Operon And Its Potential Application

Expression Regulation of metabolic pathways is one of the most important issue of Metabolic Engineering. In prokaryotes, genes are arranged as operon. Genes involved in related pathway or biological functional processes are co-regulated in an operon as a transcriptional unit and are expressed as polycistronic mRNA. The control of the operon is the common mechanisms how bacterial regulate gene expression depending on environmental conditions. And its promoter, which initiates transcription process, regulates the transcription of operon. So the nature of promoter plays important role in regulating gene expression. Structure of operon and the function of multiple promoters in operon were studied.To construct a promoter activity evaluation system, the low copy plasmid pCL1920 was employed to cloning a promoter-less gfp and lacZ expressing operon. And there is a promoter-cloning region upstream the gfp and lacZ expressing operon. By cloning certain promoter into this region, the promoter activity can be evaluated by the expression level of green fluorescence protein andβ-galactosidase. In this way, the promoter activity can be easily evaluated by monitoring fluorescence and/β-galactosidase activity. This promoter activity evaluation system can be used as a basic molecular tool of studying promoters. By emplying this system, five promoters-fic, spc, rpos, nlpD, katE and the core region of trc promoter were studied. According to the expression level of green fluorescence protein andβ-galactosidase activity, the activity of fic and katE promoter are relatively low compared to spc, rpoS and nlpD promoter.The synthetic operon was also employed to study the impact of multiple promoters in an operon and promoter locates between genes. Five copies of trc promoter core region were synthesised and cloned to the promoter-cloning region of the promoter evalution system. The activity of multiple-trc promoter is 2.47-fold stronger than the activity of trc promoter. Both spc and trc promoter were cloned to the promoter-cloning region next to each other. The activity of the spc-trc promoter is stronger than either promoter alone. It suggests the possible application of multiple promoters in increasing the expression level of genes in metabolic engineering. To accumulate Poly (3-hydroxybutyrate-co-4-hydroxybutyrate) in Escherichia coli with unrelated carbon sources, cat2 gene from Clostridium kluyveri, gabD from Eschrichia coli, gbD gene from Ralstonia eutropha and YihU gene gene from Eschrichia coli were expressed as synthetic operon. The succinate-producing strain expressing the synthetic operon was used to produce P (3HB-co-4HB). P (3HB-co-4HB) was detected.