Characterization Of Promoters From Saccharomyces Cerevisiae And Escherichia Coli And Application In Metobolic Engineering

Regulation of metabolic pathway is the core of metabolic engineering and syntheticbiology; it can be regulated at the level of transcription, translation and protein. Thepromoter is the most effective way to control gene transcription and regulate metabolic fluxas a functional element of metabolic engineering. The constitutive promoters were clonedand characterized based on the analysis of genomes sequences of Saccharomyces cerevisiaeand Escherichia coli, and the cloned promoters were applied to metabolic engineering inthis thesis; the main results were as follows:(1) Eleven constitutive promoters were cloned from glycolysis pathway and aminoacyl tRNA synthase genes in Saccharomyces cerevisiae, these promoters were characterizedusing eGFP and LacZ as report genes at transcription and translation levels. The resultsshowed that the cloned promoter had certain biodiversity in the strength. It also found thatthe constitutive promoters from different pathways behaved differently during the growth,and the strength of promoters using galactose as a carbon source were stronger than usingglucose as carbon source.(2) The effect of transcription factor binding sites (TFBSs) on promoters was found byanalysis the sequences of promoters, and verified by knockout the TFBSs of Rap1, Gcr1and Gcr2in promoter FBA1p and GPM1p. It was found that the strength of promoter wasincreased by fusion the TFBSs to other promoters, and effects were different by fusion ofdifferent copy number of TFBSs or different combination of TFBSs.(3) The pathway of xylitol production from xylan was constructed in Saccharomycescerevisiae by cloning xylanase, xylosidase, xylose reductase and promoters whichcharacterized in this thesis through the method of synthetic biology. The yield of xylitolwas increased by90%after optimization of the heterogenous pathway transcriptional levelin engineering strain. Glucose and galactose were used as co-substrates in the fermentationand two-stage fermentation was employed to improve the production of xylitol. After theoptimization of the fermentation process, the yield of xylitol could reach0.71g xylitol/gxylan. (4) Five constitutive promoters were cloned from glycolysis pathway in Escherichiacoli, these promoters were characterized using eGFP as report gene at transcription andtranslation levels. The results showed that the cloned promoter had certain biodiversity inthe strength, compared with the commonly used promoter T7, the strength of the strongestpromoter GAPAp was8.92%of T7. The pathway of xylitol production from xylose wasconstructed by promoter GAPAp and T7. After optimization of fermentation process ofengineered strain BL-GAPAp and BL-T7, it was found that the yield of BL-GAPAp wasmuch higher than BL-T7. Based on the analysis of enzyme activity assay and SDS-PAGE,xylose reductase was in the form of soluble protein and the activity was high in BL-GAPAp;xylose reductase was in the form of inclusion body and the activity was low in BL-T7.