Metabolic Engineering Of Riboflavin Operon And Respiratory Chain Of Bacillus Subtilis

Based on riboflavin biosynthetic reaction, a series of genetic engineering Bacillus subtilis for riboflavin over production were constructed in this study. The effects of promoter replacement and the amplification of ribA on riboflavin biosynthesis were studied. The respiratory chain was reconstructed by knocking out the bd oxidase. A biochemical reaction network was assembled with metabolites, and then the comparison analysis of metabolic fluxes in batch cultures was carried out between the bd mutant and the control. The traits of riboflavin overproducing Bacillus subtilis were improved by protoplast fusion. The main results presented in this work are as follows:The primary promoter of riboflavin operon was successfully substituted with the constitutive strong promoter P43 and SPO2 respectively. A series of ribP1 substituted Bacillus subtilis were constructed by different integration mechanism using different integration vectors. B. subtilis PK constructed by integration of the pUC18 carried P43-rib fragment into B. subtilis RH13 chromosome via compell-type mechanism made a showy display of its riboflavin biosynthetic ability which was about ten times higher than the host strain.A biochemical reaction network of engineered riboflavin overproducing Bacillus subtilis was constructed. The theoretical yields with riboflavin biosynthetic rate of 0.48 mmol (glucose)/g (CDW)/h and growth yield of 0.47 g cell C/g glucose C were obtained in B. subtilis PK by linear programming using Lingo software.The effects of ribA amplification on riboflavin biosynthesis of strain B. subtilis RH13,B. subtilis PK and B. subtilis PK/pXJ96 showed that the expression of ribA gene was the limit factor for riboflavin over production in the strain with lower riboflavin operon expression level.A series of cyd mutants were constructed. The mutants showed more rapidly specific growth rate, higher biomass yield, lower maintenance requirement, and higher riboflavin biosynthetic ability than the control. Compared to by-products formation, a discernible shift with less acetate and more acetoin in cyd mutant was observed.Comparative flux analysis between B. subtilis PK/pXJ96 and B. subtilis PK cyd/pXJ96 showed that the fluxes in PP pathway and also the fluxes used for biomass biosynthesis were enhanced in cyd mutant. The overflow metabolism was also...