Construction Of Genome Reduced Bacillus Subtilis And Their Application In Production Of Bio-based Chemicals

In present study,Bacillus subtilis 168 was selected to reduce genome via large scale deletion and a series of genome-reduced strain of Bacillus subtilis were constructed.We investigated changes in physiological phenotype of genome-reduced mutants.Some genome-reduced strains were engineered to produce guanosine,thymidine,acetoin,riboflavin by introducing some specific genetic modifications.Protential of genome-reduced strain as chassis cell were evaluated.The main results are as follows:We sequentially deleted all prophage,large operons for secondary metabolite biosynthesis and some nonessential regions via a scarless gene deletion method.The sizes of deleted regions finally added up to 845.6 kb.These genome reduced strains showed decreased growth rates,sporulation ratios,transformation efficiencies and maintenance coefficients,as well as increased cell yields.Changes in physiological phenotype were related to deletion of nonessential genes.Genome-reduced strains were engineered to produce guanosine and thymidine,respectively.Strain BSK814G2,in which purA was knocked out,and prs,purF and guaB were co-overexpressed,produced 115.2 mg/L of guanosine,which was 4.4 fold as much as the production of the parental strain engineered with the same gene modifications.We also constructed a thymidine producer by deleting tdk gene and overexpressing prs,ushA,thyA,dut,and ndk genes from E.coli in strain BSK756,and the resulting strain BSK756T3 accumulated 151.2 mg/L thymidine,showing a 5.2-fold increase compared with the control strain.We also explored possibility of genome-reduce strains as chassis of aceoin and riboflavin production.By introducing some modification of target pathway,we anaylysis and compared difference between genome–reduced strains and their parental strain under same modifications.The results showed that genome-reduced strains could potential producer because of lower mainance energy and higher expression efficiency of genes.