Genome Reduction Of Bacillus Subtilis And Its Effects On The Production Of Riboflavin And Thymidine

Engineering chassis cells with reduced genome is an important research aspect in synthetic biology. The aim of this study was to construct reduced genome Bacillus subtilis strain and to test its ability for the production of riboflavin and thymidine.We proceeded genome reduction from BSZ11 which was a Bacillus subtilis strain with 200 kb sequences deleted in the genome. Using double crossover recombination with upp as counter selectable marker, we deleted ynxB-dut, yoaV-yobO, pdp-rocR, ycxB-sipU, yisB-yitD, yrkS-yraK, and yybP-yyaJ genome region and finally constructed strain BSZ18 with 500 kb genome sequence deleted.We then tested the ability of strains with varied deleted genome length for the production of riboflavin. We found that riboflavin production increased as genome sized reduced. Riboflavin production in BSZ18 increased 20% compared with undeleted strain. Further introducing plasmid PMX45 containing riboflavin operon into the tested strains significantly increased riboflavin production and the results followed the same trend that riboflavin production increased as genome size reduced. With PMX45, BSZ18 yielded 2.3-fold higher riboflavin production than undeleted strain.We further tested the ability of BS745, a strain with 745 bp genome sequence deleted, for the production of thymidine. Knocking out pyrimidine anaplerotic pathway gene tdk in BS745(resulting in BST745) allowed to accumulate 12.46 mg/L thymidine while did not accumulate thymidine in undeleted strain. In BST745, overexpressing thymidine biosynthetic pathway genes(ushA, thyA, dut, and ndk) and overexpressing two key genes in the purine biosynthetic pathway(prs and ywlf) resulted in 50% and 27% improvement in thymidine production, respectively. On the other hand, the same genetic manipulations only resulted in trace amount thymidine accumulation in undeleted strains. These results indicated that cells with reduced genome had superior ability for riboflavin and thymidine production than undeleted strain and could potentially work as chassis cells for the construction of high-performance microbial cell factories.