Preliminary Study On Genome Reduction In Bacillus Subtilis

This study aims to obtain reduced genome Bacillus subtilis strain via marker-lessgene knock-out as a platform to study their ability to synthesize riboflavin.First, we selected B.subtilis168RibC+Δupp as the starting strain for genomesimplification research. According to the literature reports, we selected targetedregions including similar prophage regions skin, prophage3, pks operon which isresponsible for encoding polyketide synthesis of secondary metabolites and prophageSPβ. Finally we obtained simplified strains of BSZ10. BSZ10strain was deleted223.199Kb genome, which coverd the entire genome of5.3%.We deleted non-essential genes by upp-based counterselective method. Targetdregions can be deleted through the two-step single crossover. Step1is the integrationof the deletion plasmid into the chromosome of the starting strain, selected asresistance to chloromycetin. Depending on the location of the second recombinationalevent, the5-FuRtransformants were either wild type or had target region deletedwithout retention of the upp-cassette and could be further verified by colony PCR.Then we selected four strains with reduced genomes for fermentation, namelyBSZ5strains (strains without pks, genome reduced74.837Kb), BSZ7strains (strainswithout skin and pks, knockout fragment size121.399Kb), BSZ8strain (strainswithout skin, pks and Pro3, genome reduced132.199Kb) and BSZ10strains (strainswithout skin, pks, Pro3as well as some SPβ area, the genome is reduced223.199Kb).Compared with the starting strain, the growth rate and riboflavin synthesis of genomesimplify strains were similar to that of the staring strain, CDW were higher, glucoseconsumption rate was increased.