Preliminary Study Of Riboflavin Production In Bacillus Subtilis From Glycerol

This study focused on enhancing the riboflavin production of Bacillus subtilisgrowth on glycerol by the combination of evolution engineering and metabolicengineering. The growth characteristic of strain BSU45was optimized throughadaptive laboratory evolution. Based on the evolved strain BSU3-78-2, some genesrelated to respiratory chain, purine pathway, pentose phosphate pathway andriboflavin operon were modified for improving the biosynthesis of riboflavin in B.subtilis.The evolved strain was achieved by serial passage for78times, and the specificgrowth rate was increased from0.34h-1to0.43h-1when growth on M9medium withglycerol.The cyd encoding cytochrome bd oxidase was deleted in order to improve theefficiency of energy production in cell, and strain BSUL2was achieved. Batchcultures of strain BSUL2showed that the riboflavin concentration reached43.8mg/L,and increased by10%. At the same time, the ability of utilization of glycerol was notchanged.The purFMNHD in purine pathway was over-expressed by promoter P43. But theriboflavin production did not improved significantly, one possible reason is that themetabolic flux through purine pathway is not the bottleneck for riboflavinbiosynthesis in BSUL1.The ywjH in the non-oxidative pentose phosphate pathway was over-expressed,which led to32.9%increased riboflavin production (up to58.2mg/L) with glycerol ascarbon source in shake flask, while introduction of zwf243or gnd361in oxidativepentose phosphate pathway from Corynebacterium glutamicum did not increaseriboflavin production. It indicated that the precursor ribulose-5-phosphate forriboflavin biosynthesis was mainly supplied through non-oxidative pentose phosphatepathway in B. subtilis cultivated on glycerol.Overexpression of riboflavin operon in BSUL4by plasmid pMX45showedsignificant improvement of riboflavin production (up to226.9mg/L), which increased283%and424%compared with that of BSUL4and BSUL1.