In Vitro And In Vivo Metabolic Engineering Of Riboflavin Production By B.subtilis And Optimization Of Eco-Friendly Production Process

Riboflavin has been widely used in the fields of agriculture,medicine,and feed.Microbial fermentation is a popular method to produce riboflavin,and B.subtilis has been utilized as the chassis in riboflavin overproduction.To enhance riboflavin yield and establish an eco-friendly riboflavin production process,three sections of work were carried out in this paper.Firstly,the fermentation process are analyzed and regulated to improve riboflavin yield.The fermentation process is divided into four stages based on correlation analysis of physiological parameters in 10-L fermenters and the fermentation characteristics in every stage are elaborated.Subsequently in regulation of metabolic overflow,riboflavin yield increased by 15.3%by maintaining amino nitrogen concentration at 1-1.2 g/L.Secondly,metabolic flux of the pentose phosphate pathway(PPP),which provides precursors in riboflavin synthesis,is increased through in vitro and in vivo metabolic engineering.Sodium gluconate is substituted for glucose to increase metabolic flux of PPP in vitro.Flask tests show that the riboflavin titer increases from 636.7 IU/mL to 873.2 IU/mL,and the result proves that the direct usage of sodium gluconate can benefit for riboflavin production.The gntP(encoding gluconate permease)is overexpressed in B.subtilis RF to improve sodium gluconate uptake in vivo.The riboflavin titer ultimately reaches 1002.0 IU/mL after gntP overexpression.These results are also verified at the fermenter level.Thirdly,the recycling process of waste bacterium and wastewater are established.The acid-treated of waste bacteria are act as the nitrogen source for the next round of fermentation,which can be recycled at least three times and still promoted riboflavin synthesis;the fermentation medium of following rounds are prepared with 85%wastewater and 25%phosphate,which can be reused at least once and not interfere with riboflavin synthesis.Above processes are both validated at the shake flask and fermenter level.Based on the analysis of synthesis pathways and fermentation processes in B.subtilis,this study is conducted to improve the yield of riboflavin by metabolic engineering and fermentation regulation and establish an eco-friendly riboflavin production process.