Metabolic Engineering Of Escherichia Coli For The Production Of Riboflavin And Flavin Mononucleotide

Flavins are a set of pteridine-based organic compounds derived from the isoalloxazine ring.Flavins include riboflavin（RF）,flavin mononucleotide（FMN）and flavin adenine dinucleotide（FAD）,which have been widely used in the food,feed,pharmaceutical and cosmetic industries.In this study,we engineered Escherichia coli using metabolic engineering strategies to produce RF and FMN,and constructed RF and FMN overproducing strains with high titer and yield,respectively.First,we designed 5 target-binding sequences of ribF,purA and guaC genes,which involved in the downstream or branch reactions of RF biosynthesis pathway.Among 15 single-s RNA expression strains,WY05 expressing anti-rib F₅had the strongest RF synthesis ability,and its titer increased from 552.2mg/L to 829.7mg/L;Secondly,co-expression of double-and triple-s RNAs further increased the RF titer to1091.3mg/L（WY23 expressing anti-rib F₅and anti-gua C₃）.Thirdly,the expression levels of anti-rib F₅and anti-gua C₃were fine-tuned by using 4 different promoters.The best strain WY40 produced 1454.5mg/L riboflavin with an increase of 163.4%compared to WY0T.At the same time,suitable promoters were selected to fine-tune the expression levels of anti-rib F₅and anti-pur A₅,and WY62 produced 1346.7mg/L riboflavin with a yield of 0.158g/g glucose,which was the highest yield among all reported riboflavin producing strains.Finally,anti-rib F₅,anti-gua C₃and anti-pur A₄were simultaneously regulated by using different promoters,and the titer of WY74reached 1326.0 mg/L.Therefore,fine-tuning the expression levels of the 3 genes using synthetic s RNA could significantly improve the RF titer and yield.On the other hand,the biosynthetic ability of RF and FMN in engineered strains was significantly improved by optimizing ATP supply and reducing power balance:The ATP supply was enhanced by knocking out the low-efficiency respiratory chain genes ndh,app B and cyd B;The conversion from NADPH to NADH was promoted by overexpressing sth A and knocking out pnt AB.PH20T produced 1211.3mg/L RF,which was 119.4%higher than that of WY0T.PH04S produced 1746.8mg/L FMN,which was 408.0%higher than that of PH00S and was the highest yield among all reported FMN producing strains.