Multi-strategy engineering unusual sugar TDP-L-mycarose biosynthesis to improve the production of 3-O-α-mycarosylerythronolide B in Escherichia coli

The insufficient supply of sugar units is the key limitation for the biosynthesis of glycosylated products.The unusual sugar TDP-L-mycarose is initially attached to the C3 of the polyketide erythronolide B,resulting in 3-O-α-mycarosylerythronolide B(MEB).Here,we present the de novo biosynthesis of MEB in Escherichia coli and improve its production using multi-strategy metabolic engineering.Firstly,by blocking precursor glucose-1-phosphate competing pathways,the MEB titer of triple knockout strain QC13 was significantly enhanced to 41.2 mg/L,9.8-fold to that produced by parental strain BAP230.Subsequently,the MEB production was further increased to 48.3 mg/L through overexpression of rfbA and rfbB.Moreover,the CRISPRi was implemented to promote the TDP-L-mycarose biosynthesis via repressing the glycolysis and TDP-L-rhamnose pathway.Our study paves the way for efficient production of erythromycins in E.coli and provides a promising platform that can be applied for biosynthesis of other glycosylated products with unusual sugars.