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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-a-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. [ABSTRACT FROM AUTHOR]