Glycosyltransferase DesVII and its auxiliary partner DesVIII from Streptomyces venezulae, homologs of EryCIII and EryCII in Saccharopolyspora erythraea, have previously been demonstrated to be flexible on their substrates in vitro. Herein, we investigated their in vivo function by interspecies complementation in the mutant strains of Sac. erythraea A226. As desVII and desVIII were concomitantly expressed in the Delta eryCIII mutant, the erythromycin A (Er-A) production was restored. Interestingly, co-expression of desVII and desVIII in the Delta eryBV mutant exhibited an increased Er-A yield by 15 % in comparison to A226. Hence, DesVII/DesVIII not only replaced EryCIII to upload D-desosamine to C5 position of 3-O-mycarosyl erythronolide B (MEB) but also in vivo attached L-mycarose, not D-desosamine to C3 position of erythronolide B (EB) with a higher activity than EryBV. Furthermore, expression of desVII in Delta eryCIII and Delta eryBV-CIII partially restored the Er-A production; however, no Er-A was detected while desVII was expressed in Delta eryBV. It was implicated that DesVII coupled with EryCII to form the DesVII/EryCII complex for attaching above two deoxysugars in the absence of EryCIII in Sac. erythraea. In addition, when desVII and desVIII were co-expressed in Delta.eryBV-CII, Er-A was recovered with a lower yield than Delta eryBV-CIII. Our study presents an opportunity with Sac. erythraea as a cell factory for macrolide glycodiversification.