A lanGT4 mutant of the landomycin A producer Streptomyces cyanogenus S136 was constructed, leading to the production of landomycin D with two deoxy sugars in the side chain and proving that LanGT4 is responsible for attaching the third deoxy sugar of the hexasaccharide side chain. Heterologous expression of lndGT4 of the landomycin E producer Streptomyces globisporus 1912 in the lanGT4 mutant restored landomycin A production, indicating that LndGT4, like LanGT4, also has the ability to work iteratively. A S. cyanogenus S136 mutant with a mutation in lanGT1, encoding a d-olivosyltransferase, was shown to produce landomycin I with one deoxy sugar and, surprisingly, a new landomycin derivative (landomycin L) containing a d-olivose followed by an l-rhodinose. Heterologous expression of lndGT1 of S. globisporus 1912 in the lanGT1 mutant did not restore landomycin A production but led to the formation of a second new landomycin derivative (landomycin K) containing an unusual pentasaccharide chain (d-olivose-d-olivose-l-rhodinose-d-olivose-l-rhodinose). The formation of landomycin L and landomycin K is most probably attributed to the high substrate flexibility of the rhodinosyltransferase LanGT4.