Here we report the isolation, structure elucidation, and antimicrobial activity of two novel nikkomycin derivatives, nikkomycins Lx and Lz, synthesized as main components by a genetically engineered mutant of Streptomyces tendae Tu901. The mutant was obtained by inserting a kanamycin resistance gene in the nikkomycin biosynthesis gene nikF encoding a cytochrome P450 monooxygenase. Cultivation of the mutant in SP production medium and analysis of the culture filtrate by HPLC and agar diffusion assay against Paecilomyces variotii showed that the mutant did not synthesize nikkomycins Z, X, J, and I, but accumulated two novel compounds named nikkomycins Lz and Lx. UV/Vis diode-array spectral analyses indicated uracil as the base for nikkomycin Lz and 4-formyl-1,3-dihydro-2H-imidazole-2-one as the base for nikkomycin Lx. Structure elucidation by electrospray mass spectrometry and 1H- and 13C-NMR spectroscopy revealed that nikkomycin Lx is identical to nikkomycin X except for the lack of a hydroxy group on the pyridyl ring of the arylhomothreonine moiety, and nikkomycin Lz is the uracil analogue of nikkomycin Lx. Nikkomycins Lx and Lz were significantly more stable under alkaline conditions than nikkomycins X and Z. Antimicrobial activity assays showed that the activity of nikkomycin Lx against various test organisms was slightly lower or similar to that of nikkomycin X, while nikkomycin Lz was expected to have activities similar to those of nikkomycin Z. Synthesis of these derivatives was achieved via molecular genetic manipulation, and their peptidyl moiety, 2-amino-4-hydroxy-3-methyl-4-(2'-pyridyl)butanoic acid (nikkomycin E), is probably a biosynthetic intermediate hydroxylated by the nikF-encoded P450 monooxygenase to form the peptidyl moiety of nikkomycins I, J, X, and Z. Nikkomycins Lx and Lz complete the series of nikkomycin derivatives lacking the hydroxy group at the pyridyl ring of the peptidyl moiety.