A novel sansanmycin analogue, sansanmycin Q (1), was identified by genome mining from the fermentation broth of Streptomyces sp. SS (CPCC 200442). In comparison with other sansanmycin compounds, sansanmycin Q has an extra glycine residue at the N-terminus of the pseudopeptide backbone. The additional glycine was proved to be assembled to sansanmycin A by SsaB, a tRNA-dependent aminoacyltransferase, based on the results of rescrutiny of sansanmycin biosynthetic gene cluster, and then overexpression and knockout of ssaB in the wild-type strain. The structure of sansanmycin Q was assigned by interpretation of NMR and mass spectral data. The results of the bioassay disclosed that sansanmycin Q exhibited more potency against Mycobacterium tuberculosis H Rv and a rifampicin- and isoniazid-resistant strain than sansanmycin A.