Study On Structural Diversity Of Xiamenmycins And Polycyclic Tetramate Macrolactams From Mangrove-derived Streptomyces

Mangrove-derived actinobacteria are well-known for producing novel secondary metabolites and highly effective bioactive compounds.This study focused on the investigation of structural diversity of secondary metabolites,including xiamenmycins from Streptomyces xiamenensis 318 and polycyclic tetramate macrolactams(PTMs)from Streptomyces avicenniae 9-9.Xiamenmycins exhibit anti-fibrotic and anti-inflammatory activities.Xiamenmycin A belongs to benzopyran derivative with isoprenoid moiety and amino acid side chain.The biosynthetic pathway can be regarded as three steps,i.e.,isoprenylation and oxidation-cyclization to form benzopyran ring,as well as the post modification to form amide bond.To broaden the structural diversity of xiamenmycins,the strategy of combinatorial biosynthesis was adopted in this study.Xim A,an amide synthase,is involved in the last step of the xiamenmycin biosynthesis and responsible for incorporation of _L-threonine moiety into xiamenmycin B.We employed in vitro assays to clarify its enzymatic characteristics.Xim A exhibits specific selectivity on the amide donors.Xim A is an ATP-dependent amide synthase with both adenylation and transferase activity.In order to improve the enzyme promiscuity,we engineered Xim A by site-directed mutagenesis at specific position based on our theoretical model of Xim A.Thus,we produced diverse benzopyran derivatives with up to 15different _L-form or _D-form amino acids substitutions,catalyzed by several Xim A variants.This study provided xiamenmycin derivatives generated via combinatory biosynthesis to enlarge the structural diversity.In addition,based on genome mining,we analyzed the secondary metabolites from Streptomyces avicenniae 9-9 targeting on PTMs.We found four genes,named avi A-D involved in PTMs biosynthetic gene clusters.Two PTMs,15 and 16,were identified as ikaruganmycin and capsimycin B,respectively.Compound 17-19 also belonged to PTMs family,and their structures were proposed based on MS/MS fragmentation pattern.We proposed that compound 17 was C-3 hydroxyl substituted epoxyikarugamycin;compound 18 formed a double bond structure on the side chain of capsimycin B;compound 19 was C-30 hydroxyl substituted ikarugamycin.This study confirmed that mangrove-derived Streptomyces avicenniae 9-9 can produce PTMs compounds with the 5-6-5 fused tricyclic skeleton.