Microorganisms could produce a large variety of biologically active secondary metabolites representing a vast diversity of fascinating molecular architecture,which usually spurs particular attentions for chemical synthesis,mode of action,biosynthesis,and even drug discovery.Our lab.focus on the biosynthetic studies of the complex antitumor antibiotics.Previously,we cloned the biosynthetic gene clusters of saframycin(SFM)A from Streptomyces lavendulae and kosinostatin(KST)from Micromonospora sp.TP-A0468,and proposed their biosynthetic pathways; herein,we described the additional tailoring steps toward the fully understanding of the pathways.In SFM-A biosynthesis,we demonstrate that a membrane-bound peptidase,SfmE,hydrolyzes the fatty acyl chain to release the amino group; then,SfmCy2,a secreted oxidoreductase through covalent association of FAD,performs the following oxidative deamination extracellularly.These results not only fill in the missing steps of SFM-A biosynthesis,but also reveal a unexpected FAD-binding oxidoreductase acting on complex substrates to remove an amino group,as well as a previously unknown extracellular tailoring step in natural product biosynthesis.In KST pathway,we uncover an unprecedented four-enzyme-participated hydroxyl regioisomerization process.Firstly,KstA15 and KstA16 function together to catalyze a cryptic hydroxylation of the 4-hydroxyl-anthraquinone core yielding a 1,4-dihydroxyl product,which undergoes a chemically challenging asymmetric reduction dearomatization acted by KstA11 subsequently; then,KstAl0 catalyzes a regioselective dehydration to afford the 1-hydroxyl anthraquinone.Remarkably,the shunt products obtained by trapping of the hydroxylation and dehydration processes,detailed structural characterization of KstA11,and site-directed mutagenesis reveal mechanistic insights into the redox dearomatization and rearomatization steps.These findings provide a distinguished tailoring paradigm for type Ⅱ PKS engineering.