Biosynthesis Of Diketopiperazine Alkaloids From Aspergillus Versicolor

Indoledione piperazine alkaloids are a class of natural cyclic dipeptides produced by filamentous fungi.They have significant and extensive biological activities,such as antibacterial,antiviral,antitumor,immunomodulatory and antioxidant.Therefore,this class of compounds is important lead molecules in drug/pesticide development.Previous studies have found that different sources and types of piperazine alkaloids often have different biosynthetic mechanisms.Therefore,elucidation of the biosynthetic process of diketopiperazine alkaloids is of great significance for understanding the diversity of fungal biosynthesis strategies and exploring new catalytic elements.Additionally,it can provide the basis of gene,protein and chemical principle for rational modification and design of new derivatives.Aspergillus versicolor HBU-7 is a fungal strain isolated from the intertidal zone of the Bohai Sea,China,which can produce various indoledione piperazine monomeric compounds(mostly enantiomer)and a rare cyclobutyl skeleton-based dimer(±)asperginmlin A.It remained a mystery about how is the cyclobutyl formed and what is the molecular basis for the various isomer product-formation.In this study,whole genome sequencing and biosynthetic gene cluster prediction were performed towards the HBU-7 strain,followed by the capture of the diketopiperazine skeleton biosynthetic gene cluster asp by using NotE,a key enzyme in the synthesis of the precursor compound brevianamide F,as a probe.The biosynthetic mechanism of these compounds was investigated by a combination of in vivo gene heterogeneous expression,in vitro enzyme function reconstruction and 16 labeling experiments.According to the in vivo gene heterogeneous expression results,the catalytic function and sequence of aspA～D encoded proteins in the asp gene cluster were identified,and a catalytic pathway was initially established to brevianamide Q(BQ)from the initial substrate L-Trp and L-Pro.The catalytic function and mechanism of a-KG oxidase AspC were systematically studied by reaction reconstruction in vitro.It was found that AspC catalyzed the hydroxylation reaction of brevianamide V(B V)to generate a reaction site configuration reversal product(-)-BQ.It was found that(-)-BQ can undergo configuration isomerization under acidic conditions,such as(±)-BQ and brevianamide K(BK).Based on the catalytic principle of α-KG oxidase,the enzymatic mechanism of the atypical"configuration reversal" hydroxylation mediated by AspC and the chemical mechanism of the spontaneous racemization of(-)-BQ were speculated.It was found that AspC could catalyze a variety of diketopiperazine isomers to produce racemated products through the substrate breadth test.In addition,the production of dimer AA is a non-enzymatic reaction process under complex conditions.In conclusion,a new biosynthetic pathway of indoledione piperazine alkaloids was revealed by genome mining,biochemical characterization,and spontaneous reaction exploration.In particular,the α-KG oxidase AspC was found to be the molecular basis of the various isomer products.These results provided new insights into the biosynthetic mechanism of indoledione piperazine alkaloids,and enriched the the catalytic function of αKG oxidase,and laid catalytic and chemical mechanism-foundations for further research and modification of AspC to create more derivatives.