Piperamide biosynthesis in black pepper (Piper nigrum)

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The differential RNA-Seq approach of Piper nigrum already resulted in the identification and functional characterization of two full length transcripts encoding enzymes for piperamide synthase activities as well as the preliminary identification of a transcript encoding cytochrome-P450-type monooxygenase, required for methylenedioxy bridge formation of the aromatic part of piperine. To continue the project for an additional year beyond the remaining eight months’ time frame of the current proposal will allow us to continue the exploration of the piperamide biosynthesis and specifically complete the investigations on the identified piperamide synthases to a level where they serve as a new source of enzymes with high potential in the microbial production of a huge diversity of amides with possible applications in biotechnology and pharmacology. The proposed focus on structural aspects of piperamide synthases will enable us to correlate individual protein domains and amino acids to substrate preference, promiscuity, and stereoselectivity. Localization of piperine biosynthesis based on antibodies and the identification of piperamide related metabolites during fruit development by MALDI-MS-imaging will provide a first view of this specialized pathway at the cellular level. Finally, to further explore the diversity of piperamide biosynthesis within the Piperales we intend to collect RNA-Seq information from selected Piperales to computationally identify and functionally compare orthologous piperamide biosynthetic genes with respect to sequence identity and substrate profile.