Piperamide biosynthesis in black pepper (Piper nigrum)

黑胡椒的差异RNA-Seq方法已经得到了两个编码胡椒胺合成酶活性的全长转录本的鉴定和功能鉴定，以及一个编码细胞色素P450型单加氧酶的转录本的初步鉴定，该转录本是胡椒碱芳香部分亚甲二氧基桥形成所必需的。在当前建议的剩余八个月时间框架之后再继续一年的项目，将使我们能够继续探索胡椒胺的生物合成，并具体完成对已确定的胡椒胺合成酶的研究，使其成为微生物生产多种酰胺的高潜力酶的新来源，并可能在生物技术和药理学中应用。建议的重点放在胡椒胺合成酶的结构方面，将使我们能够将单个蛋白质结构域和氨基酸与底物偏好、混杂和立体选择性联系起来。基于抗体的胡椒碱生物合成的定位和利用MALDI-MS成像鉴定果实发育过程中与胡椒胺相关的代谢物将在细胞水平上提供这一专门途径的第一个视角。最后，为了进一步探索胡椒目中胡椒胺生物合成的多样性，我们打算从选定的胡椒目中收集RNA-Seq信息，以从序列同源性和底物谱方面对胡椒胺生物合成基因进行计算鉴定和功能比较。

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.