Excellence in Research: Biosynthetic investigation of manzamine class alkaloids

锰胺是海洋生物碱的天然产物。它们具有复杂的结构，并表现出许多生物活性，包括抗癌和抗菌活性。缺乏对生物碱分子和生化基础的了解一直是生物工程中新的生物碱衍生物的主要障碍。该项目将提供这些知识，并将作为新的曼扎胺衍生物的生物工程基础，在医学，农业和生物技术方面具有潜在的应用。该项目还将为马里兰大学东岸的本科生和研究生提供广泛的研究经验，其中许多人来自STEM领域代表性不足的群体，因此这项工作将有助于发展多元化的STEM劳动力。Manzamines是一种结构独特的海洋生物碱天然产物，与其他生物碱几乎没有相似之处。manzamine的结构独特性，包括manzamine A，来自于一个复杂的五环核心连接到一个-羰基单元的组装。虽然已经从海洋海绵中分离出一百多种具有广泛生物活性的锰胺，但生物体如何组装积木和中间体来提供复杂结构的锰胺支架仍然是一个谜。拟议项目的主要目标是确定生物合成构建块和途径中间体，并阐明生产曼扎明a的关键生物合成步骤。拟议项目采用多管齐下的实验方法，包括：(A)同位素结合实验，以确定β -卡伯碱和五环的生物合成构建块；(B)实验，以确定这两个结构单元的连接所涉及的酶；(C)对Micromonospora sp. M42的全基因组序列分析，以确定manzamine的生物合成位点；(D)异源生产和鉴定manzamine A生物合成途径所涉及的酶。通过该项目获得的结果将为通过生物信息学分析发现其他锰胺铺平道路。具有潜在新催化活性的Manzamine A生物合成酶将成为进一步推进生物合成途径工程/合成生物学研究项目的宝贵工具，特别是用于生产新的Manzamine类似物。因此，拟议的项目提供了一种生物替代品，以合成生产新的manzamine生物碱。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Manzamines are members of marine alkaloid natural products. They possess complex structures and exhibit a number of biological activities, including anticancer and antibacterial activities. Lack of knowledge of the molecular and biochemical basis for the production of these alkaloids has been a major barrier to the bioengineering of new alkaloid derivatives. This project will provide such knowledge and will serve as the foundation for bioengineering of new manzamine derivatives with potential applications in medicine, agriculture and biotechnology. This project will also provide extensive research experiences for University of Maryland Eastern Shore undergraduate and graduate students, many of whom are from groups underrepresented in STEM, and thus the work will contribute to development of a diverse STEM workforce. Manzamines represent a structurally distinct group of marine alkaloid natural products with little resemblance to other alkaloids. The structural uniqueness of manzamines, including manzamine A arises from the assembly of a complex pentacyclic core attached to a beta-carboline unit. Although over a hundred manzamines that exhibit a wide range of biological activities have been isolated primarily from marine sponges, how organisms assemble building blocks and intermediates to furnish complex structural scaffolds of manzamines has remained a mystery. The main objective of the proposed project is to identify biosynthetic building blocks and pathway intermediates and elucidate key biosynthetic steps involved in the production of manzamine A. The proposed project utilizes a multi-pronged experimental approach including: (A) isotope incorporation experiments to identify biosynthetic building blocks of the beta-carboline and the pentacyclic ring, (B) experiments to identify the enzyme involved in the attachment of these two structural units, (C) complete genome sequence analysis of Micromonospora sp. M42 for identifying manzamine biosynthetic locus, and (D) heterologous production and characterization of select enzymes involved in the manzamine A biosynthetic pathway. The results garnered through this project will pave the way for the discovery of other manzamines through bioinformatic analysis. Manzamine A biosynthetic enzymes with potentially new catalytic activities will be an invaluable addition to the existing repertoire of tools for the further advancement of research projects concerning biosynthetic pathway engineering/synthetic biology, particularly for the production of new manzamine analogs. The proposed project thus offers a biological alternative to the synthetic production of new manzamine alkaloids.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.