RUI: Directed Evolution of Cytochrome P450 for Synthesis of Pyrrole Marine Natural Products

在化学系化学合成(SYN)计划的支持下，韦恩斯堡大学的Takashi L.Suyama将致力于开发一种混合化学/生物催化方法，用于合成一类有趣的、具有生物活性的多溴芳醚。大部分研究将在本科生中进行，该项目还有一个酶工程方面的内容，将与加州大学圣地亚哥分校的布拉德利·摩尔合作进行。这条杂交路线显示了高效合成具有重要药用价值的化合物的前景。酶工程将通过定向进化的方法进行，使用化学试剂引起的随机突变，然后根据所需的化学性质选择突变的酶。许多本科生将获得该奖项的资助，以便在学年和暑假期间投入更多时间进行这一研究项目，并将获得宝贵的经验，这些经验将推动他们在科学、技术、工程和数学(STEM)领域取得成功。这个实验室的毕业生在人口统计学上是不同的，他们被安置在著名的研究生院。作为推广活动的一部分，该实验室还将在夏季接待高中生参与这项研究。拟议的资金是该大学几十年来收到的第一笔重要的联邦研究拨款，将极大地影响校园及其周围社区的研究和教育环境，这些社区位于联邦指定的阿巴拉契亚县。NSF资助的学生将有机会在同行评议的期刊上发表研究结果，并在国家科学会议上发表，并与来自经济优势地区的毕业生展开竞争。这些学生将利用该大学的美国化学学会计划和海洋生物实验室，领导和参与对当地社区和K-12学生的科学宣传。多溴芳烃，如多溴二苯醚(PBDEs)及其衍生物二苯并-对二恶英，因其毒性而引起人们的关注。海洋环境中这类化合物的来源有些含糊，因为一些多溴二苯醚及其衍生物已被证明是天然产物，而大多数多溴二苯醚是作为阻燃剂工业生产的。TBFP(2，3，5，7-四溴苯并呋喃并[3，2-b]吡咯)最近从海洋裸枝杆菌中分离出来，具有抗甲氧西林耐药金黄色葡萄球菌(MRSA)活性，并具有类似于多溴联苯醚(PBDEs)的3-苯氧基吡咯醚键。为了阐明TBFP的生物发生机制，并提供方便地获得这种抗MRSA化合物的途径，我们将继续进行TBFP的生物催化合成。为此，可能的生物合成前体五溴苏糖素(PBP)将在参与PBP生物合成的细胞色素P450酶的催化下发生分子内Ullmann型反应，导致酚环化到吡咯环上。研究小组将致力于通过化学诱变和基于LCMS分析的选择来定向进化酶，以提高该反应的产率。传统的Ullmann型反应条件与不稳定的吡咯不相容，不能有效地将PBP转化为TBFP。这种工程酶在天然产物合成中的多功能性将被用来构建其他医学上重要的代谢物的支架，如Marinopyrole B，它在吡咯的C3处具有类似于TBFP中的杂原子-C键。如果成功，该项目将使人们能够方便地获得具有重要医学意义的化学支架，使工程生物催化剂在天然产物的全合成中得到更广泛的适应。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemical Synthesis (SYN) program in the Division of Chemistry, Takashi L. Suyama of Waynesburg University will work to develop a hybrid chemo/biocatalytic entry into the synthesis of an interesting and biologically active class of polybrominated aryl ethers. Most of the research will be conducted with undergraduate students and there is an enzyme engineering aspect of the project to be conducted in collaboration with Bradley Moore of the University of California-San Diego. This hybrid route shows promise for efficient synthesis of medicinally important compounds. The enzyme engineering will be conducted via a directed evolution approach using random mutation caused by chemical reagents, followed by selection of mutated enzymes based on their desired chemical properties. A number of undergraduate students will be financially supported by this award to devote more time to this research project both during the academic year and summers and will gain invaluable experience that will propel them into successful careers in science, technology, engineering, and mathematics (STEM). Graduates from this laboratory are demographically diverse and have been placed in prestigious graduate schools. High school students will also be hosted by the lab during the summer to participate in the research as part of outreach. The proposed funds are the first significant federal research grant received by the University in decades and will drastically impact the research and education climate on campus and its surrounding communities, which are located in a federally designated county of Appalachia. NSF-supported students will have opportunities to publish findings in peer-reviewed journals and present them at national scientific conferences and compete well against graduates from economically privileged areas. These students will lead and participate in scientific outreach to local communities and K-12 students by leveraging the University’s American Chemical Society program as well as the marine biology lab.Polybrominated aromatics, such as polybrominated diphenyl ethers (PBDEs) and their derivatives dibenzo-p-dioxins have attracted attention due to their toxicity. The origin of such compounds in the marine environment has been somewhat ambiguous because some PBDEs and their derivatives have been shown to be natural products while the majority of PBDEs are produced industrially as flame retardants. Recently isolated from marine Pseudoalteromonas found on nudibranchs, TBFP (2,3,5,7-tetrabromobenzofuro[3,2-b]pyrrole) has shown anti-MRSA (methicillin resistant Staphylococcus aureus) activities and possesses 3-phenoxypyrrole ether linkage reminiscent of PBDEs. To elucidate TBFP’s biogenesis and to provide facile access to this anti-MRSA compound, a biocatalytic synthesis of TBFP will be pursued. To this end, the putative biosynthetic precursor for TBFP, pentabromopsuedilin (PBP), will be subjected to an intramolecular Ullmann-type reaction catalyzed by the cytochrome P450 enzyme involved in the biosynthesis of PBP to cause the phenolic cyclization onto the pyrrole ring. The research team will work to improve the yield of this reaction by directed evolution of the enzyme through chemical mutagenesis and selection based on LCMS-analysis. Traditional Ullmann-type reaction conditions are not compatible with labile pyrroles and are ineffective in converting PBP to TBFP. The versatility of this engineered enzyme in natural product synthesis will be examined for constructing the scaffolds of other medicinally important metabolites, such as marinopyrrole B, that possess a heteroatom-C bond at C3 of pyrrole similar to that in TBFP. If successful, this project would lead to facile access to medicinally important chemical scaffolds for wider adaptation of engineered biocatalysts in total synthesis of natural products.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.