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

RUI：细胞色素P450的定向进化用于合成吡咯海洋天然产物

• 批准号: 2102225
• 负责人: Takashi Suyama
• 金额: $ 22.5万
• 依托单位: The Waynesburg University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2102225&HistoricalAwards=false
• 关键词: RUI; Directed; Evolution; Cytochrome; P450

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.

在化学系化学合成（SYN）项目的支持下，Takashi L。Waymont大学的Suyama将致力于开发一种混合化学/生物催化剂，用于合成一类有趣的生物活性多溴芳基醚。 大部分研究将与本科生一起进行，该项目的酶工程方面将与加利福尼亚大学圣地亚哥分校的布拉德利摩尔合作进行。 这种混合路线显示出有效合成药用重要化合物的前景。酶工程将通过定向进化方法进行，使用化学试剂引起的随机突变，然后根据其所需的化学性质选择突变的酶。一些本科生将通过这个奖项的财政支持，在学年和夏季投入更多的时间到这个研究项目，并将获得宝贵的经验，这将推动他们在科学，技术，工程和数学（干）成功的职业生涯。该实验室的毕业生在人口统计学上是多样化的，并已被安置在著名的研究生院。高中学生也将在夏季由实验室主办，参加研究作为推广的一部分。拟议的资金是几十年来该大学收到的第一笔重要的联邦研究补助金，将对校园及其周边社区的研究和教育氛围产生巨大影响，这些社区位于联邦指定的阿巴拉契亚县。NSF支持的学生将有机会在同行评审的期刊上发表研究结果，并在国家科学会议上展示，并与来自经济发达地区的毕业生竞争。这些学生将通过利用大学的美国化学学会计划以及海洋生物学实验室来领导和参与对当地社区和K-12学生的科学宣传。多溴化芳烃，如多溴联苯醚（PBDEs）及其衍生物二苯并二恶英由于其毒性而引起关注。这些化合物在海洋环境中的来源有些模糊，因为有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在工业上生产的。TBFP（2，3，5，7-tetrabromobenzofuro[3，2-B]pyrrole）是最近从裸鳃类海洋假交替单胞菌中分离出来的一种新化合物，具有抗MRSA（耐甲氧西林金黄色葡萄球菌）的活性，并具有类似于PBDEs的3-苯氧基吡咯醚键。为了阐明TBFP的生物起源并提供容易获得这种抗MRSA化合物的途径，将进行TBFP的生物催化合成。为此，TBFP的推定生物合成前体，pentabromopsuedilin（PBP），将进行分子内Ullmann型反应催化的细胞色素P450酶参与PBP的生物合成，导致酚环化到吡咯环。研究小组将通过化学诱变和基于LCMS分析的选择来定向进化酶，从而提高该反应的产率。传统的Ullmann型反应条件与不稳定的吡咯不相容，并且在将PBP转化为TBFP中无效。这种工程酶在天然产物合成中的多功能性将被检查用于构建其他医学上重要的代谢物的支架，例如marinopyrrole B，其在吡咯的C3处具有类似于TBFP中的杂原子-C键。如果成功的话，这个项目将导致容易获得医学上重要的化学支架，以更广泛地适应工程生物催化剂在天然产物的全合成。这个奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。