CAREER: Investigation and Redesign of Nonribosomal Peptide Synthetases

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Intellectual merit: The project involves an investigation and redesign of biosynthetic pathways of known natural product biomolecules. The engineering of such pathways is currently one of the most promising routes toward the development and production of novel structurally diverse biologically active molecules. Efficient combinatorial biosynthesis critically depends on mechanistic information on enzymes involved in natural product formation. Incompletely understood, intriguing, and even unique enzymes are found in the gene cluster of the bisintercalator natural product thiocoraline. The project uses in vitro reconstitution of the thiocoraline biosynthetic pathway, predominantly through mutagenetic manipulation, mechanistic, and structural characterization of individual enzymes, in working towards (i) gaining the ability to manipulate the derivatization of the amino acid L-tryptophan for future production of novel bisintercalators and thiopeptide antibiotics, (ii) understanding the mechanism of stimulation of activity of adenylation domains (integral components of nonribosomal peptide synthetase assembly-lines) by MbtH-like proteins and the potential role of these enzymes in combinatorial biosynthesis, (iii) gaining insight into the function of interrupted adenylation domains, and (iv) developing novel adenylation domains with broad or altered substrate specificity to extend the ability to manipulate nonribosomal peptide synthetase assembly-lines for combinatorial synthesis. The research has the potential to advance the understanding of the mechanism of unique enzymatic transformations and is expected to contribute to the development of new tools for engineering of natural biosynthetic pathways.Broader impacts: Through the proposed activities of the project, students from local high schools surrounding the University of Michigan, as well as undergraduate and graduate students, will receive the hands-on experience of modern research in the fields of biochemistry and structural biology. Through the development of a freely accessible database on nonribosomal peptides, polyketides, and their hybrids, undergraduate and PharmD students will be introduced to research and will have the opportunity to raise their overall scientific literacy. Finally, through a high school outreach program, high school students will not only be introduced to modern and interdisciplinary science, but also will be well informed on and provided with guidance for planning a successful and rewarding scientific career. The goal is to establish an integrated educational and research program to develop future scientists interested in working at the interface of chemistry and biology and to spark and foster the interest of students in science at different stages of their education.