ORCID
https://orcid.org/0000-0002-9791-4751
Date of Award
9-14-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
The scarcity of iron in the environment has driven microorganisms to evolve diverse iron acquisition strategies. Among these strategies, the synthesis and utilization of siderophores stand out as a crucial mechanism. Siderophores are a family of low-molecular weight organic compounds with a remarkable affinity for binding iron. Microbes employ nonribosomal peptide synthetases (NRPSs) and NRPS-independent siderophore (NIS) synthetases to assemble siderophores from precursor molecules, tailoring their structures and properties to meet their specific requirements. Advancements in the understanding of the structure and mechanism of NRPS and NIS enzymes have paved the way for the in silico prediction of product structures from the nucleotide sequences of biosynthetic gene clusters. This knowledge has allowed researchers to make informed inferences about the products synthesized by these enzymes and prioritize the study of new natural product systems. However, the predictive capacity of genome mining is still limited by the knowledge base of chemical logic governing natural product scaffold assembly. This thesis describes my research along with collaborative studies related to the biosynthesis of microbial siderophores, with a focus on two hydroxamate-containing siderophores, fimsbactin and desferrioxamine. These siderophores serve as prime examples of the diverse assembly patterns employed by NRPS and NIS enzymes, respectively. Through our research, we achieved the complete in vitro reconstitution of the biosynthetic pathway for each of these siderophores using purified enzymes, enabling us to conduct functional, mechanistic, and structural analyses. Our approach involved a combination of enzymatic and chemical probe-based studies to detect biosynthetic intermediates, as well as the use of synthetic substrates and mutated proteins allowing us to meticulously dissect the catalytic mechanism behind the NRPS-mediated assembly of fimsbactin. Additionally, in-depth bioinformatic analysis provided a comprehensive understanding of the peptide branching logic and product release mechanisms for NRPS-derived natural products. We employed synthetic intermediates, inhibitors, and chemical probes to investigate the structure and mechanism of DesD, the iterative NIS synthetase responsible for desferrioxamine production. Our investigations revealed the substrate recognition pattern of DesD and related homologs and the establishment of a unified mechanism for desferrioxamine assembly. By deciphering the underlying enzyme mechanisms and biosynthetic logic of NRPS and NIS derived natural products, we aim to advance the field for the prediction and engineering of siderophore biosynthetic pathways.
Language
English (en)
Chair and Committee
John-Stephen Taylor
Recommended Citation
Yang, Jinping, "Biosynthetic Strategies for Microbial Siderophore Assembly" (2023). Arts & Sciences Electronic Theses and Dissertations. 3176.
https://openscholarship.wustl.edu/art_sci_etds/3176