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dc.contributor.advisorBalskus, Emily
dc.contributor.authorZha, Li
dc.date.accessioned2019-08-08T08:19:59Z
dash.embargo.terms2020-11-01
dc.date.created2018-11
dc.date.issued2018-07-17
dc.date.submitted2018
dc.identifier.citationZha, Li. 2018. Characterization of the Colibactin Biosynthetic Pathway. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41121247*
dc.description.abstractColibactin is a genotoxin produced by human gut Escherichia coli strains. This metabolite is biosynthesized by the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymes encoded in a 54-kilobase biosynthetic gene cluster (the pks island) where all of the NRPS and PKS genes are required for genotoxicity. E. coli strains harboring the pks island induce DNA double strand breaks in HeLa cells and are implicated in the development of colitis-associated colorectal cancer in animal models. However, despite multiple attempts, the active genotoxin has not been isolated and its chemical structure is currently unknown. In the quest to solve the structure of colibactin, in vitro analyses of the PKS and NRPS modules from the pks island have allowed us to develop strategies for isolating pks-dependent metabolites and to formulate hypotheses for structure and biosynthesis of the active genotoxin. In addition to guiding isolation, we believe application of the in vitro approach is also indispensable for gaining mechanistic insights into individual steps of colibactin biosynthesis. In the past few years, my study of pks assembly line enzymes in vitro has led to the discovery of novel assembly line enzymology and advanced biosynthetic hypotheses to better predict colibactin’s structure. The non-canonical biosynthetic logic of the pks island will be presented in the following chapters. In Chapter 2, the study of PKS modules is reported with an emphasis on the formation, recognition, and incorporation of the rare PKS extender unit aminomalonyl-acyl carrier protein in the colibactin assembly line. We demonstrated that ClbG, a freestanding acyltransferase (AT), recognizes this building block and transfers it to multiple PKS modules from the pks gene cluster, including three atypical-AT PKSs (ClbC, the PKS module of ClbK, and ClbO) and a cis-AT PKS ClbI. These findings suggest that the pks assembly line has the potential to incorporate the non-canonical extender unit more than once into colibactin’s scaffold and will inform the identification of new aminomalonate-derived candidate precolibactins. Chapter 3 details the characterization of NRPS modules in the colibactin assembly line. We confirmed the predicted substrates of all NRPS modules and discovered an unusual NRPS module, ClbH, that uses S-adenosylmethionine (SAM) as a non-proteinogenic amino acid building block for amide bond formation and ClbI-mediated cyclopropanation. Our results revealed a distinct strategy for cyclopropane formation in natural product biosynthesis that requires direct activation of the underutilized carboxylic acid of SAM. This study also shed light on the biosynthetic origin of a functional group that is likely important for colibactin’s genotoxicity. Finally, Chapter 4 describes the characterization of an unusual PKS machinery that combines an iterative PKS module and an atypical-AT PKS module to incorporate malonate units in the early stage of colibactin biosynthesis. We established that the PKS module of the NRPS-PKS hybrid ClbB is capable of catalyzing two rounds of malonate extension. Although early models proposed that the second malonate unit was loaded onto the ACP domain of ClbC, results from our site-directed mutagenesis work indicate that this domain is not required for malonate extension. Collectively, studies presented in this thesis highlight the utility of in vitro biochemistry in revealing the unusual features of NRPS and PKS assembly lines that cannot be predicted from bioinformatics and providing insights into unprecedented biosynthetic logic of natural products.
dc.description.sponsorshipChemistry and Chemical Biology
dc.format.mimetypeapplication/pdf
dc.language.isoen
dash.licenseLAA
dc.subjectnatural product biosynthesis
dc.subjectnon-ribosomal peptide synthetases
dc.subjectpolyketide synthases
dc.subjecthuman microbiome
dc.subjectcolibactin
dc.subjectDNA damage
dc.subjectcolorectal cancer
dc.titleCharacterization of the Colibactin Biosynthetic Pathway
dc.typeThesis or Dissertation
dash.depositing.authorZha, Li
dash.embargo.until2020-11-01
dc.date.available2019-08-08T08:19:59Z
thesis.degree.date2018
thesis.degree.grantorGraduate School of Arts & Sciences
thesis.degree.grantorGraduate School of Arts & Sciences
thesis.degree.levelDoctoral
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy
thesis.degree.nameDoctor of Philosophy
dc.contributor.committeeMemberKahne, Daniel
dc.contributor.committeeMemberSzostak, Jack
dc.type.materialtext
thesis.degree.departmentChemistry and Chemical Biology
thesis.degree.departmentChemistry and Chemical Biology
dash.identifier.vireo
dc.identifier.orcid0000-0002-8465-0232
dash.author.emailzhalireed@gmail.com


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