Of polyketides and fatty acids : in vitro reconstitution and analysis of metabolic pathways

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As a tool, in vitro reconstitution of metabolic pathways has a strong record of illuminating biological processes. The pathways for biosynthesis of secondary metabolite polyketides and primary metabolite fatty acids involve very similar catalytic steps and related protein machinery. Each strategy has a growing intermediate chain elongated stepwise with acetate-based building blocks. Here, two main systems were reconstituted in vitro: an unusual assembly line polyketide synthase from human pathogenicity-associated Nocardia bacteria, and the fatty acid synthase of cyanobacterium Synechococcus sp. PCC 7002, a potentially industrially relevant strain for alkane production. While polyketides have been a rich source of antibiotics and other useful molecules, the majority currently in use were discovered decades ago by searching cultured microorganisms or soil samples for produced molecules and screening for a specific function, such as antibacterial activity. Though fruitful in the past, a newer approach is to use sequenced genomes. Here the discovery method using in vitro reconstitution began the decoding process for a so-called "orphan" (natural product unknown) gene cluster. Interestingly, the cluster was found in nine related potentially pathogenic species isolated from nocardiosis-diseased patients. Proteins of the system were heterologously expressed and purified from E. coli. Adding cofactors and substrates and using a stable isotope 13C-labeling strategy, heptaketide and octaketide products were identified from in vitro reactions and characterized by 2D NMR. Looking at another system, kinetic analysis was performed on the in vitro reconstituted fatty acid synthase (FAS) of Synechococcus sp. PCC 7002. Cyanobacteria are desirable hosts for biodiesel production, because they are photosynthetic, relatively fast growing, and can secrete products. The overall FAS rate was exclusively limited by the FabH ketosynthase, which initiates product synthesis. This finding sharply contrasts with the related E. coli FAS, which is predominantly limited by its dehydratase (FabZ) and enoyl reductase (FabI) activities, showing that the FAS regulatory behavior is a property of controlling enzyme(s). In all cases, the goal was to learn something by taking Nature's complex biosynthetic machinery outside of a cell and placing it into a test tube, allowing near complete control over what components are added in a reaction.


Type of resource text
Form electronic; electronic resource; remote
Extent 1 online resource.
Publication date 2016
Issuance monographic
Language English


Associated with Kuo, James
Associated with Stanford University, Department of Chemical Engineering.
Primary advisor Khosla, Chaitan, 1964-
Thesis advisor Khosla, Chaitan, 1964-
Thesis advisor Sattely, Elizabeth
Thesis advisor Smolke, Christina D
Advisor Sattely, Elizabeth
Advisor Smolke, Christina D


Genre Theses

Bibliographic information

Statement of responsibility James Kuo.
Note Submitted to the Department of Chemical Engineering.
Thesis Thesis (Ph.D.)--Stanford University, 2016.
Location electronic resource

Access conditions

© 2016 by James C Kuo
This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).

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