The unparalleled structural and functional diversity of natural products has been possible due to the power of enzymes and multi-enzyme metabolic pathways. The fraction of this extensive biocatalytic repertoire has led to an array of natural products and natural product derivatives for use in various industries (pharmaceutical, cosmetic, agrochemicals etc.). Nevertheless, a significantly larger and more diverse universe of natural compounds and processes, as well as the enzymes and metabolic pathways that generate such molecules, remains untapped. As a result, a growing number of researchers are looking to engineer metabolic pathways to produce new compounds and novel chemistries. An in vitro strategy to manipulate these pathways offers a number of advantages and opportunities including the examination of the pathway free from cellular regulation. With the increasing availability of pure enzyme samples from genetically engineered organisms, an in vitro methodology appears to be feasible to produce novel chemistries from flexible pathways like the polyketide synthases.; The first part of this thesis research was focused on synthesizing a library of non-native polyketides taking advantage of the broad substrate specificity of a type III polyketide synthase (RppA). RppA is an iterative polyketide synthase that condenses five units of malonyl-CoA to produce an aromatic polyketide, flaviolin. Using several commercially available CoA esters, a range of 4-hydroxy-2-pyrone polyketide derivatives were synthesized. To further increase the structural diversity of polyketides, a class of broad specificity enzyme, peroxidases, was used to tailor flaviolin and other pyrones produced through RppA biocatalysis. A library of 31 novel polyketides including halogenated and coupled polyketide derivatives were synthesized. The approach developed in this study provides a new paradigm to exploit biocatalysis in the synthesis of complex natural product derivatives.; The second part of this thesis research was focused on the development of a microfluidic platform capable of performing single and multi-step enzymatic reactions to investigate in vitro tailoring of products from metabolic pathways. With the increasing popularity of microscale devices and the advantages they offer, such as improved sample handling, low reagent consumption, and ease of automation, among others, in vitro tailoring of metabolic pathways with non-pathway enzymes at the microscale offers existing opportunities. (Abstract shortened by UMI.)
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