Expanding the Toolkit for Metabolic Engineering
Expanding the Toolkit for Metabolic Engineering Yao Zong (Andy) Ng Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2016 © 2016 Yao Zong (Andy) Ng All rights reserved ABSTRACT Expanding the Toolkit for Metabolic Engineering Yao Zong (Andy) Ng The essence of metabolic engineering is the modification of microbes for the overproduction of useful compounds. These cellular factories are increasingly recognized as an environmentally-friendly and cost-effective way to convert inexpensive and renewable feedstocks into products, compared to traditional chemical synthesis from petrochemicals. The products span the spectrum of specialty, fine or bulk chemicals, with uses such as pharmaceuticals, nutraceuticals, flavors and fragrances, agrochemicals, biofuels and building blocks for other compounds. However, the process of metabolic engineering can be long and expensive, primarily due to technological hurdles, our incomplete understanding of biology, as well as redundancies and limitations built into the natural program of living cells. Combinatorial or directed evolution approaches can enable us to make progress even without a full understanding of the cell, and can also lead to the discovery of new knowledge. This thesis is focused on addressing the technological bottlenecks in the directed evolution cycle, specifically de novo DNA assembly to generate strain libraries and small molecule product screens and selections. In Chapter 1, we begin by examining the origins of the field of metabolic engineering. We review the classic “design–build–test–analyze” (DBTA) metabolic engineering cycle and the different strategies that have been employed to engineer cell metabolism, namely constructive and inverse metabolic engineering.
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