Semester of Graduation

Summer 2019

Degree

Master of Science in Mechanical Engineering (MSME)

Department

Mechanical & Industrial Engineering

Document Type

Thesis

Abstract

In E. coli, fatty acid synthesis is catalyzed by the enzyme acetyl-CoA carboxylase (ACC), which converts acetyl-CoA into malonyl-CoA. Malonyl-CoA is a major building block for numerous of bioproducts. Multiple parameters regulate the homeostatic cellular concentration of malonyl-CoA, keeping it at a very low level. Understanding how these parameters affect the bacterial production of malonyl-CoA is fundamental to maximizing it and its bioproducts. To this end, competing pathways consuming malonyl-CoA can be eliminated, and optimal nutritional and environmental conditions can be provided to the fermentation broth. Most previous studies utilized genetic modifications, expensive consumables, and high-cost quantification methods, making unfeasible the development of an economically-attractive process with high product yield. In this work, we propose a low-cost, simple, and effective method to maximize and quantify malonyl-CoA production in E. coli. The enzyme, 1,3,6,8-tetrahydroxynapthalenesynthase (THNS) catalyzes the condensation of five molecules of malonyl-CoA forming 1,3,6,8- tetrahydroxynaphthalene (THN), which is auto-oxidized into flaviolin. Flaviolin can be measured spectrophotometrically, meaning that direct quantification of malonyl-CoA is accomplished by measuring the absorbance of flaviolin. Results showed that the main parameters associated with malonyl-CoA maximization are the use of rich medium supplemented with glucose and metals, the incubation temperature of 37C, the inoculum incubation time, and the use of lactose as an inducer. Moreover, a kinetic model of the bacterium metabolism was built in order to guide the maximization of malonyl-CoA. By performing a stability analysis and a metabolic control analysis, the system was determined to be stable and flux controlled by phosphoglucose isomerase. This suggests that this enzyme should be modified to optimize the production of malonyl-CoA.

Committee Chair

De Queiroz, Marcio

Available for download on Friday, May 13, 2022

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