“I’m a greater believer in luck, and I find the harder I work the more I have of it” Thomas Jefferson
My name is Nestor Hernandez Lozada. I have a bachelor’s degree on chemical engineering from the University of Puerto Rico-Mayaguez. I am currently finishing my third year of a PhD in Chemical and Biological Engineering at University of Wisconsin-Madison under Dr. Brian Pfleger. My research focuses on the engineering of Escherichia coli for the production of medium chain fatty acids (6-12 carbons). In Escherichia coli, fatty acids are produced in the fatty acid biosynthesis pathway. One of the most important steps to engineer in the biosynthesis of fatty acids is the final step of the pathway, which involves the cleavage of a thiol bond in acyl-ACP by a thioesterase. My focus in the last year has been expressing different thioesterases in E. coli. One of my projects is working on the development of an industrially relevant strain of E. coli for the production of octanoic acid. On another project I am doing collaborations with Dr. Costas D. Maranas at Penn state to engineer a thioesterase for which the crystal structure is known, to change its substrate specificity. I am also looking at the E coli membrane and its effects on relieving of membrane stresses due to fatty acid overproduction. All these projects are different approaches to the same problem which is to improve medium chain fatty acid production in E. coli. My career objective is to work on strain development and fermentation scale-up research in the chemical industry.
Free fatty acids (FFA) are precursors to important oleochemicals such as alcohols, aldehydes, and amines. The current supply of FFA is limited by availability of oil crops such as canola, rapeseed, and coconut. Demand for oil crops have created tensions between food, fuel, and environmental stewardship. Therefore, microbial hosts are being developed as an alternative production route for FFA. Thioesterases are enzymes that hydrolyze the thiol bond in fatty acyl-ACP producing a FFA and its substrate specificity determines the chain length obtained. Moreover, since this step acts as both a product sink and a deregulation mechanism it is critical to have a thioesterase that is highly active to keep the inhibitory fatty acyl-ACP pool low, and specific towards the desired chain length to maximize its production. Here, we used a computational approach to engineer the highly active E. coli thioesterase I (TesA) to decrease its specificity to its best natural substrate myristoyl-ACP (14:0) and increase the specificity to lauroyl-ACP (12:0) and caproyl-ACP (C8:0). TesA crystal structure was used to guide the algorithm to calculate the binding energy of the substrates upon configurational changes imposed to TesA. Mutants predicted were validated experimentally on E. coli and results were used to guide subsequent iterations. After three round of predictions, seven mutants with main specificity towards lauroyl-ACP were obtained. The best two mutants exhibited 2-fold and 8-fold increases in lauroyl-ACP and caproyl-ACP specificity, respectively, compared to wild type without affecting the enzyme activity. These successful results highlight the potential of computational approaches combined with experimental validation in protein engineering.