(3dy) Engineering Cofactor-Dependent Catalytic Enzymes in Biochemical Production

Cofactor-dependent enzymes such as oxidoreductases are ubiquitous and catalyze natural and synthetic reactions. They can catalyze reactions that are difficult to complete via chemical synthesis and have been used to produce a wide range of chemicals, including biofuels, cosmetics, and pharmaceuticals. While their versatility makes them attractive for biochemical synthesis, their applicability is challenged by factors that include: native preferences for a specific cofactor (eg., NAD(P)H, NAD(P)+), need for a partner enzyme for electron transfer, and enzyme instability. For example, the cofactor availability in the host system, the compatibility of the cofactor with a regenerating enzyme, and the cofactor's cost can hinder the use of an enzyme for biosynthesis. Cytochrome P450s also show promise as biocatalysts due to their ability to activate the C-H bond, which is difficult to accomplish by chemical synthesis. However, P450s generally require a separate reductase to transfer electrons to the catalytic site, and some non-native pairs result in reduced activity. Thus, engineering cofactor-dependent enzymes is an important approach to improve biochemical productions. Furthermore, there are still many unexplored cofactor-dependent enzymes that need to be studied.

I developed my interest in protein engineering and biochemical reaction optimization during my PhD and postdoctoral research. These experiences shaped my knowledge and enthusiasm for improving enzymatic catalysis. In my postdoctoral research, I have worked with cofactor dependent enzymes such as cytochrome P450 and NADH producing GapA, IcdE, and Zwf. I have been encapsulating the NADH producing enzymes in bacterial microcompartments (MCPs) to create a privileged redox pool within the compartment. The redox pool can facilitate the reverse β-oxidation pathway's encapsulation into MCP to produce medium-chain fatty acids, as the pathway involves NADH dependent enzymes. MCPs are proteinaceous organelles that compartmentalize enzymes and cofactors necessary to metabolize niche carbon sources, such as 1,2-propanediol (Pdu). They reduce competition of the intermediate metabolic reaction steps with the cytosolic enzymes. Collaborative work with the graduate student Nolan Kennedy led us to discover that PduA and PduJ, two of the proteins that make up the Pdu MCP shell, are necessary and redundant for the protein shell assembly [1]. In a separate collaborative project with Prof. David Baker of the University of Washington, I am using protein engineering techniques to create a brighter variant of a mini-fluorescence-activating protein. In my graduate research, I also used a protein engineering approach to modify antimicrobial peptide histatin 5. I designed variants with enhanced antifungal activity against Candida albicans and/or reduced susceptibility to cleavage, and thus inactivation, by secreted aspartic proteases produced by C. albicans [2,3]. In addition to the histatin 5 work, I developed a purification-free method to immobilize in vivo biotinylated single-chain antibody fragments (scFvs) from cell lysate using the biotin-streptavidin interaction [4]. I also conceptualized and led the work to study the effect of linkers on the scFv immobilization with undergraduate students I mentored [5].

In my future lab, I plan to engineer various cofactor-dependent enzymes for applications in the biosynthesis of desirable biochemicals, including biofuels and pharmaceuticals. I can achieve this goal by 1) converting an enzyme's NADPH dependence to NADH dependence or vice versa, focusing on enzymes that have not yet been engineered or for which previous work led to reduced catalytic activity; 2) elucidating design rule for creating efficiently active pairs of cytochrome P450s and reductases using linkers; 3) enhancing biocatalytic efficiency by immobilizing cofactor-dependent enzymes on to a protein scaffold and enhancing their stability. Through these approaches, I aim to gain information on engineering not only an individual enzyme but also find patterns that will direct and simplify future enzyme engineering efforts.

Teaching Interest

Part of my teaching philosophy emphasizes the need to understand the fundamental concepts rather than memorize them in all the courses. My extensive chemical engineering training in academia and industry enables me to teach all core chemical engineering courses. Through teaching assistantship during my graduate studies, I have the experience and enjoyed teaching mass and energy balances and unit operations, for which I received a Chemical and Biochemical Engineering Department TA of the Year Award. I also co-instructed and led lectures in thermodynamics as a Future Faculty Fellow. In addition to course teaching, I have also strengthened my one-on-one teaching through the mentorship of four graduate, four undergraduate, and two high school students in laboratory research. Since my research interest involves enzyme kinetics, I would like to teach kinetics and incorporate examples from my own research into the class. In the future, I would also like to develop special topic courses on protein engineering and enzyme kinetics.

Postdoctoral Projects:

1. “Elucidating assembly and permeability of 1,2-propanediol utilization bacterial microcompartments for heterologous pathway incorporation”
2. “Enhancing the brightness of mini-fluorescence-activating protein using comprehensive codon mutagenesis”

Postdoctoral Advisor: Prof. Danielle Tullman-Ercek, Chemical and Biological Engineering, Northwestern University, Evanston, IL

PhD Dissertation: “Protein engineering approaches to improving diagnosis and treatment of Candida albicans infections”

PhD Advisor: Prof. Amy J. Karlsson, Chemical and Biomolecular Engineering, University of Maryland, College Park, MD.

Patent: Jabra-Rizk MA, Karlsson AJ, and Ikonomova SP. “Histatin-5 based synthetic peptides and uses thereof,” US Patent 10,464,977, 2019.

Selected publications:

1. Kennedy NW*, Ikonomova SP*, Lee MS, Raeder HW, Tullman-Ercek D. Self-assembling shell proteins PduA and PduJ have essential and redundant roles in bacterial microcompartment assembly. Submitted. *Equal contributions.
2. Ikonomova SP, Moghaddam-Taaheri P, Jabra-Rizk MA, Wang Y, and Karlsson AJ. Engineering improved variants of the antifungal peptide histatin 5 with reduced susceptibility to Candida albicans secreted aspartic proteases and enhanced antimicrobial potency. FEBS J. 2018 285:146-159.
3. Ikonomova SP, Moghaddam-Taaheri P, Wang Y, Doolin MT, Stroka KM, Hube B, and Karlsson AJ. Effects of histatin 5 modifications on antifungal activity and kinetics of proteolysis. Protein Sci. 2020 29(2):480-493.
4. Ikonomova SP, He Z, Karlsson, AJ. Simple and robust approach to immobilization of antibody fragments. J Immunol Methods. 2016 435, 7 – 16.
5. Ikonomova SP, Le MT*, Kalla N*, and Karlsson AJ. Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction. Biotechnol Appl Biochem 2018 65(4):580-585. *Equal contributions