Title: Engineering Electron Transfer Processes in Oxidoreductases
Description: As the demand for cost-efficient and environmentally friendly processes increases in the chemical industry, impact of biocatalysis, which is the utilization of enzymes and whole microorganisms for production of fine chemicals, has become more predominant. From pharmaceuticals to cosmetics, biocatalysts are widely used in various sectors, and their significance have dramatically intensified with the introduction of initial protein engineering techniques in 1980s. As the field of protein engineering has evolved over the last few decades, its integration with other disciplines such as process engineering and synthetic biology is now more critical for establishing non-natural pathways and reactions to produce broader range of chemicals. While developing an interdisciplinary approach, few strategies have emerged to be more prevalent: (i) better integration of biocatalysts with (nano)devices, and (ii) use of protein based scaffolds for creating novel synthetic multienzyme cascades. Throughout this doctoral thesis, applicability of these ideas with oxidoreductases was investigated.

Oxidoreductases are a class of under-utilized enzymes that catalyze the electron transfer between different metabolites, while at the same time use cofactors (NAD(P)(H), molecular oxygen, etc.) as the electron supplier. In Chapter 2, the electron transfer mechanism of a monooxygenase, cytochrome P450 27B1 (CYP27B1), was mimicked for electrochemical sensing of a vitamin D form (25(OH)D) in solution. Natural electron transfer pathway of this enzyme uses NADPH and two electron transfer proteins for conversion of 25(OH)D to its product. Inspired by this mechanism, this enzyme was mixed with an artificial redox mediator and immobilized on an electrode surface. As a result of rigorous experiments, CYP27B1-modified electrode was found to detect 25(OH)D in its physiological range. This is a significant result as it opens a new way for development of a vitamin D biosensor that can diminish the amount of required cost and time for testing. In the next chapter of the thesis, effects of changing the size of cofactor on catalysis of dehydrogenases were studied in detail.

Natural cofactors of two different redox enzymes were chemically modified with PEG, and kinetic experiments were conducted in order to better understand the relation between transport phenomena and biocatalysis. It was found that when the size of the cofactor was increased, two enzymes were affected differently; while efficiency of one enzyme was not altered significantly, that of the other dropped dramatically. Through comprehensive analysis, dominant impact of PEGylation was determined to be due to the differences in the interactions of PEGylated cofactors and enzymes. This study showed that protein engineering methods can be utilized to gain insights into better understanding of the relationship between mass transfer and catalysis in engineered bioprocesses and biocatalytic cascades. In Chapter 4, PEGylated cofactors were used to create artificial multienzyme complexes. In this study, SpyCatcher-SpyTag scaffold was utilized for wiring two redox enzymes and by tethering with PEGylated cofactors, a new biocatalyst with self-contained redox chemistry was obtained.

Detailed kinetic analysis showed that this new multienzyme cascade was able to catalyze a reaction that was thermodynamically downhill but kinetically very slow in the absence of any enzyme. This also proved that attached cofactor acts as a ‘swing-arm’, carrying electrons from one enzyme to another; similar to the unique mechanism of pyruvate dehydrogenase complex. Generality of this methodology was investigated by constructing an immobilized three-enzyme-containing biocatalyst, which was hypothesized to catalyze an industrially important reaction under very mild conditions. This work is a significant contribution to the field, and a good demonstration of use of protein engineering for process engineering applications. Chapter 5 concludes this thesis with a study that investigates the practicability of a collagen mimetic peptide as a novel way of constructing multiprotein cascades. Collagen mimetic peptides are composed of three individual strands that might (homotrimer) or might not (heterotrimer) have identical sequences, and in this work, we have utilized a recently designed hydroxyproline-free sequences of a heterotrimer collagen mimetic peptide.

Individual strands were attached to different proteins by genetic fusion, and optimum experimental conditions for self-assembly of a multiprotein complex were investigated. Initial results suggested formation of such a complex, but further experiments are required to finalize the confirmation. This new collagen-based platform studied in this chapter is a crucial step towards development of cofactorless multienzyme cascades. Finally, this doctoral thesis demonstrates the prominence of protein engineering in biocatalysis applications by utilizing various strategies together with the electron transfer mechanisms of oxidoreductases. By expanding and building upon these methodologies, it is possible to obtain more improved biosensors and functional artificial multienzyme cascades with industrial applications. Hence, this study is a promising example to exhibit the impact of interdisciplinary approach on industrial biotechnology.