Enzymes offer a transformative solution to traditional chemical catalysts, providing both highly selective and highly pure compounds for the fine chemical, pharmaceutical, and insecticide industries. Despite significant progress, the enzymatic toolbox remains somewhat limited, spurring scientists in the biocatalysis field to strive for the expansion of chemical reactivities. The aim of this thesis was to study the Old Yellow Enzymes (OYEs) chemical versatility, in an attempt to broaden their reaction portfolio. This work is organized into five research chapters: four focusing on the chemical reactivities of OYE, and the fifth on a scaled-up reaction. We begin with an introduction outlining the state-of-the-art of OYEs and conclude with a conclusion and outlook. A detailed overview of each chapter is provided in the following paragraphs.

Chapter one provides an overview of our current understanding of OYEs, covering their history, physiological roles, classification among enzymes, structural characteristics, coenzymes, chemical reactivities, and their potential applications within industry.

In chapter two, we reveal how versatile OYEs are, by exploring an unknown reactivity, the stereoselective monoreduction of α,β-dicarbonyls towards chiral α-hydroxycarbonyls. We investigated ten aromatic, cyclic, aliphatic α,β-dicarbonyl compounds and evaluated their reduction using five OYEs and one flavin-independent double bond reductase (DBR). The most effective substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, which was converted to phenylacetylcarbinol with 91% conversion using OYE3 (R-selectivity >99.9% ee).

In chapter three, we continue to highlight OYEs’ versatility by yet another reactivity, the semireduction of allenes. Six activated allene substrates were screened against eighteen enzymes, including sixteen OYEs and two DBRs. The best results occurred using a class I OYE, PETNR, with 99% conversion of 10 mM ethyl-2,3-pentadienoate to ethyl-pent-3-enoate (E:Z ratio, 49:51). High selectivity was observed with class II OYE3 using methyl 2-methyl-2,3-pentadienoate as a substrate (81% conversion, E:Z ratio, 11:89), as well as with another class II OYE, EBP1 and ethyl 2-methyl-2,3-butadienoate (87% conversion, 97% ee).

Chapter four covers our re-examination of OYEs’ oxidative reaction, developing a new method for selective desaturation without requiring high temperatures. We show that by a simple pH adjustment, the active site tyrosine is deprotonated and serves as a catalytic base. Several OYEs and substrates were screened to demonstrate this desaturation method. This study expands the range of biocatalytic applications for OYEs, introducing an elegant approach to synthesizing chiral α,β-unsaturated carbonyl compounds.

In chapter five we further examined the intricacies of oxidation, by assessing whether redox potential influences desaturation. We measured the redox midpoint potential of eleven OYEs and their mutants from various classes, focusing on specific active site mutations that might shed light on desaturase activity. Our findings revealed a range of redox potentials across the different OYE classes, but no clear correlation between desaturation activity and redox potential. We examined the active site’s threonine/cysteine near the flavin N5 position and the proton-donating tyrosine with mutant enzymes to understand their role in desaturation.

In chapter six we demonstrate that OYEs are well suited for industrial use, by carrying out a 150 g/L scale-up for monoterpene asymmetric reduction. Until now, OYEs have rarely been applied in scale-up reactions, with limited turnover numbers of 102-104. We present a preparative scale using the thermostable OYE from Thermus scotoductus (TsOYE) for the asymmetric reduction of activated alkenes achieving a record turnover number of 123,000 with 1 M of (S)-carvone (98% conversion, 90% isolated yield) towards product (2R,5S)-dihydrocarvone with a diastereomeric excess of 92% (>99% ee).

In general this work advances the understanding of the biocatalytic reactivity of the OYE family, demonstrating their capacity to catalyze diverse and novel chemical reactions towards industrial applications.
... Read more

Despite the increasing demand for efficient and sustainable chemical processes, the development of scalable systems using biocatalysis for fine chemical production remains a significant challenge. We have developed a scalable flow system using immobilized enzymes to facilitate flavin-dependent biocatalysis, targeting as a proof-of-concept asymmetric alkene reduction. The system integrates a flavin-dependent Old Yellow Enzyme (OYE) and a soluble hydrogenase to enable H2-driven regeneration of the OYE cofactor FMNH2. Molecular hydrogen was produced by water electrolysis using a proton exchange membrane (PEM) electrolyzer and introduced into the flow system via a designed gas membrane addition module at a high diffusion rate. The flow system shows remarkable stability and reusability, consistently achieving >99% conversion of ketoisophorone to levodione. It also demonstrates versatility and selectivity in reducing various cyclic enones and can be extended to further flavin-based biocatalytic approaches and gas-dependent reactions. This electro-driven continuous flow system, therefore, has significant potential for advancing sustainable processes in fine chemical synthesis. ... Read more

Ene-reductases from the old yellow enzyme (OYE) family have been traditionally employed in the reduction of conjugated C═C double bonds. This study explores the underutilized oxidative potential of OYEs, demonstrating their capability to catalyze the enantioselective desaturation of carbonyl compounds. Utilizing a deprotonated tyrosine residue as a catalytic base, we developed a method to enable OYE-catalyzed desaturation at ambient temperature and alkaline pH without the need for high-temperature conditions. Through screening of various OYE enzymes, we identified several candidates from different genera with enhanced desaturase activity across different substrates. This work broadens the scope of biocatalytic applications for OYEs, introducing a novel approach to the synthesis of chiral α,β-unsaturated carbonyl compounds. ... Read more

Ene reductases (EREDs) catalyze asymmetric reduction with exquisite chemo-, stereo-, and regioselectivity. Recent discoveries led to unlocking other types of reactivities toward oxime reduction and reductive C–C bond formation. Exploring nontypical reactions can further expand the biocatalytic knowledgebase, and evidence alludes to yet another variant reaction where flavin mononucleotide (FMN)-bound ERs from the old yellow enzyme family (OYE) have unconventional activity with α,β-dicarbonyl substrates. In this study, we demonstrate the nonconventional stereoselective monoreduction of α,β-dicarbonyl to the corresponding chiral hydroxycarbonyl, which are valuable building blocks for asymmetric synthesis. We explored ten α,β-dicarbonyl aliphatic, cyclic, or aromatic compounds and tested their reduction with five OYEs and one nonflavin-dependent double bond reductase (DBR). Only GluER reduced aliphatic α,β-dicarbonyls, with up to 19% conversion of 2,3-hexanedione to 2-hydroxyhexan-3-one with an R-selectivity of 83% ee. The best substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, with 91% conversion to phenylacetylcarbinol using OYE3 with R-selectivity >99.9% ee. Michaelis–Menten kinetics for 1-phenyl-1,2-propanedione with OYE3 gave a turnover kcat of 0.71 ± 0.03 s–1 and a Km of 2.46 ± 0.25 mM. Twenty-four EREDs from multiple classes of OYEs and DBRs were further screened on 1-phenyl-1,2-propanedione, showing that class II OYEs (OYE3-like) have the best overall selectivity and conversion. EPR studies detected no radical signal, whereas NMR studies with deuterium labeling indicate proton incorporation at the benzylic carbonyl carbon from the solvent and not the FMN hydride. A crystal structure of OYE2 with 1.5 Å resolution was obtained, and docking studies showed a productive pose with the substrate. ... Read more

Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN⁺). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN⁺ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN⁺ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA⁺), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)⁺. ... Read more