Engineering of Heme-Dependent Monooxygenases Towards Heterocycle Conversion

Engineering of heme-dependent monooxygenases towards heterocycle conversion

von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Gustavo de Almeida Santos

Master of Science (Molecular Genetics)

aus Aveiro, Portugal

Berichter: Univ. Prof. Dr. rer. nat. Ulrich Schwaneberg

Univ. Prof. Dr. Ing. Lars M. Blank

Tag der mündlichen Prüfung: 02.10.2020

Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar.

Abstract

Abstract

Aromatic oxygen and nitrogen-containing heterocycles (O- and N- heterocycles) are significantly abundant in nature as they are an important class of bioactive molecules and are involved in a variety of fundamental biological functions. Benzo-1,4-dioxane and indole are O- and N- heterocycles respectively, and their regioselective hydroxylation can produce small to macrocyclic building blocks with great importance for drugs with antimicrobial, antigrastic, spasmolytic, antipsychotic, anxiolytic and hepatoprotective. Additionaly, these heterocycles can be used for the production of pesticides and dyes. However, the traditional chemical syntheses of said derivatives requires at least one of the following: rare and costly catalyst, heating, active cooling and multi-step reactions. The usage of P450 monooxygeanses can be used to hydroxylate these heterocycles in a more efficient and sustainable way. Three different monooxygenases were selected to investigate the structural determinants of activity over the benzo-1,4-dioxane and indole heterocycles. Cytochrome P450 BM3 monooxygenase from Bacillus megaterium, P450 Cand_1 monooxygenase from Pseudomonas sp. 19-rlim and P450 Cand_10 from Phenylobacterium zucineum. Each P450 was subjected to different engineering strategies: P450 BM3 to epPCR, P450 Cand_1 to sequence saturation method (SeSaM) and P450 Cand_10 to site-saturation-mutagenesis (SSM) to improve their activity towards benzo-1,4-dioxane and/or indole. Product-specific screening systems were developed and applied to efficiently identify improved variants in all generated libraries. Significantly improved variants from P450 Cand_10 and P450 Cand_1 were not found, P450 Cand_1 exhibited poor and inconsistent expression in 2.2 mL deep-well plates and for P450 Cand_10 the determinants for activity over the tested substrates are likely to be present outside the active site, in the active site tunnel access and/or protein shell. Regarding P450 BM3, the phenol detection 4-AAP assay, as well as the developed multiplex capillary electrophoresis (MP-CE), enabled simultaneous detection and quantification of the target product and side products in a 96-well format. Employing both methods allowed for the identification of position R255, which when substituted by leucine in the P450 BM3 WT leads to ≈140-fold increase in the initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), ≈21-fold increase in total turnover number (TTN) (WT: 40 ± 3; R255L: 860 ± 15), and, ≈2.9-fold increase in coupling efficiency (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis revealed that when R255 is substituted by leucine (substitution distant from the heme-cofactor) the previously existing salt-bridge between R255 and D217 (in WT) ceases to exist, introducing

i Abstract flexibility into the I-helix and rearranging the heme, thus allowing a more efficient hydroxylation. This improvement was not limited to hydroxylation of benzo-1,4-dioxane and ≈20-fold improvement in conversion of the O-heterocycles, phthalan, isochroman, 2,3- dihydrobenzofuran, benzofuran, and dibenzofuran was found. The improvement observed in variant R255L provides useful routes to produce pharmaceutical precursors in a selective and environmentally friendly way via late-stage hydroxylation.

ii Acknowledgments

Acknowledgments

I would like to use this section for showing my short but meaningful appreciation for all people that have been, in way or the other, helpful and supporting during my time at Lehrstuhl für Biotechnologie. Thank you, it was great meeting all of you.

This research was funded by European Union (EU) project OXYtrain (grant agreement no. 722390) under the EU’s Horizon 2020 Programme Research and Innovation actions H2020-EU.1.3.1. The views and opinions expressed in this work are only those of the authors and do not necessarily reflect those of the European Union Research Agency. The European Union is not liable for any use that may be made of the information contained herein.

iii Publications and Poster Presentations

Publications and Poster Presentations

Parts of this thesis have been published: de Almeida Santos, G.; Dhoke, G. V; Davari, M.D.; Ruff, A.J.; Schwaneberg, U. Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles. Int. J. Mol. Sci. Artic. 2019, 20.

Gärtner, A.* and de Almeida Santos, G.*; Ruff, A.J.; Schwaneberg, U. A screening method for P450 BM3 mutant libraries using multiplexed capillary electrophoresis for detection of enzymatically converted compounds. Methods in Molecular Biology; Humana Press Inc. 2020.

*shared co-authorship

Posters: de Almeida Santos, G., Ruff, A. J., Schwaneberg, U., “Evolution of heme-dependent monooxygenases toward robust industrial biocatalysts”. Enzymes, biocatalysis and chemical biology: The new frontiers, Pavia Italy, 9-12 September 2018 de Almeida Santos, G., Ruff, A. J., Schwaneberg, U., “Evolution of heme-dependent monooxygenases toward robust industrial biocatalysts”. ACIB 2017, Graz, Austria, November 2017

iv Table of Contents

Table of Contents

Abstract ...... i Acknowledgments ...... iii Publications and Poster Presentations...... iv Table of Contents...... v 1. Introduction ...... 1 1.1. Biocatalysis ...... 2 1.2. Monooxygenase Classes ...... 4 1.3. Heme-Dependent Monooxygenases ...... 4 1.4. P450 Monooxygenases as Biocatalysts ...... 7 1.4.1. P450 Bacillus megaterium 3 ...... 8 1.4.2. P450 Cand_1 & P450 Cand_10 ...... 10 1.5. Protein Engineering ...... 10 1.6. Screening Systems ...... 13 1.7. Aromatic heterocyclic compounds ...... 15 1.7.1. Indole and Indigo ...... 16 1.7.2. Benzo-1,4-dioxane and derivatives ...... 17 2. Objectives ...... 18 3. Materials and Methods ...... 20 3.1. Chemicals ...... 20 3.2. Enzyme and Kits ...... 20 3.3. Machines and Equipment ...... 21 3.4. Cultivation Media, Additives and Buffers ...... 22 3.5. Bacterial Strains, Vectors and Genes...... 23 3.6. Software ...... 24 3.7. Microbiological Methods ...... 25 3.7.1. Preparation of Escherichia coli Competent Cells...... 25 3.7.2. Transformation of Plasmid DNA into Escherichia coli Competent Cells ...... 25 3.7.3. Cryo-Culture Preparation ...... 26 3.7.4. Shake Flask Expression ...... 26 3.7.5. Production of Indigoids ...... 26 3.7.6. Cell Lysis via Sonication ...... 27 3.7.7. Mutant Library Preparation and Expression in Multi Well Plates ...... 27 3.7.8. Preparation of Cell Lysates in Multi Well Plate ...... 28 3.8. Molecular Biology Methods ...... 28 3.8.1. DNA Extraction, Storage and Sequencing ...... 28 3.8.2. Polymerase Chain Reaction (PCR) ...... 29 3.8.3. Oligonucleotide Design for PCR Amplifications ...... 30 3.8.4. Library Generation by Error-Prone PCR...... 30 3.8.5. Library Generation by Sequence Saturation Mutagenesis (SeSaM) ...... 30 3.8.6. Culture PCR ...... 30 3.8.7. Circularization of Linearized DNA ...... 30 3.8.8. Agarose Gel Electrophoresis ...... 31 3.8.9. Phosphorothioate-Based Ligase-Independent Gene Cloning (PLICing) ...... 31 3.8.10. Site Directed and Site Saturation Mutagenesis ...... 33 3.9. Biochemical Methods ...... 33 3.9.1. NADPH Depletion Assay for Screening of Mutant Libraries in Multi Well Plate ...... 33 3.9.2. 4-AAP screening system for product based quantification of 2,3-dihydro-1,4-benzodioxin-5-ol and 2,3- dihydro-1,4-benzodioxin-6-ol ...... 34 3.9.3. Product Based Screening System for Quantification of Indigo and Indirubin ...... 34 3.9.4. Capillary Electrophoresis for Side Product Quantification of 2,3-dihydro-1,4-benzodioxin-2-ol ...... 35 3.9.5. Statistical Evaluation and Selection of Variants ...... 36 3.9.6. Purification and Lyophilisation of P450 BM3 ...... 36 3.9.7. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) ...... 37 3.9.8. P450 Quantification by Carbon Monoxide Difference Spectrum ...... 38 3.9.9. Determination of NADPH Oxidation Rates and Coupling Efficiency of P450 BM3 Wild-type and Variants .... 38 3.9.10. Long Term Substrate Conversions ...... 40 v Table of Contents

3.10. Analytical and Chemical Methods ...... 41 3.10.1. Two-Phase Solvent Extraction of Reaction Products...... 41 3.10.2. Quantification of Indigo and Indirubin by High-Performance Liquid Chromatography ...... 41 3.10.3. GC-FID Measurements ...... 42 3.10.4. GC-MS Measurements ...... 42 3.11. Molecular Modeling ...... 42 3.11.1. Molecular Docking ...... 42 3.11.2. Molecular Dynamics Simulation...... 43 4. Results and Discussion ...... 44 4.1. P450 BM3 ...... 44 4.1.1. Selection of P450 BM3 starting variant and its substrate ...... 44 4.1.2. Using the 4-AAP Screening System for Product-Based Quantification ...... 45 4.1.3. Development of CE Screening System for Product-Based Quantification ...... 47 4.1.4. Development of a Gas Chromatography method for benzodioxins quantification...... 49 4.1.5. P450 BM3 Library Generation and Screening ...... 51 4.1.6. Characterization of P450 BM3 WT and Variants R255G and R255L in Respect to Hydroxylation of the Six Selected O-Heterocycles ...... 55 4.1.7. Rationale behind the Activity Improvement of R255G and R255L Variants over the WT ...... 61 4.1.8. Conclusion ...... 64 4.2. P450 Cand_1 ...... 65 4.2.1. Development of a Screening Systems for Product-Based Detection of Indigo and Indirubin ...... 65 4.2.2. Quantification of Indigo via High-Performance Liquid Chromatography ...... 67 4.2.3. Analysis of P450 Cand_1 tunnels ...... 68 4.2.4. P450 Cand_1 Library Generation and Screening ...... 69 4.2.5. Conclusion ...... 71 4.3. P450 Cand_10 ...... 72 4.3.1. Analysis of P450 Cand_10 tunnels ...... 72 4.3.2. P450 Cand_10 Library Generation and Screening ...... 73 4.3.3. Conclusion ...... 75 5. Final Summary ...... 75 6. References ...... 77 7. Appendix ...... 85 7.1. List of Tables ...... 85 7.2. List of Figures ...... 86 7.3. List of Equations ...... 90 7.4. List of Abbreviations ...... 91 7.5. Additional Experimental Information and Data ...... 91 7.5.1. Gene and Protein Sequences ...... 92 7.5.2. Primers and Vectors map ...... 94 7.5.3. Gas-chromatography programs and compound retention time ...... 96 7.5.4. Additional figures and graphs ...... 96

vi Introduction

1. Introduction

Since the beginning of human history, it is estimated that more than 6 × 106 chemical compounds have been isolated and/or created and between 60 000 and 95 000 are in current commercial use [1]. Chemical industry plays a key role in the economy and in future technologies, however, such a role has an environmental impact mainly due to the processes that: are energetically unfavorable requiring low/high temperature and/or low/high pressure for long periods of time (sometimes over 24 hours), form undesired by-products (nitrogen oxides, carbon monoxide, sulfur compounds, chlorinated and other organic compounds), and need catalysts and/or organic solvents which can be toxic, flammable and corrosive. With the acknowledgement of this impact in the last few decades, the term “Green Chemistry” and its 12 principles came to rethink the future of chemical synthesis. The latter can be defined as the “design of chemical products and processes to reduce or eliminate the use and generation of hazardous substances” [2]. Simple, yet with a significant impact on the industry as it has demonstrated how “fundamental scientific methodologies can protect human health and the environment in an economically beneficial manner” [3], furthermore it keeps pushing forward the “design of safer chemicals and environmentally benign solvents, and the development of renewable feedstock” [3], this opened the door even more to the use of biocatalysts and biotransformants in the chemical industry. In fact, the use of biocatalysts can selectively deliver high value products and reduce the environmental burden during chemical synthesis [4].

1 Introduction

1.1. Biocatalysis

Enzymes are highly specific biological catalysts that accelerate the rate of chemical reactions in the cells of living organisms. These natural catalysts are biodegradable, fast, efficient and selective, and produce low amounts of by-products while also being less demanding with respect to process energy, raw materials and toxic components than many traditional chemical catalysts. Their exquisite catalytic power, specificity of action and reduced environmental footprint makes them seemingly ideal tools for numerous biotechnological applications. Indeed, a society without industrial enzymes is difficult to imagine as they are present in almost every aspect of our lives, the manufacturing of food and feedstuff, textiles, detergents, cosmetics, medicinal products, biofuels and as tools for research and development. The enzyme industry has experienced significant growth during the last decades due to increasing activity in research and development, increasing population, and growing global growing for cleaner and greener technologies to safeguard the environment. In fact, the global market for industrial enzymes has evolved from €720 million in 1995 to an estimated € 2 800 million in 2011, € 5 400 million in 2018 and it is projected to reach € 7 000 to 10 000 million by 2024 [5,6]. The (unknowingly) enzyme usage in daily routines dates back to ancient times when yeast or other microorganisms were used to produce and preserve products such as cheese, beer, vinegar, and wine. Today, we describe the latter as modern biocatalysis which is described as the use of whole cells, enzymes (purified or as part of cell lysate) to convert a molecular substrate into a product [7]. Although it was known by the end of the 19th century that enzymes were producing interesting products, for a long time enzyme catalysis was not an option for chemists because enzymes had a few disadvantages, namely the observed/assumed narrow substrate scope, limited stability, low efficiency and diluted product leading to low space-time yields [8]. However, with the progress of molecular biology techniques, computational power, enzyme discovery (via genome sequencing/ mining), enzyme engineering (directed evolution, rational-design or de novo design) and process development, many of the disadvantages can be surpassed [8–10]. Furthermore, a broad range of biotransformations that are relevant in organic chemistry can now be catalyzed, including redox reactions, carbon-carbon bond formation and hydrolytic reactions to name a few [11]. Each aforementioned reaction requires an enzyme or group of enzymes that belong to different classes (are different in their mode of action) and this can be plotted with their application area (Table 1.1). The majority of enzymes used industrially are hydrolytic in action (e.g. amylases, cellulases, xylanases, proteases, pectinases, etc.) for the

2 Introduction degradation of natural substrates, therefore they have application in all segments: 1) in the detergent segment mainly for stain removal; 2) in the pulp & paper for bleaching; 3) in the food and feed segment with different purposes and sectors, e.g. reducing proofing time in bakery clarification of juices in beverages, tenderization of meat and fish to name a few; 4) in

Table 1.1. Classification and application areas of enzymes. Based on Averill et al [12]

Enzyme Class Detergent Pulp & paper Food & feed Waste treatment Specialties Research Hydrolases • • • • • • Lyases • • • Oxidoreductases • • • Isomerases • • Transferases • Ligases • the waste treatment for the degradation of polymers and 5) in the specialties segment for the production of fine chemicals and pharmaceuticals [5,12]. Hydrolases are by far the best- studied class of enzymes and have been more successfully applied in commercial processes (Table 1.2). However, other enzymes such as oxidoreductases are of crucial importance for the food and feed, specialties (biotechnological and pharmaceutical) and research segment. They catalyze oxidation and reduction reactions and despite being a large Table 1.2. Examples of biocatalysis use in commercial processes for the production of fine chemicals. Yield/Annual Product Substrate Enzyme Reference production Acrylamide Acrylonitrile Nitrile hydratase 600 000 tons [4,13] L-Aspartate Fumarate Aspartase 7 000 tons [4,12,14]/DSM L-DOPA Pyruvate, catechol, NH4+ Tyrosine-phenol lyase 200-250 tons [4,12] L-Alanine L-Aspartate L-Aspartate-β-decarboxylase 100-150 tons [4,12] Enantiopure alcohols Racemic alcohols Lipases Thousands tons [4]/BASF 6-APA Penicillin G/V Penicillin acylase Thousands tons [4]/DSM 6-Hydroxynicotinic Niacin Niacin hydroxylase 65 g/L [4]/Lonza 6-Hydroxy-S-nicotine (S)-Nicotine Hydroxylase 30 g/L [4]/Lonza

class, few members are known to be used in commercial processed. Nevertheless, examples do exist, especially in the food & feed segment for instance for off-flavor scavenging in soybean, production of flavors or acidification of cheeses [15]. There are already several processes where enzymes are used at the industrial level as shown in Table 1.2 but the success of biocatalysis (using enzymes or whole-cells) depends ultimately on the economics of the processes.

3 Introduction

1.2. Monooxygenase Classes

Monooxygenases (EC 1.13.x.x and EC 1.14.x.x) are oxidoreductases that catalyze the insertion of one oxygen atom into an organic substrate. To perform this reaction, molecular oxygen needs to be activated, this is done by donation of electrons after which, oxygenation of the organic substrate can occur. The type of reactive oxygen-intermediate that is formed depends on which cofactor is present in the monooxygenase. In some cases, no cofactor is present. Thus the necessity to divide monooxygenases into classes. So far seven classes have been created: 1) Heme-dependent monooxygenases also referred to as cytochrome P450 monooxygenases or CYPs (EC 1.14.13.x, EC 1.14.14.x and EC 1.14.15.x); 2) Flavin-dependent monooxygenases (EC 1.13.12.x and EC 1.14.13.x) where the flavins utilized by these enzymes are either FMN or FAD (for their structural properties) and are either bound tightly to the enzyme (prosthetic group) or function as a substrate (coenzyme) [16]. Flavin-dependent monooxygenases are able to catalyze reactions such as epoxidations, Baeyer-Villiger oxidations, and halogenations; 3) Copper-dependent monooxygenases (EC 1.14.17.x) which require copper ions for hydroxylation of their substrates; 4) Non-heme iron-dependent monooxygenases utilize two iron atoms as cofactor for their oxidative activity and are also referred as bacterial multicomponent monooxygenases (BMMs) because they consist of three components: a monooxygenase, a reductase and a small regulatory protein [17,18]; 5) Pterin- dependent monooxygenases (EC 1.14.16.x) are known to hydroxylate phenylalanine, tyrosine and tryptophan at their aromatic ring [19]; 6) Other cofactor-dependent monooxygenases and 7) cofactor-independent monooxygenases, which do not require any cofactors/coenzymes. For this particular class, it is proposed that the molecular oxygen is activated by the substrate. This reflects the limited substrate acceptance of these enzymes and highlights the importance of substrate nature for the catalytic activity of these monooxygenases. This work will only focus on the heme-dependent class of monooxygenases.

1.3. Heme-Dependent Monooxygenases

Heme-dependent monooxygenases can also be referred to as cytochrome P450 monooxygenases (P450) or CYPs (EC 1.14.13.x, EC 1.14.14.x and EC 1.14.15.x). They earned their name due to light absorption at λ 450 nm of the reduced CO-bound heme- complex [20,21]. CYPs are more commonly found in eukaryotes (mammals, plants, fungi) but are also present in prokaryotes (a wide variety are expressed in bacteria) and perform a diverse variety of functions in living organisms on primary and secondary metabolism [22,23]. This enzyme family is one of the oldest and largest gene families with more than

4 Introduction

300 000 P450 sequences described [24], this abundance is then latter reflected on the diverse physicochemical properties, primary sequences, folds, and overlapping but different specificities that lead to a classification based on CYPs architecture, cellular location and their electron transport that is summarized in (Table 1.3).

Table 1.3. Organization of different cytochrome P450 systems. Based on Hannemann et al. and Cook et al. [25,26] Class Origin/Example Organization of electron transport chain Bacterial NAD(P)H ▸ [FdR] ▸[Fdx]a▸ [P450] I Mitochondrial NADPH ▸[FdR] ▸[Fdx] ▸[P450] II Bacterial & Microsomal NADPH ▸[CPR] ▸[P450] III Bacterial / P450cin NAD(P)H ▸[FdR] ▸[Fldx] ▸[P450] IV Bacterial / Sulfolobus tokadaii Pyruvat, CoA ▸[OFOR] ▸[Fdx] ▸[P450 V Bacterial / Methylococcus capsulatus NADH ▸[FdR] ▸ [Fdx–P450] VI Bacterial / Rhodococcus rhodochrous NAD(P)H ▸[FdR] ▸[Fldx–P450] VII Bacterial / Ralstonia metallidurans NADH ▸[PFOR–P450] VIII Bacterial / P450 BM3 NADPH ▸[CPR–P450] IX Soluble eukaryotic NADH ▸[P450] X Independent eukaryotic [P450] Abbreviated protein components contain the following redox centers: Fdx (iron–sulfur-cluster); FdR, Ferredoxin reductase (FAD); CPR, cytochrome P450 reductase (FAD, FMN); Fldx, Flavodoxin (FMN); OFOR, 2-oxoacid:ferredoxin oxidoreductase (thiamin pyrophosphate, [4Fe–4S] cluster); PFOR, phthatate-family oxygenasereductase (FMN, [2Fe–2S] cluster). a Fdx containing iron–sulfur-cluster of [2Fe–2S], [3Fe–4S], [4Fe–4S], [3Fe–4S]/[4Fe–4S] type. Such is the diversity of physicochemical properties, primary sequences, and folds that they are involved in more than 20 different reactions such as hydroxylation, N-, O- and S- dealkylation, sulphoxidation, epoxidation, deamination, desulphuration, dehalogenation, peroxidation, and N-oxide reduction to name a few [27,28]. These reactions are catalyzed by the insertion of a single oxygen atom from molecular oxygen (O2) in a highly chemo-, regio-, and/or enantioselective manner where the remaining oxygen atom is reduced to water (H2O). To activate the molecular oxygen, 2 electrons need to be donated by NAD(P)H. The simplified reaction is shown below in Equation 1.1.

Equation 1.1. The simplified reaction of a heme-dependent monooxygenase

+ + R-H + O2 + NAD(P)H + H → R-OH + H2O + NAD(P)

5 Introduction

The more detailed overview of the proposed catalytic cycle of heme-dependent monooxygenases is depicted in Figure 1.1 and can be condensed into six steps: In the first step, the substrate binds to the active site at the heme domain, prompting the release of a water molecule that covers the active site in the resting state of the enzyme. (2) Ferric cytochrome P450 is reduced to ferrous cytochrome P450 with one electron transferred by the reductase from NAD(P)H. (3) Molecular oxygen (O2) will bound to the ferrous cytochrome and the complex ferrous P450-dioxygen will be formed. (4) The second electron transfer occurs and the protonation leads to a Fe3+-hydroperoxy complex. (5) The O-O bond is protonated and cleaved leading to the release of a water molecule producing an iron-oxo intermediate. And (6), the last step, one oxygen is transferred from the iron-oxo complex to the substrate, culminating in its oxidation and release, returning the enzyme to its resting

Figure 1.1. Proposed catalytic cycle of P450 monooxygenases Based on Cook et al. 2016 [26]. state (1) [26,29,30]. However, it has been observed that the amount of oxidized NAD(P)H cofactor does not generate stoichiometric amounts of product, especially when using non-natural substrates [31,32]. This is described as “coupling”, it describes the efficiency of a P450-catalyzed reaction and it depends on the target substrate [32,33]. Three types of uncoupling can occur during the catalytic cycle and have been defined as- “shunts”: 1) auto-oxidation shunt that leads to O2. formation, 2) peroxide shunt that leads to H2O2 formation and finally 3) oxidase shunt leading to H2O formation.

6 Introduction

1.4. P450 Monooxygenases as Biocatalysts

Monooxygenases (EC 1.13.x.x and EC 1.14.x.x) have been receiving great attention because they can hydroxylate organic substrates with highly chemo-, regio-, and/or enantioselectivity in water and at ambient temperatures, making them an attractive biocatalyst for the production of value-added products. Therefore, the pharmaceutical, agrochemical and biorefinery industry will benefit the most of P450 power (e.g. precursors of drugs, anticancer and antioxidant agents, fine chemicals, fragrance, and flavoring compounds) [34]. In fact, this enzyme family is involved in the biotransformation of xenobiotics, metabolism of chemical carcinogens and the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins, and bile acids [23]. Additionally, monooxygenases are known to convert different substrates such as alkanes, terpenes, and aromatic compounds as well as the degradation of herbicides, insecticides, and heterocyclic compounds. These reactions are of particular interest because many of the products formed cannot be synthesized by the standard chemical way [34]. However, the application of P450s in industry is restricted to very high priced compounds such as pharmaceuticals, such as 11-ß-hydroxylation of 11-deoxycortisol to hydrocortisone using P450s (CYP11A1, CYP17A1, CYP21A und CYP11B1) from the fungi Curvularia sp. [35], production of artemisinin using on one of the steps using CYP71AV1 isolated from Artemisia annua [36,37], conversion of progesterone to cortisone by P450s expressed by Rhizopus sp. for the conversion of progesterone to cortisone [38,39]. Other examples are collected in several reviews [23,35,37,40]. Despite their synthetic potential, P450 use is limited in industrial applications to high priced compounds due to the associated production costs [35], such as the need of expensive cofactors NAD(P)H, the reduced coupling efficiency for relevant industrial compounds, self-inactivation via formation of reactive oxygen species and poor stability under industrial conditions. To overcome these disadvantages several strategies have been used: provide a constant supply of NAD(P)H or its regeneration using enzymatic [41], chemical [42,43] or electrochemical [44] methods; protein engineering campaigns [45– 48] to increase coupling efficiency; and to tackle (ins)stability issues, immobilization in gel matrixes [49] or metal surfaces by using selectively binding peptides [50] and also covalently immobilize P450s on iron oxide nanoparticles [51], however in all cases, despite improvement of the enzyme storage stability, P450 activity was significantly reduced.

7 Introduction

1.4.1. P450 Bacillus megaterium 3

Cytochrome P450 from Bacillus megaterium also known as CYP102A1 or P450 Bacillus megaterium 3 (BM3) is a self-sufficient natural fusion protein in a single polypeptide chain (1048 amino acids, ≈ 119 kDa). It’s composed of a cytochrome P450 or HEME domain (BMP, amino acids 1–470, ≈ 55 kDa), a flavin mononucleotide domain (FMN, amino acids 471–664 and a flavin adenine dinucleotide domain (FAD, amino acids 665–1048) (≈ 64 kDa). Although the majority of P450s are expressed independently of their redox partners, P450 BM3 (CYP102A1, BM3) from Bacillus megaterium is fused to its redox partner, and this unique structural feature facilitates efficient electron transfer. Under natural conditions, P450 BM3 is predominantly a dimer (monomer, trimer, and tetramer are found as a minority) and electrons from the NADPH cofactor are transferred between the domains of two different monomers. Diflavine reductase domain of monomer 1 transfers electrons to the P450 domain of monomer 2 and vice-versa (Figure 1.2) [40,52]. The heme is composed of an iron ion coordinated by four nitrogen atoms of porphyrin and it is linked to the apo-protein via a conserved cysteine.

Figure 1.2. Schematic of putative electron transfer pathways in flavocytochrome P450 BM3 in its dimeric form. CPR: FAD and FMN. Reductase domain represented in yellow and heme domain in red. Based on Girvan and Munro 2016 and Neeli et al. 2005 [40,52]. P450 BM3 typically binds and oxidizes several mid- to long-chain fatty acids at the ω-1, ω-2, and ω-3 positions and it is an attractive biocatalyst due to its water solubility, self- sufficiency and relatively high catalytic rates for P450’s ( ~17,000 min−1 for arachidonic acid as substrate [53]). Although, the wild-type P450 BM3 has low catalytic activity toward industrially relevant substrates, it has been studied extensively and was the subject of intense protein engineering campaigns to fully apply and exploit its catalytic power, generating variants of this enzyme capable of binding and oxidizing diverse compounds, including steroids, terpenes and various human drugs (Table 1.4). Several key-residues are known that influence the activity and selectivity of P450 BM3 such as F87, R47, Y51, T268 and A330, and through the last decades, researchers reported variants with increased activity, better coupling efficiency, expanded substrate scope, and even the ability to perform abiotic reactions (oxidative deamination of alkyl azides, olefin 8

Introduction cyclopropanation via carbene transfer, carbene N–H insertion to create C–N bonds, and intramolecular C–H amination reactions to name a few [46-57]). Additionally, P450 BM3 was also engineered for increased activity in organic solvents such as dimethyl sulfoxide (DMSO) [66] and for improved organic solvent resistance [67]. A key example of P450 oxidation technology applied to the pharmaceutical industry is the selective and environmentally friendly route towards the synthesis of 4-hydroxy-α-isophorone on a kilogram scale [68]. The latter demonstrates how the application of protein engineering to perform chemo- enzymatic syntheses of chemical derivatives in a synthetic late-stage fashion significantly extends the synthetic toolbox, offering chemists an attractive alternative to the conventional chemical strategies [69].

Table 1.4. A glimpse into P450 BM3 selectivity-directing and activity-enhancing mutations compiled. A table of positions substituted, improvements and references A more comprehensive overview of P450 BM3 substitutions is described by Whitehouse et al [70] and is also available online in the muteinDB [www.MuteinDB.org] [71]. Substitution. Improvements Ref.

R47Q/S A 2-3-fold decrease in binding affinity for N-myristoyl-L-methionine as a substrate. [72]

A74G/ Higher activity in the hydroxylation of highly branched fatty acids; Indole hydroxylation [73][74] F88V/L188Q

800-fold binding affinity for laurate as substrate. Increased indole hydroxylation. Significantly A83F/W [57] higher rates of NADPH consumption in the absence of substrate. This R255L Increased hydroxylation of aromatic heterocycle work Substrate binding affinity increases 5-10 fold and the turnover number increases 2-8-fold for palmitate as substrate compared to the wild-type. It has a very different product distribution A329V [75] favoring greatly oxidation at the omega-1 position and shows almost no oxidation at the omega- 3 position. A 10-fold increase in binding affinity for lauric acid. Catalytic activity rates accelerate across a I402P range of hydrophobic non-natural substrates, including (+)-alpha-pinene, fluorene, 3- [76] methylpentane, and propylbenzene.

Many of the research performed on P450 BM3 rely on its crystal structure (PDB: 1BU7 [77]) to infer/predict structural changes that might lead to the desired improvement. Nearly 80 crystal structures of P450 BM3 were deposited in the Protein Data Bank (PDB; www.rcsb.org) the majority of them being crystals with the heme domain only.

9 Introduction

1.4.2. P450 Cand_1 & P450 Cand_10

P450 Cand_1 and P450 Cand_10 are less known bacterial class I monooxygenases of ∼50 kDa each belonging to the CYP153 family that have been recently isolated from Pseudomonas sp. 19-rlim and Phenylobacterium zucineum respectively, with crystal structures solved in 2018 (PDB code 6HQD and 6HQG respectively) [78]. Fiorentini et al. in 2018 [78] predicted P450 Cand_1 and P450 Cand_10 to be multidomain protein comprising an N- terminal CYP domain, an FdR domain, and a C-terminal Fdx domain which correlated well with the best expression results: co-expression with Fdx and FdR from Mycobacterium sp. HXN-1500 (the redox partners of CYP153A6) [79]. Using the latter strategy, Fiorentini et al. (2018) observed that P450 Cand_1 exhibited activity towards octane, dodecanoic acid, N-BOC pyrrolidine, and indole and P450 Cand_10 towards octane, dodecanoic acid, N-BOC pyrrolidine [78]. The observed activity towards indole by P450 Cand_1 was promising however the conversion did not go beyond 50%, probably because the rapid accumulation of indigo, being highly hydrophobic, hampers the stability of the enzyme in solution to the point of stopping its activity after a short time [78]. To date, there are no protein engineering reports for P450 Cand_1 and P450 Cand_10.

1.5. Protein Engineering

With the realization of “monooxygenase power” for fine chemical synthesis, there is the need to meet the industry demands that often focus on enhanced activity, stability or expression. P450s biocatalysts for industrial with the desired features can be achieved via molecular biology methods combined with a medium or high throughput screening system. Generally, three main protein engineering approaches are followed: rational design, semi-rational design and random design (also known as directed-evolution) (Figure 1.3). Rational design requires structural

Figure 1.3. Overview of approaches for protein engineering by random, rational and combined methods. 10 Introduction knowledge and alignment data to perform well-defined amino acid substitutions executed by site-directed mutagenesis at the DNA level [80] or by simply manufacturing a new molecule of DNA by a DNA synthesis company. By contrast, the random approach, also known as directed evolution, does not require any previous structural knowledge or alignment data and relies on diversity generation methods such as error-prone PCR [81] where PCR conditions are designed to change the error rate of the polymerase. Usually, Taq polymerase is commonly used due to naturally occurring high error rate which can be further increased with varying 2+ concentrations of Mn , unbalanced dNTPs and/or increasing the concentration of MgC12, and/or increasing polymerase concentration and/or increasing extension time [82]. A typical directed evolution experiment begins with the gene for the parent protein being randomized by using error-prone PCR or equivalent. The generated gene library is then used to produce mutant proteins, which can be screened/selected for the desired target property (e.g., expanded substrate scope or increased stability). Variants that do not exhibit improvements during screening/selection are discarded, whereas the genes for the improved mutants are used as the parents for the next round of mutagenesis and screening. This procedure is generally repeated until the evolved protein exhibits the desired level of the target property (Figure 1.4). Another possibility for gene randomization is DNA shuffling where it is possible to recombine parts of

Figure 1.4. Schematic outline of a typical directed evolution experiment. Based on Bloom and Arnold (2009) [215]. related but diverse enzymes from nature generating chimeras that are beyond the horizontal exchange of genetic information between organisms [83]. A third possible approach is semi- rational design, it combines the rational and the random approach and is often referred as smart or knowledge-based library generation, where the information from protein sequence alignments, structural knowledge as well as computational power are used to select promising positions to be randomized thus reducing the library size. Randomization is usually done by

11 Introduction site-saturation-mutagenesis (SSM) allowing for the identification of the best amino acid for a specific position by applying a NNN (64 codons) or NNK degeneracy (32 codons), covering all 20 canonical amino acids. However some limitations do exist in the randomization of the gene of interest: for instance, in epPCR it is often observed a mutational bias of employed polymerase (C vs. T or G vs. A are not identical), the lack of subsequent mutations in a codon, as well as the organization of the genetic code which can lead to conservative amino acid exchanges [82]. In DNA shuffling, regions with medium to high degree of homology are needed for the randomization to occur efficiently [84]. By contrast, DNA synthesis can allow for complete randomization of a sequence (including randomization of subsequent positions) and can overcome the drawbacks and limitations of epPCR and DNA shuffling. In any case, the complete randomization of an enzyme comprising of 100 amino acids would theoretically result in 20100 = ≈1.3130 variants (20 amino acids possible per position), a diversity that is impossible to completely screen. Therefore, approaches that reduce library size are valued. More recently, a knowledge gaining directed evolution approach (KnowVolution) [85] was published as a strategy to speed up directed evolution, by including computational analysis to gain molecular understanding in the process, which reduces screening efforts and time consumption (Figure 1.5). It has proven itself as a valid strategy to evolve and improve enzyme properties as documented by a glucose oxidase for diabetes analytics [86], a phytase for the feed industry [87], proteases for industrial applications and cellulases with improved ionic- resistance [88], to name a few. It is composed of four phases: Phase I, the identification of potential beneficial positions by directed evolution techniques, followed by the determination of beneficial substitutions (Phase II). In Phase III, the investigation and determination of the cooperative effects of the substitutions are done by structural analysis. Lastly, in Phase IV recombination of beneficial substitutions is performed to maximize improvements. The

Figure 1.5. Overview of the KnowVolution strategy which comprises four phases: (I) Identification of potentially beneficial amino acid positions, (II) determination of beneficial amino acid positions and substitutions, (III) computational analysis and a group of amino acid substitution which might interact with each other, and (IV) recombination of beneficial substitutions in a simultaneous or iterative manner. The KnowVolution strategy can also be performed in an iterative manner to further improve targeted enzyme properties. 12 Introduction recombination can occur by site-directed mutagenesis (SDM) or via OmniChange which is a focused library generation method that can simultaneously saturate up to five independent amino acid positions, allowing for investigation of synergetic effects [89]. Additionally, other successfully applied methods do exists, such as Mutagenic Organized Recombination Process by Homologous In Vivo Grouping (MORPHING) [90] or Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) [91]. More recently, a Computer-assisted Recombination (CompassR) strategy provided a selection guide for beneficial substitutions that can be recombined to gradually improve enzyme performance by analysis of the relative free energy of folding (ΔΔGfold). It allows to recombine beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time-efficient manner to reduce the library size and screening effort. The final goal is that with the deeper understanding of the molecular enzyme properties, time and screening efforts can be reduced and protein engineering can/will become a “standard tool in biocatalysis process design”

1.6. Screening Systems

Employing diversity generation methods can generate thousands of variants on a single round of directed evolution thus leading to a screening effort, especially in the case of monooxygenases [92]. Here, the major challenge is the development of product-based screening systems to identify better performing catalysts with reliability, i.e., the screening system has to be of high throughput, reproducible, and optimized for the sensitivity of the desired function. In all cases, when using directed evolution campaigns, the screening method of choice is related to the library size and to the product to be detected (see Figure 1.4 in the previous section). The existing screening strategies range from selective media in agar-plates, gas chromatography or high-performance liquid chromatography (low-throughput) to multi- well plates in combination with analytical methods (medium throughput) and finally to cytometry using fluorescence-activated cell sorting (FACS) systems (high throughput) [92–94]. Regarding P450s evolution, the methods for diversity generation have been used to improve the industrial use of P450s for increased activity towards industrially relevant substrates (improved oxidation rate, turnover number, chemo-, regio-, and/or enantioselectivity, or substrate specificity to name a few [95,96]). Traditionally, P450 enzyme activity is determined in 96-well plates using either crude cell lysates or purified enzyme to perform either NADPH depletion assay [97] or product-based colorimetric or fluorometric assays (e.g., 4- aminoantipyrine for phenolic compound detection (Figure 1.6) [98], NpCN for the detection of

13 Introduction specific hydroquinones [99], pNTP for styrene epoxidation [100], or fluorescence for the detection of steroid hydroxylation [101]). Flow cytometry offers the opportunity to screen with high throughput (up to 1.8 × 107 events per hour), nevertheless, a detection system based on the desired function is still needed. Few published examples exist for P450s, more notably a

Figure 1.6. Under alkaline condition and catalyzed by potassium peroxodisulfate, phenols (1) react with 4-APP (2) forming a product (3) that displays strong absorbance maxima at λ 509 nm. fluorescence-based continuous-flow enzyme affinity detection system was reported for investigation of the activity of P450 BM3 variants e.g., in the presence of organic solvents [102] and a more general FACS screening system using the BCCE as surrogate substrate [103]. A generally applicable and emerging possibility is 96 multiplex-capillary electrophoresis (CE) which is product specific and suitable for substrates that are not colorimetric or fluorescent and has recently been added to the range of suitable screening systems for P450- directed evolution campaigns [104]. The CE works by applying a voltage potential and separating the analytes present in a sample based on their electrophoretic mobility and interaction with the walls of the capillary (Figure 1.7). The CE is a powerful and versatile technique as different detection systems can be coupled (UV-vis spectrophotometric detection, laser-induced fluorescence (LIF), contactless conductivity detection (CCD), or even mass spectrometers (MS) [48]). It can be also be automated and used for the separation and analysis of several charged substances and biological macromolecules such as amino acids, peptides and proteins, chiral drugs, whole cells, and virus particles to name a few [105,106].

Figure 1.7. Schematic outline of a typical capillary electrophoresis experiment

14 Introduction

1.7. Aromatic heterocyclic compounds

Aromatic heterocyclic compounds also referred to as heterocycles, are characterized by having one or more atoms in their ring structure that is not carbon (C) (Figure 1.9). In nature, they are present in several naturally occurring compounds such as vitamins, hormones, antibiotics, sugars, pigments, and antioxidants (e.g. tryptophan, serotonin, histamine, thiamine, nicotinic acid, protoporphyrin IX, vitamin B12, to name a few) and the most common heteroatoms are oxygen (O), nitrogen (N) and sulfur (S). Of particular interest for this work are

Figure 1.9. Examples of oxygen, nitrogen and sulphur-based heterocycles building blocks. aromatic oxygen (O) and nitrogen (N) containing heterocycles as they are an important class of bioactive molecules. For instance the antibacterial fosfomycin (contains oxirane), ovalicin, a fungal product that prevents angiogenesis (contains two oxirane rings) [107] or paclitaxel, a four-membered O-heterocycle oxetane for anticancer treatment [108]. Moreover, five- membered O-heterocycles form the cores of various naturally-occurring sugars and six- membered O-heterocycle tetrahydropyran forms the fused core of the vitamin E group compounds. Regarding N-heterocycles, they occur widely in nature in the same fashion as O- heterocycles being involved in a variety of biological functions as well [109]. More notably, tryptophan (contains indole) for being an essential amino acid, monocyclic pyridine aldehyde in the coenzyme form of vitamin B6 or pyrimidines in both DNA and RNA, additionally some piperidine and pyrrolidine alkaloids are a major group of natural products isolated from plants or microbes that have diverse functions [109,110]. Efforts have been made to develop general, chemo-, regio- and enantioselective methods and strategies to construct small rings to macrocyclic oxygen and nitrogen-containing heterocycles as lead and attractive scaffolds for the development of new drugs [111–114]. Here , we will focus on two aromatic heterocycles: indole and benzo-1,4-dioxane (see Figure 1.8).

Figure 1.8. 2D chemical structure of the tested aromatic heterocycles in this work.

15 Introduction

1.7.1. Indole and Indigo

Indole is a precursor of indigo dye, one of the oldest dyes known to man. It is a water- insoluble pigment originally extracted from flowering woad plants and tropical of the Indigofera genus and became a principal item of commerce between Europe and the Far East. The yield of the colorant from Indigofera was 2 % (w/w), from 100 kg of leaves one could obtain 2 kg of indigo [115]. Later on, Baeyer's elucidation of the structure of indigo in 1883 was followed by the development of a synthetic and commercially practical synthesis. Since then, the demand has increased to 50 000 tons in 2011 [116–118]. This enormous amount presents a serious sustainability menace for two reasons: first, the chemicals and conditions used to manufacture requires aniline (4) to couple chloroacetic acid (5) and produce sodium phenylglycinate (6), that when treated with a alkaline melt of sodium and potassium hydroxides containing sodamide under temperatures exceeding 200 °C produce indoxyl (7), which is then oxidized in air to form indigo (8) (Figure 1.10) Alternatively, indigo can also be produced by using aniline, formaldehyde and, HCN as starting materials [119–122]. Second, indigo is

Figure 1.10. Manufacturing process of indigo. (4) aniline, (5) chloroaceticacid, (6) sodium phenylglycinate, (7) indoxyl and (8) indigo. Based on Yamamoto, Inoue and Takaki et al. [120]. insoluble in water, meaning it needs to be reduced for the dyeing process where sodium dithionite is used and later decomposes into sulfate and sulfite corroding equipment and pipes in the dye mill and wastewater facilities [116,117,122,123]. Furthermore, if those wastewaters are not treated and dumped directly into rivers and water streams they have a serious negative ecological impact. Considerable advantages exist when using enzymatic synthesis of indigo as comes as an alternative to the harsh chemical synthesis described above and has been studied by several researchers and the most successful approaches are presented in Table 1.5 [132–136]. In all cases, substantial environmental improvements are present by the usage of monooxygenases or

Table 1.5. Best bacterial strains and best enzyme resources with the ability of producing indigo from indole. Enzyme Organism Indigo Yield Reference

- P. aeruginosa HOB1 246 mg/L [124] - A. sp ST-550 292 mg/L [125] - A. sp. PP-2 203 mg/L [126] Naphthalene dioxygenase Comamonas sp. MQ 205 mg/L [127] Flavin monooxygenase Methylophaga aminisulfidivoran 920 mg/L [128–130] Flavin monooxygenase Corynebacterium glutamicum ATCC13032 685 mg/L [131]

16 Introduction dioxygenases as biocatalysts. The setup requires a balance of electron donors, which can be provided either by the cell in case of whole cell or by NADPH if in a free-cell scenario and,

Figure 1.11. Tryptophan (9) is converted to indole (10) by the a tryptophanase which is then oxygenated to indoxyl (7) by a mono- or dioxygenase which spontaneously oxidizes to indigo (8). Based on Hsu et al. [116]. indole (10) is obtained by cleavage of tryptophan (9) by tryptophanase (Figure 1.11) [116]. By comparison, the most recent publication for the production of indigo starting from indole requires cumene hydroperoxide, acetic acid and, molybdenum hexacarbonyl in cumene to react in a reflux of tert-butyl alcohol at 85.7 - 86.5 ℃ for for 7h [120]. A process that in neither “user- friendly” and due to a maximum of 81 % yield is not environmentally sustainable.

1.7.2. Benzo-1,4-dioxane and derivatives

Benzo-1,4-dioxane, a bicyclic heterocyclic compound consisting of a benzene ring fused to a heterocyclic dioxane ring represents a series of synthetic and natural compounds [4–11] of considerable medicinal importance and various biological activities [137–139]. It has been known since the 19th century but only after a publication 1933 by Fourneau and Bovet which described the adrenoblocking properties of 2-alkylaminomethylbenzodioxanes [140] interest in derivatives of this heterocyclic system increased [141]. Derivatives of benzo-1,4-dioxane with other medical applications have been found since then: antimicrobial [142], antigrastic [143], spasmolytic [144], antipsychotic [145], anxiolytic [146], hepatoprotective [147], or α- adrenergic blocking agent activity [137,148,149]. Additionally, the benzo-1,4-dioxane ring is present in drug precursors such as eltoprazine [150,151] which is itself a precursor for S-15535 and Lecozotan (Phase III trials completed in 2008 [152]). More recently, other activities have been described for 1,4-benzodioxane lignans: insecticidal, anti-cancer, anti-angiogenic and anti-oxidant activities to name a few [153,154]. Several synthetic chemical routes are published and/or patented for the production of 3- dihydrobenzo-1,4-dioxin-5-ol [155–158], 3-dihydrobenzo-1,4-dioxin-6-ol [159–161] and, 3- dihydrobenzo-1,4-dioxin-2-ol [162–164] Figure 1.12 . In all available routes at least one of the following is required: rare and expensive catalysts, heating, active cooling, and multi-step reactions. For instance, the most recent routes of 3-dihydrobenzo-1,4-dioxin-5-ol, mix 1,2- dibromomethane and benzene-1,2,3-triol in a heating oil bath at 90 °C for 16 h in a inert atmosphere to yield a reported 45%-56% of 3-dihydrobenzo-1,4-dioxin-5-ol (Figure 1.12a).

17 Objectives

Figure 1.12. Available chemical routes for the production of (13) 3-dihydrobenzo-1,4-dioxin-5-ol, (15) 3-dihydrobenzo-1,4- dioxin-6-ol and, (18) 3-dihydrobenzo-1,4-dioxin-2-ol. (11) dibromomethane, (12) benzene-1,2,3-triol, (14) 2,3-dihydro-1,4- benzodioxine-6-carbaldehyde, (16) Alternatively, pyrogallol can be dissolved in 2-butanone containing potassium carbonate at 90 °C [158,165]. For 3-dihydrobenzo-1,4-dioxin-6-ol, the highest reported chemical route yielded 94 % - 98 % was patented and uses 2,3-dihydro-1,4-benzodioxine-6-carbaldehyde as starting compound to react with meta-chloroperoxybenzoic acid (mCPBA) and potassium floride in dicloromethane overnight at room temperature (Figure 1.12b) [166]. The production of 3-dihydrobenzo-1,4-dioxin-2-ol requires active cooling with liquid nitrogen to keep temperateures under -70 °C while a solution of diisobutylaluminum hydride (DIBAL-H) in toluene is added dropwise to a solution of 1,4-benzodioxan-2-one in dry toluene (Figure 1.12c) [163]. These chemical routes are labourious and costly from a sustainability point of view. To date, there are no reports for targeted enzymatic hydroxylation of benzo-1,4-dioxane and, thus opening such enzymatic route would offer an user-friendly and sustainable reaction. These O- and N-heterocycles (and their derivatives) are, in most cases, synthesized and functionalized by the traditional chemical route to serve as building blocks for relevant pharmaceuticals, pesticides and, dyes. Using the chemical oxygenation to functionalize these heterocycles is still challenging as it involves weary and costly steps that are catalyzed in the presence of expensive, toxic heavy metals and chemicals [69,167] and often occur with little chemo-, regio-, and/or enantioselectivity leading to sustainability problems [69]. To overcome these challenges, the use of monooxygenases, well known for their ability to hydroxylate non- activated carbon atoms [168–170], can provide a powerful tool for the functionalization of aromatic O- and N-heterocycles with high chemo-, regio-, and/or enantioselectivity.

2. Objectives

18 Objectives

This work is part of the EU-funded Innovative Training Network (ITN) OXYTRAIN “Harnessing the power of enzymatic oxygen activation” a joint academic/non-academic training initiative supporting the convergence of biochemistry, enzyme engineering, and biotechnology” (project reference: 722390). The focus of this work is the directed evolution of P450s, to provide powerful tools for the functionalization of aromatic O- and N-heterocycles with high chemo-, regio-, and/or enantioselectivity. Identifiyng new P450 variants can provide new biosynthtetic routes that lead to advancements in biocatalysis generating drug precursors of high pharmaceutical importance in a selective and environmentally friendly way. Three different heme-dependent monooxygenases, P450 Cand_1, P450 Cand_10 and P450 BM3, are subjected to directed evolution for the conversion of the heterocyclic compounds benzo-1,4-dioxane and indole. Further objectives include the usage of different methodologies to generate the gene libraries and the development of high-throughput product-specific screening assays, specifically a 96- multiplexed capillary electrophoresis system and absorbance based 96-well microtiter plate system. Additionally, to develop gas chromatography and high-performance liquid chromatography methods to evaluate by-product and total product formation and characterization of the improved variants via coupling efficiency, NADPH oxidation rate, and the total turnover number. Finnaly, the elucidation at the molecular level on structure-function relationships that are responsible for the catalytic performance improvement observed in the tested monooxygenases for benzo-1,4-dioxane and indole.

19 Materials and Methods

3. Materials and Methods

3.1. Chemicals

Chemicals used in this study were of analytical grade or higher and purchased from Sigma-Aldrich Chemie (Steinheim, Germany), AppliChem (Darmstadt, Germany) and Carl Roth (Karlsruhe, Germany), ABCR (Karlsruhe, Germany), Molekula (München, Germany) and Angene Chemical (Eching, Germany) unless otherwise specified. Salt-free and HPLC grade DNA oligonucleotides were purchased from Eurofins MWG Operon (Ebersberg, Germany). Substrates used in this study are listed below in Table 3.1. Table 3.1. List of substrates used in this study and their respective supplier.

Name CAS Supplier 2,3-dihydro-1,4-benzodioxin-5-ol 10288-36-5 Sigma-Aldrich Chemie 2,3-dihydro-1,4-benzodioxin-6-ol 10288-72-9 Sigma-Aldrich Chemie 2,3-dihydrobenzofuran 496-16-2 Sigma-Aldrich Chemie Benzo-1,4-dioxane 493-09-4 Sigma-Aldrich Chemie Benzofuran 71-89-6 Sigma-Aldrich Chemie Dibenzofuran 132-64-9 Sigma-Aldrich Chemie Indigo 482-89-3 Sigma-Aldrich Chemie Indirubin 479-41-4 TCI Indole 120-72-9 Sigma-Aldrich Chemie Indoline 496-15-1 Sigma-Aldrich Chemie Isochroman, 493-05-0 Sigma-Aldrich Chemie Phthalan 496-14-0 Sigma-Aldrich Chemie Piperidine 110-89-4 Sigma-Aldrich Chemie Pyridine 110-86-1 Sigma-Aldrich Chemie Pyrrolidine 123-75-1 Sigma-Aldrich Chemie Tetrahydrofuran 109-99-9 Sigma-Aldrich Chemie

3.2. Enzyme and Kits

Restriction enzymes were purchased from New England Biolabs (Frankfurt, Germany). PfuS DNA polymerase and Taq DNA polymerase were prepared in-house. Glucose dehydrogenase from Pseudomonas sp., catalase from bovine liver and lysozyme from chicken egg white were provided by Sigma Aldrich (Steinheim, Germany). Plasmid purification, PCR purification, and gel extractions were performed using QIAGEN® (Hilden, Germany) kits (QIAprep® Spin Miniprep Kit, QIAquick® PCR Purification Kit, and QIAquick® spin Gel Extraction Kit) respectively.

20 Materials and Methods

3.3. Machines and Equipment

Table 3.2. Machines and equipment used during this work.

Name Manufacturer

Biophotometer plus Eppendorf, Hamburg, Germany Centrifuge 5424, 5810R Eppendorf, Hamburg, Germany CO-lecture bottle station Sigma Aldrich, Steinheim, Germany Deep well plates (96 well-round bottom) Brand GmbH, Wertheim, Germany DNA electrophoresis chamber Bio-Rad, München, Germany Experion Automated Electrophoresis System Bio-Rad, München, Germany GC-FID 2010 and 2010 Plus Shimadzu GmbH, Duisburg, Germany GCMS-QP2010S Shimadzu GmbH, Duisburg, Germany Gel Doc™ XR+ Gel Documentation System Bio-Rad, München, Germany Micro plate reader Infinite M200 pro Tecan Group, Männedorf, Switzerland Micro plate reader Sunrise Tecan Group, Männedorf, Switzerland Microtiter plate shaker Microtron INFORS, Heinsbach, Germany Microtiter plate (F or V bottom) Corning, Kaiserslautern, Germany Mini-Protean Tetra Cell system Bio-Rad, München, Germany NanoDrop Spectrophotometer 1000 NanoDrop Technologies, Wilmington, USA PCR thermocycler Mastercycler Pro S Eppendorf, Hamburg, Germany Themoblock Themomixer 5436 Eppendorf, Hamburg, Germany Ultrasonicator Vibra-Cell VCX-130 Sonics & Materials, Newton, USA Varian Spectrophotometer Cary 50 UV AgilentTechnologies, Darmstadt, Germany

21 Materials and Methods

3.4. Cultivation Media, Additives and Buffers

LB medium: 10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl were dissolved in dH2O and sterilized by autoclaving (121 °C, 20 min, 1.05 kg/cm2) and stored at room temperature until further use. Agar plate preparation was performed in the same way but with the addition of 20 g/L agar-agar. The corresponding antibiotics were added once the media cooled down to ≈ 50 °C and then the media was poured into plastic petri dishes. After solidifying the LB-Agar plates were stored at 4 °C until further use. TB medium: Solution A: 12 g tryptone, 24 g yeast extract, and 4 g glycerol were dissolved in

950 mL dH2O. Solution B; 2.31 g 12.54 g K2HPO4 were dissolved in 50 mL dH2O. Solution A was sterilized by autoclaving (121 °C, 20 min, 1.05 kg/cm2) and solution B was filter sterilized through a 0.2 µm filter, afterward they were mixed together. TB media was stored at room temperature until further use.

Phosphates (20x concentrated): 23.1 g KH2PO4 and 125.4 g K2HPO4 were dissolved in 2 500 mL dH2O and sterilized by autoclaving (121 ºC, 20 min, 1.05 kg/cm ).

Kanamycin solution (1000x concentrated): Prepared in dH2O as 50 mg/mL stock solution and filter sterilized through 0.2 μm filter. 1 mL aliquots were stored at -20 °C.

Ampicillin solution (1000x concentrated): Prepared in dH2O as 100 mg/mL stock solution and filter sterilized through 0.2 μm filter. 1 mL aliquots were stored at -20 °C.

Trace element solution (1000x concentrated): 0.5 g/L-1 CaCl2.2H2O, 0.18 g/L ZnSO4.7H2O,

0.10 g/L MnSO4.H2O, 20.1 g/L Na2-EDTA, 16.7 g/L FeCl3.6H2O, 0.16 g/L CuSO4.5H2O, and

0.18 g/L CoCl2.6H2O were dissolved in dH2O, sterilized by autoclaving (121 °C, 20 min, 1.05 kg/cm2) and stored at 4 °C. ALA (5-Aminolevulinic acid) solution (1000x concentrated): Prepared as a 0.5 M stock solution in dH2O and filter sterilized through 0.2 μm filter. 1 mL aliquots were stored at -20 °C. Thiamine hydrochloride solution (1000x concentrated): 100 g/L thiamine hydrochloride was dissolved in dH2O and filter sterilized through 0.2 μm filter. 1 mL aliquots were stored at -20 °C. Isopropyl-β-D-thiogalactopyranoside solution (1000x concentrated): Prepared as 1 M stock solution in dH2O and filter sterilized through 0.2 μm filter. 1 mL aliquots were stored at -20 °C.

Phosphate buffer (50 mM, pH 7.5): 50 mM solutions of K2HPO4 and KH2PO4 were mixed until the desired pH was achieved. Lysis buffer: 8 g/L lysozyme in phosphate buffer (50 mM. pH 7.5).

22 Materials and Methods

3.5. Bacterial Strains, Vectors and Genes

Bacterial strains, vector DNAs and genes employed in this work are listed in Table 3.3, Table 3.4 and Table 3.5 respectively.

Table 3.3. Strains employed in this study and their genotypes. Strain Genotype Reference F–ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 E. coli BL21-Gold (DE3) LacIQ1 gene 1 ind1 sam7 nin5]); pGRG36-lacIQ1 was employed for site- [171] specific integration of lacI into the chromosome

Table 3.4. Vectors employed in this study for genetic manipulation and recombinant protein expression. Vector Promotor Selective Marker Replicon Reference

pALXtreme-1a T7 Kanamycin ColE1(pBR322) Derived from pET28a(+) pBIDI231a tac/trp Ampicillin ColE1(pBR322) Provided by TU Graz

Table 3.5. Genes employed in this study and their origins. Gene and protein sequences are shown in appendix. Gene Full name Source

P450 BM3 Cytochrome P450 monooxygenase Bacillus megaterium P450 Cand_1 Cytochrome P450 monooxygenase Pseudomonas sp. 19-rlim P450 Cand_10 Cytochrome P450 monooxygenase Phenylobacterium zucineum

23 Materials and Methods

3.6. Software

SnapGene v.1.1.3 (GSL Biotech LLC, USA) for analysis of sequencing data, primer and construct design.

OriginPro v9.1 software for Windows (Originlab, Northampton, USA) for statistical analysis, plot and calculation of kinetic parameters.

GCsolution Chromatography Workstation v2.31.00 (Shimadzu Corporation, Duisburg, Germany) for analysis of GC chromatograms.

Yet Another Scientific Artificial Reality Application (YASARA Structure Version 17.4.17) for generation of homology models and substrate docking.

PYMOL (v.1.3) for generation of high resolution protein structure figures

24 Materials and Methods

3.7. Microbiological Methods

3.7.1. Preparation of Escherichia coli Competent Cells

Competence is the ability of a cell to take up extracellular DNA from its environment. Some strains are naturally competent and do not require any kind of treatment to be able to uptake foreign DNA. Laboratory cloning strains and production strains have to undergo special treatment in order to increase their competency and make them transiently permeable to DNA. During this study, to achieve a high amount of transformants, the natural competence of E. coli cells was increased by using a variation of the protocol by Hanahan [172]. E. coli cells from cryo-stock were transfered to inoculate 100 mL LB media in a 500 mL shake flask. The culture was grown until OD600 ≈0.4 – 0.8 (250 rpm, 30 °C) and then rested on ice for 15 to 20 min. The culture was divided into 50 mL falcons and media was removed by centrifugation (1200 g, 15 min, 4 °C). The obtained cell pellets were gently resuspended in ice-cold 25 mL TFB1 solution

(30 mM K-Acetate, 50 mM MnCl2, 100 mM CaCl2, 15 % (v/v) glycerol; pH 6.8; sterile filtered) combined and incubated for 15 min on ice. Resuspended cells were centrifuged (1200 g, 15 min, 4 °C). and gently resuspended in 2-4 mL ice-cold TFB2 solution (10 mM MOPS, 100 mM

CaCl2, 15 % (v/v) glycerol; pH 6.8; sterile filtered). TFB1 and TFB2 were sterilized using a sterile 0.2 μm filter. Cells resuspended in TFB2 solution were rested on ice for 15 min and aliquoted (100 μL) into ice-cold and sterile 1.5 mL tubes. The aliquots were immediately stored at -80 °C until further use. The competence of each batch of competent cells was always confirmed by transforming cells with pUC18 plasmid DNA. The transformed and recovered cells were plated in triplicate on LB agar plates containing the corresponding antibiotics and incubated overnight at 37 °C. The following day colonies were counted and the competence of the prepared cells was calculated.

3.7.2. Transformation of Plasmid DNA into Escherichia coli Competent Cells

Q1 Chemically competent E. coli BL21-Gold (DE3) lacI cells stored at -80 °C were thawed on ice for 15 min prior to transformation. DNA from ligation (≈ 25 ng), DNA hybridization from PLICing (≈ 200 ng) or plasmid DNA (≈ 10 ng) was mixed with 100 μL competent cells and incubated on ice for 15 min. The heat-shock was carried out at 42 °C for 45 s. Tubes were immediately placed on ice and incubated for 10 min. Afterwards 900 µL of LB media was added and cells were incubated (37 °C, 45 min, 250 rpm) to allow cell regeneration. After regeneration the cells were spread on LB agar plates with the corresponding antibiotics and incubated overnight at 37 °C.

25 Materials and Methods

3.7.3. Cryo-Culture Preparation

Although bacteria can be stored on LB agar plates at 4 °C for a few weeks, for a stable long-term storage, glycerol stocks at -80 °C should be prepared. The glycerol stabilizes the bacteria by preventing cell membrane damage at negative temperatures. Storage of obtained strains was carried out by striking one colony from a plasmid transformation (section 3.7.2) inoculating 5 mL LB media with the appropriate antibiotic and incubating overnight (37 °C, 250 rpm, 16 h). The following day, 900 μL of E. coli BL21-Gold (DE3) lacIQ1 overnight culture were mixed with 500 μL of sterile glycerol (50 % (w/v) in dH2O) in a cryo-vial and stored at - 80 °C. For storage of generated libraries in MTP format, to 150 μL of overnight culture (37 °C,

900 rpm, 14 h, 70 % RH) 100 μL sterile glycerol solution (50 % (v/v) in dH2O) was added to each well. The MTP was mixed in a MTP shaker (400 rpm) for c.a. 5 min and then stored at - 80 °C.

3.7.4. Shake Flask Expression

Pre-cultures for expression in shake flasks were prepared by inoculating a sterile glass tube containing 5 mL LB media with cells from the glycerol stock (see 3.7.3) following overnight incubation (37 °C, 250 rpm, 16 h). Protein expression was carried out in 500 mL flasks containing 100 mL TB medium supplemented with the appropriate selective marker and trace element solution (1 X final conc.). The overnight pre-culture was used to inoculate the production culture at an OD600 of 0.1 and incubated at 30 °C, 250 rpm until OD600 reaches 2.5. Protein expression was induced by addition of IPTG (1 mM final conc.) and supplemented with thiamine hydrochloride (100 mg/L final conc.) and 5-Aminolevulinic acid (0.5 mM final conc.) and carried out at 25 °C, 250 rpm for 18 h. Cells were harvested by centrifugation (3220 g, 20 min, 4 °C) washed with phosphate buffer (50 mM, pH 7.5), harvested again by centrifugation (3220 g, 20 min, 4 °C) and stored at -20 °C until further use. As a control, the expression protocol was performed in the same way with the empty vector plasmid containing no gene for heterologous expression.

3.7.5. Production of Indigoids

Pre-cultures for expression in shake flasks were prepared by inoculating a sterile glass tube containing 5 mL LB media with E. coli BL21-Gold (DE3) lacIQ1 cells co-expressing FdR- Fdx from Mycobacterium sp. HXN-1500 and P450 Cand_1 from Pseudomonas sp. 19-rlim in the pBIDI231a plasmid from the glycerol stock (prepared as in section 3.7.3) following overnight incubation (37 °C, 250 rpm, 16 h). Protein expression was carried out in 100 mL flasks containing 20 mL TB medium supplemented with the appropriate selective marker. The

26 Materials and Methods

overnight pre-culture was used to inoculate the production culture at an OD600 of 0.1 and incubated at 30 °C, 250 rpm until OD600 reaches 4.5. Protein expression was induced by addition of IPTG (1 mM final conc.) and L-tryptophan (2.5 g/L final conc.) being also supplemented, thiamine hydrochloride (100 mg/L final conc.) and 5-Aminolevulinic acid (0.5 mM final conc.) and carried out at 25 °C for 24 h.

3.7.6. Cell Lysis via Sonication

To free the over expressed proteins, frozen cell pellets from shake flask expressions were thawed on ice and ressuspended thoroughly in phosphate buffer (50 mM, pH 7.5) until no solid particles were visible (10 % culture volume). Efficient cell disruption was achieved by employing a sonication program (5 cycles: 50 % amplitude, 30 s ON, 30 s OFF) and removal of cell debris was achieved by centrifugation (>16 000 g, 30 min, 4 °C). The supernatant was used for P450 quantification with the CO binding assay (section 3.9.8) and stored at 4 °C until further use.

3.7.7. Mutant Library Preparation and Expression in Multi Well Plates

Constructed libraries were achieved by transferring single colonies with sterile toothpicks from agar plate into 96-well flat bottom plates (Greiner Bio-One GmbH, Frickenhausen, Germany) filled with 150 μL LB medium supplemented with the appropriate antibiotic. Plates were tightly sealed and incubated overnight in a humidified plate shaker (Multitron II, Infors GmbH, Einsbach, Germany) at 37 °C (900 rpm, 70 % RH). Libraries were stored as master plate as explained above in section 2.7.3. Expression in multi well plates was performed preparing a pre-culture by duplicating a master plate with a 96-pin replicator to 150 μL of LB medium supplied with the appropriate antibiotic in 96-well flat bottom multi well plates. Plates were tightly sealed and incubated overnight at 37 °C (900 rpm, 70 % RH). Protein expression was achieved by transferring 25 µL of the overnight culture into in 96-deep well plates (Polypropylene plates, Brand GmbH, Wertheim, Germany) which contained 600 μL TB media supplemented with the appropriate antibiotic, trace elements solution (1 X final conc.), IPTG (1 mM final conc.), thiamine hydrochloride (100 mg/L final conc.) and 5-Aminolevulinic acid (0.5 mM final conc.) and incubating at 30 °C, 900 rpm, and 70% RH. Expression cultures were harvested by centrifugation (3200 g, 4 °C, and 20 min), the supernatant removed and the obtained cell pellets were stored at -20 °C until further use.

27 Materials and Methods

3.7.8. Preparation of Cell Lysates in Multi Well Plate

The previously frozen pellets of the expressed libraries in 96-deep well plates were thawed at room temperature for 10 to 15 min and then 150 µL of phosphate buffer (50 mM, pH 7.5) were added. Resuspension was achieved by vortexing the deep-well plates. Afterwards, 150 μL of phosphate buffer (50 mM, pH 7.5) containing 8 g/L lysozyme were added and the plate was sealed with lid and insulation tape. Lysis occurred during incubation of deep-well plates (900 rpm, 37 °C, 1 h, 70 % RH). Lysates were transferred to a 96-well V bottom plate and centrifuged to remove cell debris (3220 g, 4 °C, and 20 min).

3.8. Molecular Biology Methods

3.8.1. DNA Extraction, Storage and Sequencing

All plasmid extractions from E. coli strains were performed using the QIAprep® Spin Miniprep Kit, as recommended by the manufacturer. For extraction, E. coli BL21-Gold (DE3) lacIQ1 cells were grown in 5 mL LB media supplemented with corresponding antibiotics as overnight culture (250 rpm, 37 °C, 16 h,). After purification, plasmids were quantified using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, USA) prior to storage at -20 °C. For sequencing, tubes were prepared as recommended, mixing 5 µL of plasmid at 100 ng/µL with 5 µL of primer at 5 µM. Primers used for sequencing can be found in appendix section 7.5 of this thesis. The sequencing files were analysed using SnapGene software v.1.1.3 (GSL Biotech LLC, USA).

28 Materials and Methods

3.8.2. Polymerase Chain Reaction (PCR)

Amplification of DNA was achieved by in vitro polymerase chain reaction (PCR) using either in-house Taq DNA or high fidelity PfuS DNA polymerase. Reaction mixtures and cycling conditions employed are shown in Table 3.6 and Table 3.7. Briefly, it contained in a final volume of 50 μL: 1 X DNA Polymerase Buffer; 3 U or 7.5 U of DNA Polymerase (Taq or PfuS respectively); 0.2 mM dNTP mix; 5 ng plasmid DNA; 0.2 μM forward and 0.2 μM reverse primer. Amplification of DNA was achieved in a PCR thermocycler Mastercycler Pro S (Eppendorf, Hamburg, Germany). Culture PCR was done using the same master mix for Taq DNA polymerase with a total volume of 12.5 µL (10 µL master mix plus 2.5 µL of boiled overnight culture).

Table 3.6. PCR master mix composition. Reagent Initial conc. Volume for Taq Volume for PfuS per reaction

Ultra-pure H2O - Up to 50 µL Up to 50 µL - Taq DNA polymerase buffer 10 X 5.0 µL - 1 X Pfus DNA polymerase buffer 10 X - 5.0 µL 1 X DNTP's mix 10 mM 1.0 µL 1.0 µL 200 µM Forward primer 20 µM 1.25 µL 1.25 µL 0.5 µM Reverse primer 20 µM 1.25 µL 1.25 µL 0.5 µM DNA template 5 ng/μL 1.0 µL 1.0 µL 5 ng

MnCl2 2.5 mM 1.0 or 1.5 µL - 0.05 or 0.075 mM Pfus DNA polymerase 2 U/µL - 1.5 µL 3 U Taq DNA polymerase 5 U/μL 1.5 µL - 7.5 U Total volume - 50 µL 50 µL -

Table 3.7. PCR cycling condition. PCR stage Temp. For Taq Time for Taq Temp. for PfuS Time for PfuS Cycles Initial denaturation 94 °C 3 min 98 °C 2 min 1 Denaturation 94 °C 30 s 98 °C 15 s Annealing Calculator 30 s Calculator 20 s 25 Extension 68 °C 1 min/kb 72 °C 30 s/kb Final extension 68 °C 1 min/kb 72 °C 1 min/kb 1 Storage 4 °C ∞ 4 °C ∞ 1

29 Materials and Methods

3.8.3. Oligonucleotide Design for PCR Amplifications

Primers (Table 7.1) for PCR were designed according to general rules for efficient amplification of DNA fragments (GC content: 40 to 60 %; Tm: > 55 °C; avoiding primer self- or hetero-dimers and hairpins to name a few). Designed oligonucleotides were analysed online using OligoAnalyzer Software from Integrated DNA Technologies.

3.8.4. Library Generation by Error-Prone PCR.

Random mutant libraries were generated employing PCR in the presence of manganese ions, known to increase the error rate of Taq DNA polymerase, using an adapted version of the protocol published by Cadwell et al. in 1992 [82]. Two different concentrations of MnCl2, 0.05 and 0.075 mM, were used to amplify the heme domain of monooxygenases while the remaining part of the plasmid was amplified by regular PCR with PfuS DNA polymerase. Afterwards PCR reaction was carried out in PCR thermocycler Mastercycler Pro S (Eppendorf, Hamburg, Germany). The PCR mix and PCR cycling conditions are displayed above in section 3.8.2.

3.8.5. Library Generation by Sequence Saturation Mutagenesis (SeSaM)

Sequence Saturation Mutagenesis (SeSaM) was performed using proprietary protocols, knowledge and polymerases from SeSaM Biotech, GmbH. A description of the method is published [173]. All PCR reactions were carried out in PCR thermocycler Mastercycler Pro S (Eppendorf, Hamburg, Germany).

3.8.6. Culture PCR

Culture PCR was performed to control the generated DNA constructs. Therefore, a single colony grown on LB agar was transferred to 5 mL of LB media with the corresponding antibiotic and grown overnight (37 °C, 250 rpm, 16 h). On the following day 50 µL of the grown culture was transferred to PCR tubes and boiled (95 °C, 15 min). Cell debris were removed by centrifugation (20 000 g, 5 min, RT) and 2.5 μL from the supernatant were added to 10 µL of Taq PCR Master Mix (section 3.8.2).

3.8.7. Circularization of Linearized DNA

Recircularization of linearized DNA was achieved by using a polynucleotide kinase (T4 PNK from Thermo Scientific) that catalysed the transfer of the γ-phosphate from ATP to the 5’-OH group of double-stranded DNA and a ligase (T4 DNA ligase) to ligate the 3’-hydroxyl group to the 5’ end of the linear product. Briefly, 6 µL of linear DNA (10 to 30 ng/μL) were mixed with 11.5 µL of dH2O, 1.5 µL of T4 DNA ligase buffer (10 X), 1 µL of PNK (10 U/µL)

30 Materials and Methods and 1 µL of T4 DNA ligase (400 U/µL). They were gently mixed by pipetting and briefly centrifuged before a 2 hours incubation at 37 °C. Afterwards the enzymes were heat inactivated at 65 °C for 15 min and the tubes stored at -20 °C until further use.

3.8.8. Agarose Gel Electrophoresis

Quality control of the purified/digested plasmids and PCR amplifications was achieved by performing a 1 % (w/v) agarose gel electrophoresis. Briefly, solid agarose powder was solubilized in TEA buffer (40 mM Tris; 2 mM EDTA; pH 8.0) by heating and after cooling to ≈ 60 °C, DNA staining solution Roti-Safe Gel Stain from Carl Roth (Karlsruhe, Germany) was added (0.01 % (v/v) final conc.). The solution was then poured into a tray with combs. The samples consisted of 5 μL of DNA samples mixed with 1 µL of 6 X loading buffer (60 % (v/v) glycerol, 0.03 % (m/v) Bromophenol Blue, 0.12 % (m/v) Orange G, 10 mM Tris-HCl and 60 mM EDTA). The prepared samples were loaded on individual wells and a potential differential was applied (8 V/cm for 45 min). Visualization of DNA samples on agarose gels was achieved with Gel Doc™ XR+ Gel Documentation System (Biorad).

3.8.9. Phosphorothioate-Based Ligase-Independent Gene Cloning (PLICing)

Phosphorothioate-based ligase-independent gene cloning (PLICing) is an enzyme free, nearly sequence independent method for cloning DNA fragments, with high efficiency and very low background of false positive colonies. It relies on a chemical cleavage reaction of phosphorothioate bonds of the PTO primers in an iodine/ethanol solution [45] that generates specific DNA overhangs for later hybridization, thus circumventing the limitations of restriction enzymes for cloning.

Figure 3.1. A schematic representation of the PLICing method. A) DNA backbone with a normal phosphodiester bond (top) and a phosphorothiodiester bond (bottom) in which one oxygen at the α-phosphate is replaced by a sulfur. B) Three step scheme of the PLICing method: amplification, cleavage, and hybridization. The figure was based on Blanusa et al. [171]

31 Materials and Methods

In the first step, the amplification of insert and vector was achieved with PTO (phosphorothiolated) primers employing a standard PCR protocol (section 3.8.2). The products were analyzed by agarose gel electrophoresis for quality of the amplified DNA (section 3.8.8). After DpnI digest and purification with QIAquick® PCR Purification Kit, the PCR products were diluted to 0.05 pmol/μL and 0.1 pmol/μL, vector and insert respectively. Cleavage of phosphorothiolated nucleotides was achieved by mixing 8 μL of insert DNA and 8 μL vector DNA each individually with 2 μL of iodine cleavage solution (30 mM Iodine in EtOH, 250 mM Tris-HCl p). The samples were then heated in a thermocycler (70 °C, 10 min) for cleavage of PTO nucleotides from the 5’endings. The mechanism for cleavage of PTO containing DNA was elucidated by Eckstein and Gish [174] and is shown below in Figure 3.2. Cleaved DNA fragments were then mixed and transformed into E. coli cells by heat shock (section 3.7.2). The obtained clones were analysed by culture PCR and agarose gel electrophoresis (section 3.8.5 and 3.8.8, respectively) and correct assembly of the plasmid was verified by sequencing of the generated DNA constructs.

Figure 3.2. Chemical cleavage of phosphothiolated nucleotides in the presence of iodine and ethanol (I2/EtOH) under alkaline conditions. In step one the sulfur atom is alkylated by iodoethanol leading to an instable intermediate which releases the phosphorothiolated nucleotide from the DNA. Based on Eckstein and Gish [174].

32 Materials and Methods

3.8.10. Site Directed and Site Saturation Mutagenesis

Focused mutagenesis is a standard approach in protein engineering for the substitution of single amino acids in protein sequences in a controlled and precise manner. The most common protocol for site directed mutagenesis (SDM) and site saturation mutagenesis (SSM) is the whole plasmid amplification using complementary synthetic oligonucleotides also known as the “QuikChange protocol” however, since the primers are complementary to each other, efficient DNA amplification is not always achieved due to formation of strong heterodimeric structures. All primers were designed according to recommendations provided in the QuikChange SDM manual and using OligoAnalyzer Software from Integrated DNA Technologies. Amplification of DNA was achieved by in vitro polymerase chain reaction (PCR) using high fidelity PfuS DNA polymerase as in section 3.8.2 and by the end of the 25 cycles confirmation of correctly amplified products was achieved by an 1 % (w/v) agarose gel electrophoresis (section 3.8.8). After that, samples were supplemented with CutSmart buffer (1 X final conc.) and 10 U of DpnI restriction enzyme to remove methylated template DNA (2 h at 37 °C) and samples were purified using QIAquick® PCR Purification Kit following the manufacturer’s instructions. DNA was quantified by using a NanoDrop Spectrophotometer 1000 (NanoDrop Technologies, Wilmington, USA) and directly transformed into chemically competent E. coli BL21-Gold (DE3) lacIQ1 cells (section 3.7.2). Obtained colonies were used to prepare variant libraries for screening in multi well plate format (section 3.7.7).

3.9. Biochemical Methods

3.9.1. NADPH Depletion Assay for Screening of Mutant Libraries in Multi Well Plate

The domain of P450 monooxygenases requires a constant supply of electrons from the reductase domain to hydroxylate substrates and NADPH is known to be a frequent electron donor during catalysis. [25]. Therefore, monitoring the depletion of NADPH at λ 340 nm can be to assess substrate conversion [97] however, due to the uncoupling effect often observed in P450 monooxygenases [25,26,29,35], the relation between NADPH depletion and product formation is not always direct and product based detection systems are favored. Nevertheless, recording NADPH depletion is still a screening method widely used. In this work, all screening procedures to identify P450 variants with improved activity towards the target substrate started with this assay. Lysate from mutant libraries was prepared as described in section 3.7.8 and two F-bottom multi well plates are prepared: one without substrate and one containing the target substrate for hydroxylation. The detailed reaction mixture can be seen below on Table 3.9.1. Immediately after supplementing

33 Materials and Methods

Table 3.9.1. Preparation of MTP plates for screening using the NADPH depletion assay. Compound Sample MTP Control MTP Substrate in ethanol (125 mM) 2 µL - Ethanol - 2 µL Phosphate buffer (50 mM, pH 7.5) 148 µL 148 µL Crude cell lysate 50 µL 50 µL NADPH (1 mM) 50 µL 50 µL Total Volume 250 µL 250 µL NADPH the absorption at λ 340 nm was recorded using a MTP reader (TECAN Sunrise, Tecan Group AG) for 20 cycles (10 s interval). NADPH-depletion values were calculated as slope of the absorption decrease during the linear range. To correct for the background signal, for each well the corresponding blank value (reaction with ethanol) was subtracted from the absorption value of the same well with substrate. Furthermore, depending on P450 activity and target substrate the total number of cycles was adjusted accordingly to monitor the linear depletion stage.

3.9.2. 4-AAP screening system for product based quantification of 2,3-dihydro-1,4- benzodioxin-5-ol and 2,3-dihydro-1,4-benzodioxin-6-ol

The 4-aminoantipyrine assay was introduced in the 1940s as a sensitive and reliable method for the detection of phenols (μg/L) in aqueous solutions.[175] The assay is based on the formation of a phenol-4-aminoantipyrine dye complex by oxidative coupling, leading to an extended conjugated electron system with strong absorbance at λ 509 nm [175]. The 4-AAP assay conditions were adjusted from Wong et al. (2005) for the application in phosphate buffer (KPi 50 mM, pH 7.5) and in MTP format. Briefly, the 4-AAP assay was performed after complete depletion of the NADPH during the NADPH depletion assay (section 3.9.1) and consisted of adding 25 µL of quenching solution (4 M Urea and 0.1 M NaOH) followed by 20

µL of 4-AAP (5 mg/mL in dH2O) and finally 20 µL of potassium peroxodisulphate (5 mg/mL in dH2O). The MTP was then incubated for 30 min (600 rpm, RT) and absorbance was measured at λ 509 nm using a MTP reader (TECAN Sunrise, Tecan Group AG).

3.9.3. Product Based Screening System for Quantification of Indigo and Indirubin

Products from P450 Cand_1 monooxygenase conversion of indole are indigo with the possibility of indirubin also being formed. Indigo is an organic compound with a distinctive blue color due to its strong absorbance at λ 610 nm allowing for an easy way to screen for its production. The assay conditions were adjusted for the application in phosphate buffer (KPi 50 mM, pH 7.5) and in MTP format. Briefly, it consisted of adding 100 μL of cell lysate with expressed P450 Cand_1 plus 2.5 mM indole, 2 % (v/v) EtOH, 3 U/mL GHD, 1200 U/mL

34 Materials and Methods catalase, 60 mM glucose in a total volume of 200 μL. MTPs were incubated for 5 min before supplementation with 50 μL NADPH (1 mM) and let to react for 20-24 hours (500 rpm, RT). Absorbance was measured at λ 610 nm using a MTP reader (TECAN Sunrise, Tecan Group AG).

3.9.4. Capillary Electrophoresis for Side Product Quantification of 2,3-dihydro-1,4- benzodioxin-2-ol

A generally applicable and emerging possibility for product based quantification is 96 multiplex-capillary electrophoresis (CE). It is a powerful, versatile and automated technique for the separation and analysis of charged substances and biological macromolecules such as amino acids, peptides and proteins, chiral drugs, whole cells and virus particles to name a few [105,106]. In this study the CE was used in parallel and for the rescreening only to investigate the formation of side products not detected by the 4-AAP assay. Briefly, each reaction contained per well: 50 μL cell lysate with expressed P450 BM3, 1.2 mM benzo-1,4-dioxane, 2 % (v/v) EtOH, 3 U/mL GHD, 1200 U/mL catalase, 60 mM glucose in KPi (50 mM, pH 7.5) in a total volume of 200 μL. MTPs were incubated for 5 min before supplementation with 50 μL NADPH (1 mM) and let to react for 4 hours (500 rpm, RT). Afterwards, 50 μL of a quenching solution (30 mM SDS, 15 mM NaPi, 6 mM benzyl alcohol in 4 M Urea) were added and the plate centrifuged from 15 min, 3220 g at RT). Afterwards, 100 μL of the supernatant were transferred to a 96 well PCR plate (VWR, USA) and sealed with a transparent film to avoid evaporation. Electrophoretic measurements were performed on 96 uncoated fused-silica capillaries (Advanced Analytical cePRO9600, USA) equipped with a UV diode array detector set for 214 nm. Data acquisition was performed with pKa Analyzer v.1.2 (Advanced Analytical, USA). Prior to their first use, the capillaries were conditioned with 1 M NaOH and deionized water for 40 min, and before measurement were conditioned with running buffer (30 mM SDS/15 mM NaPi) for 30 min. The capillary was flushed for 5 min with running buffer between runs. A pre- run at -11 kV for 1 min followed by hydrodynamic sample injection (-0.70 psi for 45 s). Separation was performed applying a voltage of -11 kV for 40 min. The standard deviation of the electrophoretic measurements was determined using 92 replicates of active P450 BM3 WT.

35 Materials and Methods

3.9.5. Statistical Evaluation and Selection of Variants

For reliable evaluation of large data sets from the NADPH depletion assay and 4-AAP assay (for P450 BM3) and Indigo screening assay (for P450 Cand_1), 2 formulas were used to identify improved P450 variants. Improved variants are characterized for increased NADPH depletion rate and higher amount of product formation relative to the starting variant or wild- type. P450 variants were only considered improved if the conditions below were satisfied:

Equation 3.1. Condition to consider a variant improved for NADPH depletion.

[ ] [ ] Improved variantNADPH= VariantNADPH ∆Abs+Sub - ∆Abs-Sub > WTNADPH ∆Abs+Sub - ∆Abs-Sub × T

Equation 3.2. Condition to consider a variant improved for product formation.

[ ] [ ] Improved variant푥= Variantx AbsVariant - AbsEV > WT푥 AbsWT - AbsEV × T

ΔAbs: NADPH depletion velocity (slope) at 340 nm; +/-Sub: reaction sample with (+) or without (-) supplemented substrate; Abs: Absorbance at λ 509 nm for 4-AAP or at λ 610 nm for indigo; WT: wild-type; EV: empty vector; T: threshold. x: assay to be used

The threshold for selection of improved variants is dependent on the deviation in activity for the starting variant or wild-type under identical screening conditions. Variants with increased NADPH depletion rate and/or increased product formation were selected rescreening. For P450 Cand_1 variant s only the criteria from Equation 3.2 were used using the absorbance values from section 3.9.3.

3.9.6. Purification and Lyophilisation of P450 BM3

Shake flasks expression ocurred as described above in 3.7.4, and purification of the P450 BM3 monooxygenase variants was performed adapting the original protocol described by Nazor et al. (2007). Briefly, for the purification frozen cell pellets from a 250 mL culture were resuspended in 15 mL Tris/HCl buffer (100 mM, pH 7.5). Cells were homogenized by sonication for 5 min (with 30 s interval, 50 % amplitude, Vibra-Cell VCX-130; Sonics, Newtown, CT, USA). After centrifugation (30 min, 16 000 g at 4 °C), the supernatant was filtered with a 0.22 μm filter membrane. Purification of the P450 BM3 variants was performed by anion exchange chromatography with a Toyopearl DEAE 650S matrix (Tosoh Bioscience, Griesheim, Germany) and an ÄKTA prime chromatography system (GE Healthcare, Solingen, Germany) using a variation of the established protocol [177]. The purified P450 BM3 enzyme was concentrated with an Amicon centrifugation tube (50 kDa cut-off; Merck Millipore, Darmstadt, Germany) and desalted using a PD-10 desalting column (GE Healthcare)

36 Materials and Methods equilibrated with KPi (50 mM, pH 7.5). For long-time storage, enzyme samples were shock- frozen in liquid nitrogen and lyophilized (Alpha 1–2 LD plus freeze-dryer Christ, Osterode am Harz, Germany). For long-term conversions cell-free lysates were used by ressuspending the frozen cell pellets in KPi (50 mM, pH 7.5) (10 % of culture volume) and lysed by sonication for 5 min (with 30 s interval, 50 % amplitude, Vibra-Cell VCX-130). Cell debris was removed by centrifugation (30 min, 16 000 g at 4 °C).

3.9.7. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a technique generally used for separation of proteins based on their size. SDS (an anionic detergent) in conjunction with β-mercaptoethanol (used to reduce disulphide bonds) linearizes all proteins and sets a negative charge on the linearized proteins. This ensures that, when an electric field is applied, samples migrates according to their size through a gel matrix of acrylamide/bisacrylamide. Separated protein bands can then be visualized either by positive (e.g. coomassie brilliant blue) or negative (e.g. copper) staining. For this work, a 10 % SDS- PAGE was used to evaluate protein production and purification. The composition of the gels used can be seen on Table 3.8.

Table 3.8. Composition of the gels for the SDS-PAGE. Reagent Stacking Gel Running Gel 40 % Acrylamide mix 3750 µL 1.75 mL

H2O 1.6 mL 2.0 mL 0.25 M Tris-HCl, SDS 0.2 % (m/v) pH 6.8 750 µL - 0.75 M Tris-HCl, SDS 0.2 % (m/v) pH 8.8 - 2.8 mL APS 10 % (m/v) 30 µL 12.5 µL TEMED 5.0 µL 5.0 µL

All gels were prepared as described in the original protocol. Sample preparation was done incubating 15 μL of protein samples (lysate or purified protein) in 5 μL SDS-PAGE loading buffer (1 x Roti-Load) in a heat block (15 min, 98 °C) for entire denaturation of proteins. The samples plus a pre-stained reference protein ladder were loaded individually on the well SDS- PAGE gel. Peptides were separated in Tris-glycine buffer (0.3 % (m/v) Tris, 1.5 % (m/v) glycine, 0.1 % (m/v) SDS in dH2O, no pH adjustment) by applying a differential potential of 16 mA/gel for 45 min. Visualization of protein bands was achieved by coomassie-brilliant blue

(CBB) staining (0.25 % (m/v) CBB, 45.5 % (v/v) methanol, 9.2 % (v/v) acetic acid, in dH2O).

37 Materials and Methods

3.9.8. P450 Quantification by Carbon Monoxide Difference Spectrum

To measure productivities, the quantification of active P450 monooxygenase in cell lysates and purified lysates. The quantification was performed according to the published protocol from Omura and Sato [20] where the reduced heme-iron has an absorption maximum at 450 nm and thus the concentration of properly folded P450s can be determined spectrophotometrically. The protein sample (crude cell lysate or purified or lyophilized protein) was diluted (1:4) in 50 mM phosphate buffer to 1 mL final volume in spectrophotometer cuvettes. Afterward, a few milligrams of sodium dithionite were added to reduce the heme-iron of P450s, mixing was achieved by pipetting. The absorbance spectrum was measured from λ 400 nm to λ 500 nm in spectrophotometer cuvettes using a Varian Cary 50 spectrophotometer (Agilent Technologies, Darmstadt, Germany) as a baseline measurement. The sample was saturated with CO for 20 sec using a bottle station (Sigma Aldrich, Steinheim, Germany) and the absorbance spectrum was recorded again from λ 400 nm to λ 500 nm. The amount of active P450 BM3 concentration was calculated by using the following formula:

Equation 3.3. The equation for calculating the P450 sample concentration.

Abs450 C = ×DF P450 ε ∙ d

ε: Extinction coefficient Home-CO-complex (91 mM-1 cm-1) d: light path length (1 cm) DF: dilution factor

3.9.9. Determination of NADPH Oxidation Rates and Coupling Efficiency of P450 BM3 Wild-type and Variants

The domain of P450 monooxygenases requires a constant supply of electrons from the reductase domain to hydroxylate substrates and NADPH is known to be a frequent electron donor during catalysis. [25]. Therefore, monitoring the depletion of NADPH at λ 340 nm can be to assess substrate conversion [97]. Measurement of NADPH depletion by P450 monooxygenases and variants was done in cuvette format using a Varian Spectrophotometer Cary 50 UV (Agilent Technologies, Darmstadt, Germany). Reactions were assembled in the cuvette with a final volume of 5 mL and distributed to the cuvettes. The reaction mixture contains the following components that were adjusted accordingly depending on the substrate and variant employed for the measurements: KPi buffer (pH 7.5, 50 mM), purified P450, substrate dissolved in EtOH (1 mM) and NADPH (500 µM). For every substrate class investigated in this thesis a small experimental section is provided to underline specific

38 Materials and Methods alterations in the reaction mixtures. After 5 min incubation the reaction is started by addition of NADPH. Depletion of NADPH is monitored at λ 340 nm using intervals of 1 sec. To correct for the background signal, for each reaction the corresponding blank value (reaction with ethanol) was subtracted from the activity with substrate. The equation (displayed below) was used to determine NADPH oxidation activity of P450.

Equation 3.4. The equation for determining the NADPH oxidation rate.

-1 ∆Abs340 min NADPH oxidation rate [min-1] = εNADPH × CP450 × d

-1 ΔAbs340 min : decrease in absorbance per minute at 340 nm wavelength; -3 -1 -1 εNADPH = specific extinction coefficient of NADPH at 340 nm [6.22 x 10 μM cm ]; CP450 = concentration of P450 monooxygenase in the reaction [μM]; d = path length [cm].

After full depletion of NADPH, the products were extracted by partitioning using two- phase extraction (section 3.10.1). Products were quantified on GC-FID applying calibration curves of the commercially available standards or using internal standards. The coupling efficiency was calculated from the quantity of depleted NADPH and yielded product amount. Following equation was used for calculation of the coupling efficiency:

Equation 3.5. The equation for determining coupling efficiency.

Product [µM] Coupling efficiency [%]= ×100 Consumed NADPH [µM]

39 Materials and Methods

3.9.10. Long Term Substrate Conversions

To access productivity (substrate conversion, product yield, and concentrations, TTN) and regioselectivity of the monooxygenases and engineered variants, long term reactions with a NADPH regeneration system using glucose dehydrogenase (GDH) and glucose were performed using either cell-free lysate (3.7.6) or purified protein (section 3.9.6). All reactions were performed assembled in 1 mL total volume in glass tubes sealed with a septum, a lid and with 50 % gaseous volume for oxygen supply. The composition of the reaction can be seen in Table 3.9. The reaction began by the addition of NADPH and continued for 1 h (250 rpm, RT). By the end of 1 hour, 100 µL of quenching solution was added (HCl 37 % (v/v)) and product

Table 3.9. Preparation of monooxygenases long-term substrate conversion.

Compound Sample Control Reaction Final Conc. Substrate in ethanol (125 mM) 12 µL 12 µL 1.5 mM Glucose (1 M) 60 µL 60 µL 60 mM Catalase (50.000 U/mL) 24 µL 24 µL 1.200 U/mL Glucose Dehydrogenase (1.000 U/mL) 3.0 µL 3.0 µL 3 U/mL Phosphate buffer (50 mM, pH 7.5) 501 µL 501 µL - Monooxygenase (3.33 µM) 300 µL - 1 μM Crude cell lysate from Empty Vector - 300 µL - NADPH (4 mM) 100 µL 100 µL 0.4 mM Total Volume 1000 µL 1000 µL - extraction followed (section 3.10.1) before analysis by GC and GC-MS (section 3.10.3 and 3.10.4). All reactions were performed in triplicate and a control reaction with crude cell lysate from EV was also performed. A full list of employed standards, their specific retention times, employed GC-column and GC heating programs can be found as in the appendix section of this thesis (section 7.5).

40 Materials and Methods

3.10. Analytical and Chemical Methods

3.10.1. Two-Phase Solvent Extraction of Reaction Products

To access productivity, the products from P450 BM3 monooxygenase conversions were identified by gas-chromatographic mass analysis (GC-MS) and quantified on GC-FID using calibration curves prepared with commercially available standards. The generated products were extracted by partitioning (two-phase extraction) employing methyl tert-butyl ether (MTBE) or ethyl acetate (EtOAc) as an extraction solvent. Commonly MTBE was used due to the low toxicity and easy handling during extractions (0.74 g/cm³ density). A compilation of all substrates, internal standards, and extraction solvents can be found in the appendix section (Table 7.2). Reaction mixtures and extraction solvent were mixed in a ratio of 2:1 thoroughly on a vortex (5 min, RT) to achieve a high level of reproducibility. Both phases were separated by centrifugation (20 000 g, 7 min, RT) and the organic phase was transferred to a 2 mL tube containing anhydrous MgSO4 to remove residual water. In the last step, the water-free organic phase was centrifuged to pellet the salts. Liquid supernatants were filled into GC glass vials containing 200 μL glass inlets.

3.10.2. Quantification of Indigo and Indirubin by High-Performance Liquid Chromatography

The detection and quantification of Indigoids are generally done via HPLC, UHPLC/MS or UV-spectroscopy [134,178–180]. Products from P450 Cand_1 monooxygenase conversion of indole were quantified by High-Performance Liquid Chromatography (HPLC), by preparing calibration curves with the available commercial standards. Indigo has low solubility in water but is soluble in solvents such as dimethyl sulfoxide (DMSO) or trifluoroacetic acid (TFA). Briefly, After the indigoid production (section 3.7.5), the culture broth was centrifuged at 10 000 g for 15 min to yield dark blue pellets, which were collected and washed with water. The material was then suspended in 10 ml of DMSO and subjected to repeated sonication with a microprobe for 5 min (30 s ON, 30 s OFF). The solutions was then centrifuged again to separate the cell debris (10 000 g for 15 min). The amount of bio-indigo produced in the supernatant was determined using a standard indigo solution dissolved in DMSO. An HPLC equipped with a photodiode array detector (Shimadzu GmbH, Duisburg, Germany), was used with an eluent flow rate of 1 mL/min and monitored at λ 620 nm, indirubin was monitored at 540 nm. A column used was a Nucleosil 100-5 (C18, 250mm×4.6 mm) with isocratic elution of methanol and water (70:30, v/v) [181].

41 Materials and Methods

3.10.3. GC-FID Measurements

Substrate depletion and product formation were measured on a GC-FID 2010 (gas chromatography with flame-ionization detector from Shimadzu GmbH, Duisburg, Germany). Calibration curves were prepared with commercially available analytical standards (Table 7.2). Products resulting from P450 BM3 conversions were separated using the following program: (100 °C for 1 min, heating 10 °C/min up to 200 °C, heating 20 °C/min up to 250 and hold for 10 min unsing an Optima-17MS column from Macherey-Nagel). All reactions were performed in triplicate. Data was processed as linear regression for product quantification. All extractions were performed in triplicate.

3.10.4. GC-MS Measurements

Initial identification of product masses was done on GCMS-QP2010S (Shimadzu GmbH, Duisburg, Germany) using helium as the carrier gas. Extraction samples were injected (1 μL; injector temperature: 300 °C) and separated on an Optima-17 MS column using the same heating programs as above (3.10.3). Obtained fragmentation patterns were analyzed with the provide compound library and by injecting commercially available standards.

3.11. Molecular Modeling

3.11.1. Molecular Docking

The starting coordinates of the P450 BM3 WT were taken from the crystal structure of cytochrome P450 BM3 with the heme domain (PDB ID: 1BU7 [53]). The models of the P450 BM3 variants R255G and R255L were constructed using the swap function in YASARA Structure Version 17.4.17 [61] and optimized using SCWRL [62] rotamer library search for the designated substitutions. The protein residues were treated using the AMBER ff99 [63] and substrate (benzo-1,4-dioxane) was treated employing GAFF [64,65] with AM1-BCC partial charges [66] with particle mesh Ewald [67] for long-range electrostatic interactions and a direct force cutoff of 10.5 Å. The crystal water molecules present in the crystal structure were deleted except the one that is coordinated to the Fe2+ ion of the heme domain. The constructed models were minimized using a water box first with the steepest descent and then simulated annealing (timestep of 2 fs, atom velocities scaled down by 0.9 every 10 steps) starting from 98K, 198K, and 298K with a time-averaged Berendsen thermostat until convergence was reached. The minimized models were further used for molecular docking studies of the substrate benzo-1,4-dioxane. A grid box of 12 Å around the active site was applied by centering heme iron of P450-BM3. Molecular docking calculations were performed using Autodock4.2 plugin

42 Materials and Methods within YASARA with a fixed protein backbone. 100 docking runs were carried out and the docking solutions were clustered applying a RMSD cutoff of 0.5 Å and using the default settings provided within the YASARA dock_run macro file. Molecular docking results were analyzed by considering the distance between the iron-bound water molecule and the closest C-atom (C5) of benzo-1,4-dioxane.

3.11.2. Molecular Dynamics Simulation

Molecular dynamics simulations were carried out using the enzyme-substrate complex obtained from molecular docking of the substrate benzo-1,4-dioxane in the binding pocket of P450-BM3 WT and variants (R255G and R255L). The PROPKA 3.1 program [182] was used to determine the protonation states of titratable residues on the basis of pKa values and visual inspection. The Amber ff14SB force-field parameters [183,184] for the protein and general Amber force field (GAFF) [185] for heme were used. The required heme parameters were taken from the literature [186] and substrate benzo-1,4-dioxane was optimized with B3LYP method [187] and 6-311G(d,p) [188] basis set using Gaussian09 [189]. Moreover, RESP charges were calculated using the Antechamber module in Amber14 [190]. The whole system was neutralized by adding 15 Na+ ions in WT and 16 Na+ ions in R255G and R255L variants. Hydrogen atoms were added using tleap module of AmberTools14 [190]. The protein was solvated in an octahedral TIP3P water box centered at the center of mass to ensure a water layer of 12 Å around the protein. The systems contained ≈ 67 000 atoms in total, including ≈ 6623 TIP3P [191] water molecules. Initially, the solvent and the ions were minimized using whole system minimization with 10 000 steps of steepest descent followed by 3000 steps of conjugate- gradient minimization. Afterwards, the system was heated slowly from 0 to 300 K for 50 ps. Constant pressure periodic boundary conditions using the particle mesh Ewald (PME) [192] method were employed during MD simulations. The electrostatic interactions were calculated using a cutoff of 10 Å. After heating step, the systems were equilibrated for 1000 ps at 300 K. Three independent production runs each for 50 ns were carried out to have reasonable statistics. All classical molecular dynamics (MD) simulations were performed using the Amber14 program [190]. The obtained MD simulations trajectories were visualized and analyzed with Pymol [193], VMD [194], and AmberTools 14 [190].

43 Results and Discussion

4. Results and Discussion

This section is divided into 3 parts, one for each monooxygenase tackled. First, the directed evolution of P450 BM3 towards functionalization of the aromatic o-heterocycles benzo-1,4-dioxane, then P450 Cand_1 from Pseudomonas sp. 19-rlim for increased indigo production and third, the active site engineering of P450 Cand_10 and its effect on the activity towards benzo-1,4-dioxane and indole.

4.1. P450 BM3

4.1.1. Selection of P450 BM3 starting variant and its substrate

P450 BM3 wild type (WT) has low to no conversion of benzo-1,4-dioxane, therefore the P450 BM3 engineering strategy started with the selection of a starting variant. We have picked variants from the institute strain collection that have been previously reported increased hydroxylation of non-natural substrates such as phenols or cycloalkanes (Table 4.1). All variants were expressed as in 3.7.7 and 3.7.8, and the NADPH depletion assay was performed (section 3.9.1) using different O- and N-heterocycles as substrate (Figure 4.1). From the figure

Table 4.1. The P450 BM3 variants used for the selection of the starting variant. Substitution Improvement reported Reference L188P Alkane hydroxylation [195] R47S/Y51W (M1) R47S/Y51W/I401M (M2) Hydroxylation of phenols [196] R47S/Y51W/A330F/I401M (M3) F87A Changed Regioselectivity for: Propylbenzene & 3-chlorostyrene [197] F87A/A328I Nootkatone production [197] R255P/P329H (CM1) Hydroxylation of cycloalkanes [198,199]

Figure 4.1. NADPH consumption rate comparison between P450 BM3 WT and the selected variants (bars). M1 (R47S/Y51W), M2 (R47S/Y51W/I401M), M3 (R47S/Y51W/A330F/I401M), CM1 (R255P/P329H). Error bars represent one SD of the mean from three replicates.

44 Results and Discussion it is possible to observe that P450 BM3 WT has no detectable activity (i.e. NADPH depletion) towards the tested substrates and that the most active variants are P450 BM3 M3 (R47S/Y51W/A330F/I401M), F87A/A328I and CM1 (R255P/P329H) especially towards the tested O-heterocycles 2,3-dihydrobenzofuran and benzo-1,4-dioxane. For instance, M1 and M2 have identical activity over benzo-1,4-dioxane and only when the substitution from M3 A330F is present there is an increase in NADPH depletion. By contrast, variant CM1 (R255P/P329H) does not share any of the M3 substitutions and has a similar activity profile, especially towards benzo-1,4-dioxane. In a similar fashion, the variant F87A/A328I also has distinct substitutions from CM1 and M3 and has the highest activity towards benzo-1,4-dioxane. This suggests that several different positions play a role in increasing the activity towards the tested O- heterocycles. We choose CM1 to be the starting variant for the evolution campaigns because compared to F87A/A328I, it exhibited a more harmonious activity towards the tested substrates and, compared to M3 it possessed fewer substitutions (2 vs. 4).

4.1.2. Using the 4-AAP Screening System for Product-Based Quantification

The two major products of the biotransformation of benzo-1,4-dioxane with P450 BM3 wild type (WT) were identified to be 2,3-dihydro-1,4-benzodioxin-5-ol and 2,3-dihydro-1,4- benzodioxin-6-ol, in a 70/30 ratio (Figure 4.2). Since hydroxylation occurred on the benzene

Figure 4.2. The hydroxylation of benzo-1,4-dioxane by cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) leads to the formation of 2,3-dihydrobenzo-1,4-dioxin-5-ol and 2,3-dihydrobenzo-1,4-dioxin-6-ol at a 70/30 ratio. ring, an assay showing color formation in the presence of phenolic compounds would offer itself as a simple means for high-throughput screening. 4-aminoantipyrine (4-AAP) is a compound that was first introduced for the reliable and sensitive detection of phenols (µg/L) in aqueous solution assays in the 1940s [175]. The interaction between phenols and 4-AAP through oxidative coupling leads to an extended conjugated electron system with strong absorbance at λ 509 nm [98,175]. The 4-AAP assay conditions were adjusted from Wong et al. (2005) [98] for application in phosphate buffer (KPi 50 mM, pH 7.5) and in multi-well plate format. Under the new conditions, 2,3-dihydro-1,4-benzodioxin-5-ol and 2,3-dihydro-1,4- benzodioxin-6-ol concentrations showed a linear response from 16 to 500 µM at

45 Results and Discussion

λ 509 nm (Figure 4.3). The nicotinamide adenine dinucleotide phosphate (NADPH) depletion assay [97] was applied in combination with the 4-AAP assay to assess both NADPH depletion rates and total product formation. The standard deviation of the 4-AAP assay after full depletion of NADPH was 6.8 % using the P450 BM3 CM1. After subtraction of the background (EV lysate), a true standard deviation of 9.6 % was obtained (Figure 4.4). Standard deviations below 15% are routinely employed in successful directed evolution campaigns [98,200]. The 4-AAP assay can detect phenolic compounds, but it cannot detect products hydroxylated at the heterocycle ring, thus the need to develop a screening assay to overcome that limitation.

Figure 4.3. The 4-AAP linear detection range of 2,3-dihydrobenzo-1,4-dioxin-5-ol and 2,3-dihydrobenzo- 1,4-dioxin-5-ol in 96-well MTP format.

Figure 4.4. The standard deviation of the 4-AAP assay with P450 BM3 WT. Measured absorption values at λ 509 nm in descending order of P450 BM3 WT catalyzed conversion of benzo-1,4-dioxane in a 96-well plate. The apparent standard deviation (6.8%) is depicted with white triangles. The white hexagons show the true standard deviation (9.4%) after subtraction of the empty vector background.

46 Results and Discussion

4.1.3. Development of CE Screening System for Product-Based Quantification

Here, the development of a 96-well capillary electrophoresis for the separation and UV detection of benzo-1,4-dioxane products hydroxylated by P450 at the heterocycle ring is described. The benzene ring in benzo-1,4-dioxane has conjugated π-electron systems that strongly absorb in the UV range. The conditions used for CE separation and detection via UV spectroscopy were adapted from Anna et al. (2019) [104] where we were able to detect and separate the substrate benzo-1,4-dioxane from the products 2,3-dihydro-1,4-benzodioxin-5-ol and 2,3-dihydro-1,4-benzodioxin-6-ol (Figure 4.5). Minimal difference in the retention time is possible to observe due to differences in the capillary length. We used the CE in parallel with the NADPH depletion assay (section 3.9.1), however, the NADPH concentration used (200 µM) was not sufficient to detect a significant formation of 2,3-dihydro-1,4-benzodioxin-5-ol and 2,3-dihydro-1,4-benzodioxin-6-ol. To achieve higher product amounts, a NADPH regeneration solution containing glucose dehydrogenase (GDH) (3 U/mL), glucose (60 mM) and catalase (1200 U/mL) was used. We investigated different NADPH regeneration times (0.5–20 h), and 4 h turned out to be suitable for complementing the rescreening. Furthermore, the CE detector showed a linear response between 50 µM and 2 mM (Figure 4.7) with a standard deviation of 15.6 % after 4 h of reaction using the P450 BM3 CM1 (Figure 4.6).

Figure 4.5. Substrate and product separation via CE. EOF—electro-osmotic flow; i.s.— internal standard (benzyl alcohol); a—2,3-dihydrobenzo-1,4-dioxin-6-ol; b—2,3-dihydrobenzo-1,4- dioxin-5-ol; c—benzo-1,4-dioxane; Mix-mixture of 2,3- dihydrobenzo-1,4-dioxin-6-ol, 2,3-dihydrobenzo-1,4-dioxin-5-ol and benzo-1,4-dioxane.

47 Results and Discussion

Figure 4.6. The CE linear detection range of 2,3-dihydrobenzo-1,4-dioxin-5-ol in a 96-well MTP format.

Figure 4.7. The standard deviation of the CE with P450 BM3 WT. Detected signal amplitude (mAU) at λ 214 nm in descending order of P450 BM3 variant WT catalyzed conversion of benzo-1,4-dioxane in a 96-well plate. The standard deviation (15.6%) is depicted with black triangles.

48 Results and Discussion

4.1.4. Development of a Gas Chromatography method for benzodioxins quantification

The analysis of engineered P450 BM3 variants, via identification and quantification of formed products during conversion benzo-1,4-dioxane is essential. Here, the development of a gas chromatography (GC) method for separation of products and educts converted by P450 is described. Standards of benzo-1,4-dioxane, 2,3-dihydro-1,4-benzodioxin-5-ol and 2,3-dihydro- 1,4-benzodioxin-6-ol were commercially available. The method was developed with a GC coupled to a flame ionization detector (FID), different temperature gradients ranging from 40 °C to 300 °C were tested as well as different GC columns with different coatings. The column Optima 17 MS columns (Macherey-Nagel) was the one producing the best separation profile. On top of that, the Optima 17 MS column was also coupled to mass spectroscopy (MS) detection and was therefore chosen for GC analysis. A satisfactory separation was observed for the benzo-1,4-dioxane derivatives 2,3-dihydro-1,4-benzodioxin-5-ol and 2,3-dihydro-1,4- benzodioxin-6-ol as seen in Figure 4.8 when using MTBE as solvent. MTBE was used as solvent and extraction solvent due to the low toxicity and easy handling during extractions (0.74 g/cm³ density). Using the established method, calibration curves of 2,3- dihydro-1,4-benzodioxin-5-ol and 2,3-dihydro-1,4-benzodioxin-6-ol and internal standard were prepared with the established GC-method to enable quantification of all products (Figure 4.9 and Figure 4.10).

Figure 4.8. The GC-FID chromatograms of benzo-1,4-dioxane derivates separation. A thermal gradient (were separated using the following program: (100 °C for 1 min, heating 10 °C/min up to 200 °C, heating 20 °C/min up to 250 and hold for 10 min) was applied on a Optima 17 MS column which yielded in separation of benzo-1,4-dioxane (a), 2,3-dihydro-1,4- benzodioxin-5-ol (b) and 2,3-dihydro-1,4-benzodioxin-6-ol (c). (a) MTBE – (methyl tert-Butyl ether).

49 Results and Discussion

Figure 4.9. The standard curve of benzo derivatives 2,3-dihydro-1,4-benzodioxin-5-ol and 2,3-dihydro-1,4-benzodioxin-6- ol. The samples were prepared in MTBE with concentrations ranging from 0.125 mM to 3 mM.s

Figure 4.10. The standard curve of the internal standard cyclododecanol. The Samples were prepared in MTBE with concentrations ranging from 0.2 mM to 2 mM

50 Results and Discussion

4.1.5. P450 BM3 Library Generation and Screening

The P450 BM3 engineering strategy is summarized in Figure 4.11, where screening of previously prepared in-house epPCR and site-saturation-mutagenesis (SSM) libraries of P450 BM3 WT yielded P450 BM3 CM1 (R255P/P329H) (see section 4.1.1), which was subjected to two rounds of epPCR to identify additional beneficial positions. The epPCR was performed on the heme domain of P450 BM3 CM1 using a MnCl2 concentration of 0.05 mM was confirmed by agarose gel electrophoresis (Figure A5 Appendix Section 7.5). The P450 BM3 CM1 (R255P/P329H) gene libraries were cloned into the vector backbone (pALXtreme-1a) via

Figure 4.11. Summary of P450 BM3 engineering strategy. Starting from the top to the bottom, in-house libraries of P450 BM3 were screened yielding P450 BM3 CM1 (R255P/P329H), which was subjected to epPCR, and P450 BM3 GS2 (R255S/P329H/F331L) was generated. P450 BM3 GS2 was subjected to another epPCR round yielding P450 BM3 GS3 (I122V/R255S/P329H/F331L). P450 BM3 WT was subjected to single site saturation mutagenesis (SSM) in the four identified positions and double SSM at positions I122 and R255, which led to the most active variants, P450 BM3 R255G and R255L. PLICing and subsequently transformed into chemically competent Escherichia coli BL21-Gold Q1 (DE3) lacI cells (Figure A6). Following DNA extraction of randomly picked clones and DNA sequencing at Eurofins Genomics (Germany) we observed an average of 5.2 mutations per kb. The number of active clones/mutational load was adjusted to the 96-well microtiter plate (MTP) screening format, to efficiently screen mutant libraries and minimize screening efforts. The percentage of active clones was determined by expressing 88 clones in 96-well MTPs and subsequent activity determination (NADPH depletion only). The variants were considered active if they exhibited a higher NADPH consumption rate than that of the empty vector (plus standard deviation of EV and the method itself). Using this, the sample plate used for checking activity exhibited an average of 75 % active clones (Figure 4.12). Screening of the P450 BM3-

51 Results and Discussion

Figure 4.12. The relative NADPH consumption of the clones present in the sample plate from 0.05 mM MnCl2 P450 BM3- epPCR library. Green bar represents P450 BM3 CM1 (positive control), EV – Empty Vector (negative control), red line represents the threshold for classifying the clones active and green line the threshold for classifying the clones improved. epPCR library (0.05 mM MnCl2) was performed via NADPH depletion assay in combination with a 4-AAP assay in a 96-well format. The lysate of E. coli BL21-Gold (DE3) lacIQ1 expressing P450 BM3 CM1 (CM1) in pALXtreme-1a served as a positive control and the lysate of E. coli BL21-Gold (DE3) lacIQ1-pALXtreme-1a (EV) as a negative control in each MTP (in quadruplicates). The activity was determined by measuring the decrease in absorbance (NADPH depletion) and taken as an absolute value of the slope, plus measuring the absorbance at λ 509 nm after performing the 4-AAP assay. Variants exhibiting significantly higher absolute values of the slope (i.e., activity) and/or higher absorbance at λ 509 nm (after 4-AAP assay) than CM1 were selected for rescreening (Equation 3.1 and Equation 3.2). In total, nearly 4000 clones from the two rounds of epPCR were screened, and the most promising variants were selected for re-screening. Re-screening results revealed that from each round of epPCR, the variants exhibited improved activity and/or higher product formation in comparison to the WT (Figure 4.13). This led to the identification of four positions in total (I122, R255, P329,

Figure 4.13. Comparison between P450 BM3 WT and resulting variants from the 2 rounds of epPCR of NADPH consumption rate (bars) and product formation (circles) using a 4-AAP assay. Error bars represent one SD of the mean from three replicates. CM1 (R255P/P329H), GS2 (R255S/P329H/F331L), and GS3 (I122V/R255S/P329H/F331L). Error bars represent one SD of the mean from three replicates.

52 Results and Discussion and F331) that were selected for individual site saturation mutagenesis (SSM) using WT as a template (Figure A4 in Appendix Section 7.5). The selected variant P450 BM3 GS3 (I122V/R255S/P329H/F331L) exhibited the highest product formation and was subsequently sent for sequencing analysis. To guide the recombination of beneficial substitutions, the crystal structure of P450 BM3 WT (PDB ID: 1BU7 [77]) was visually inspected to locate substitutions (Figure 4.14). All four positions (I122, R255, P329, and F331) were selected for individual site saturation mutagenesis (SSM) using the WT as a template. Each SSM library was screened using the 4-AAP screening assay in the same way as performed for the epPCR libraries. After the screening of 528 clones, 11 P450 BM3 WT-SSM variants showed significantly increased activity in comparison to WT (Figure 4.15) and were selected for rescreening by CE to evaluate the formation of additional products. The rescreening revealed that the most active variants had substitutions at positions 122 and 255. Therefore, a double SSM library was prepared (P450

Figure 4.14. The crystal structure of P450 BM3 WT illustrating the 4 identified positions (I222, R255, P329, and F331) from the two rounds of epPCR. The positions selected are represented as ball and sticks. The heme cofactor is depicted in red sticks.

Figure 4.15. Comparison of product formation by the colorimetric 4-aminoantipyrine (4-AAP) assay (λ 509 nm). Results show the best-performing P450 BM3 from SSM at the positions 122, 255, 329, and 331. Error bars represent one SD of the mean from seven replicates. CM1 (R255P/P329H), GS2 (R255S/P329H/F331L), and GS3 (I122V/R255S/P329H/F331L). EV—negative lysate control. Error bars represent one SD of the mean from three replicates. 53 Results and Discussion

BM3 WT-dSSM), but no variant with a relative product formation higher than P450 BM3 R255G (R255G) was found after screening approximately 800 clones. Variants R255G and R255L exhibited at least a 4.0- and 3.5-fold improvement, respectively, in total product formation (analyzed via 4-AAP assay). Furthermore, no additional product formation was detected (analyzed via CE, Figure 4.16). These results led to the selection of R255G and R255L variants for further characterization.

Figure 4.16.Product analysis via CE after conversion of benzo-1,4-dioxane with lysate from EV (negative control), P450 BM3 WT, and variants R255G and R255L. The data was obtained from 4 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. EOF—electro-osmotic flow; i.s.— internal standard (benzyl alcohol); a—2,3- dihydrobenzo-1,4-dioxin-6-ol; b—2,3-dihydrobenzo-1,4- dioxin-5-ol; c—benzo-1,4-dioxane.

54 Results and Discussion

4.1.6. Characterization of P450 BM3 WT and Variants R255G and R255L in Respect to Hydroxylation of the Six Selected O-Heterocycles

The obtained P450 BM3 R255G (R255G) and P450 BM3 R255L (R255L) variants were expressed, purified, quantified as in section 3.7.4, 3.9.6, and 3.9.8 respectively (Figure 4.17), and characterized in detail by performing conversions of benzo-1,4-dioxane in 1 mL volume in

A B

Figure 4.17. A) Absorption spectra for the produced variants of P450 BM3 in their ferrous carbon monoxide complex. A typical absorption spectrum for the WT of P450 BM3 is shown in blue. EV—empty vector (negative control), WT—wild- type, GS2 (R255S/P329H/F331L), and GS3 (I122V/R255S/P329H/F331L). B) SDS-PAGE (10% Acrylamide) analysis of the purified P450 BM3 WT and variants R255G and R255L. The molecular weight of P450 BM3 WT and variant proteins are ca. 118 kDa. the presence of a cofactor regeneration system (GDH). Product formation was assessed with GC-FID. We observed solubility issues for benzo-1,4-dioxane as its limit was 1.2 mM when using ethanol as a co-solvent. After 1 h of conversion, under constant NADPH regeneration, R255G and R255L produced 0.80 ± 0.02 mM and 0.86 ± 0.02 mM of 2,3-dihydrobenzo-1,4- dioxin-5-ol, respectively, whereas P450 BM3 WT (WT) produced 0.040 ± 0.003 mM (Figure 4.18a). This is a ≈20- and ≈22-fold improvement over WT for R255G and R255L. The formation of the 2,3-dihydrobenzo-1,4-dioxin-6-ol was also improved by R255G and R255L (Figure 4.18b), and the ratio of their formation remained the same as for the

Figure 4.18. Product formation of different P450 BM3 variants measured with GC-FID. (a) 2,3-dihydrobenzo- 1,4-dioxin- 5-ol detected by GC-FID after 1 h of benzo-1,4-dioxane conversion. (b) 2,3-dihydrobenzo- 1,4-dioxin-6-ol detected by GC- FID after 1 h of benzo-1,4-dioxane conversion. Error bars represent one SD of the mean from three replicates.

55 Results and Discussion

WT (73 ± 1/27 ± 1). The coupling efficiencies of R225G and R255L were similar, 23.7 ± 0.5 % and 25.7 ± 1.0 % respectively (Table 4.2) and significantly improved when compared to the WT (8.8 ± 0.1%). Furthermore, the variants R255G and R255L reached a total product

Table 4.2. Catalytic performance of benzo-1,4-dioxane conversion with purified P450 BM3 WT and variants R255G and R255L. Variant NADPH oxidation rate [min-1] Coupling Efficiency [%] TTN WT 8.3 ± 1.3 8.8 ± 0.1 40 ± 3 R255G 1719 ± 231 23.7 ± 0.5 798 ± 24 R255L 1168 ± 163 25.7 ± 1.0 860 ± 15 NADPH oxidation rate (min-1) was determined spectrophotometrically at λ 340 nm; Coupling efficiency (%) = ratio between 2,3-dihydrobenzo-1,4-dioxin-5-ol formation [μM] and oxidized cofactor [μM]. NADPH oxidation rate and coupling efficiency were determined using purified P450 BM3. Reaction was supplemented with 1 mM NADPH and activity of P450 BM3 was measured as initial NADPH oxidation rates at λ 340 nm. The TTN was determined with cell free lysate and calculated based on 2,3-dihydrobenzo-1,4-dioxin-5-ol formation after 1 h. Products were quantified using GC-FID and commercial standards. All reactions were performed in triplicate. concentration of 121 mg/L and 131 mg/L, corresponding to a total turnover number (TTN) of 798 ± 24 and 860 ± 15, respectively. Indeed, GC-FID analysis revealed that R255L is able to convert over 95% of the loaded substrate (1.2 mM) in 1 h of reaction, whereas the WT converts < 7%, making R255L a better catalyst for benzo-1,4-dioxane hydroxylation, as shown in Figure 1.19. From the latter, we can also observe the detection of two additional products that are likely to be 1,4-benzodioxin, the dehydrated form of 2,3-dihydro-1,4-benzodioxin-2-ol (hydroxylated product at the dioxane ring) due to the heating program in the GC-FID. However, the amounts produced were not sufficient for identification using GC-MS (data not shown). The capability of P450 BM3 R255L/G to hydroxylate benzo-1,4-dioxane at the 5’ and 6’ carbon is of great advantage for chemists as it it performed without the need of rare catalysts, at room temperature and with high selectivity. Several synthetic chemical routes are published and/or patented for the production of 2,3-dihydrobenzo-1,4-dioxin-5-ol [155–158]

Figure 4.19. Product analysis using GC-FID after conversion of benzo-1,4-dioxane with lysate from EV (negative control), P450 BM3 WT, and from variants R255G and R255L. The data was obtained from 1 h conversion reactions employing glucose dehydrogenase (GDH) for efficient NADPH cofactor regeneration. a—benzo-1,4-dioxane, d—2,3-dihydrobenzo-1,4-dioxin- 5-ol, e—2,3-dihydrobenzo- 1,4-dioxin-6-ol, i.s.—cyclododecanol, b, c—unknown product. An improved 2,3-dihydrobenzo- 1,4- dioxin-5-ol formation is visible with R255G and R255L compared to WT.

56 Results and Discussion and 2,3-dihydrobenzo-1,4-dioxin-6-ol [159–161], however, in all routes at least one of the following is required: rare and expensive catalysts, heating, active cooling, and multi-step reactions. The most recent route and with highest yield of 3-dihydrobenzo-1,4-dioxin-5-ol, mixes 1,2-dibromomethane and benzene-1,2,3-triol in a heating oil bath to yield a reported 45%-56% of 3-dihydrobenzo-1,4-dioxin-5-ol and hydrogen bromide as by-product [158,165]. The most recent and with highest yield (94%-98%) chemical route for 3- dihydrobenzo-1,4-dioxin-6-ol was patented and uses 2,3-dihydro-1,4-benzodioxine-6- carbaldehyde as starting compound to react with mCPBA and potassium floride in dicloromethane overnight at room temperature[166]. These chemical route are costly and labourious.

The influence of residue R255 on the ability of P450 BM3 to convert other O-heterocycles was also investigated, namely phthalan, isochroman, benzofuran, 2,3-dihydrobenzofuran, and dibenzofuran. Indeed, during a 1 h reaction, the variants R255G and R255L achieved high conversion of phtalan (R255G: 82 ± 7%; R255L: 90 ± 1%) and dibenzofuran (R255G: 77 ± 0%; R255L: 85 ± 3%) and full conversion of isochroman (R255G and R255L: ≥ 99%), 2,3- dihydrobenzofuran (R255G and R255L: ≥ 99%), and benzofuran (R255G: 90 ± 2%; R255L: ≥ 99%) (Figure 4.20, Figure 4.21, Figure 4.22, Figure 4.23 and Figure 4.24). In contrast, P450 BM3 WT had low conversion numbers for phtalan (≤7%), isochroman (≤2%), 2,3- dihydrobenzofuran (≤1%), benzofuran (19 ± 6%), and dibenzofuran (16 ± 4%) as seen in Figure 4.25.

Figure 4.20. Product analysis via GC-FID after conversion of 2,3-dihydrobenzofuran with EV, P450 BM3 WT, and variants R255G and R255L. The data was obtained from 1 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. a—2,3-dihydrobenzofuran, i.s.—cyclododecanol, b, c, d, e, f, g—unknown product. A total 2,3- dihydrobenzofuran depletion is visible with R255G and R255L, whereas the WT does not convert the substrate.

57 Results and Discussion

Figure 4.21. Product analysis via GC-FID after conversion of isochroman with lysate from EV (negative control), P450 BM3 WT, and from variants R255G and R255L. The data was obtained from 1 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. a—isochroman, i.s.—cyclododecanol, b, c, d—unknown product. A complete isochroman depletion is visible for R255G and R255L, whereas WT is virtually unable to convert isochroman (≤1.5 ± 0.2% conversion).

Figure 4.22. Product analysis via GC-FID after conversion of phthalan with lysate from EV (negative control), P450 BM3 WT, and from variants R255G and R255L. The data was obtained from 1 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. a—phthalan, i.s.—cyclododecanol, b, c, d, e, f—unknown product. A nearly 90% phthalan depletion is visible for both R255G and R255L, whereas WT can only convert 6 ± 1%.

58 Results and Discussion

Figure 4.23. Product analysis via GC-FID after conversion of benzofuran with lysate from EV (negative control), P450 BM3 WT, and from variants R255G and R255L. The data was obtained from 1 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. a—benzofuran, i.s.—cyclododecanol. A nearly 90 ± 2% benzofuran depletion is visible with R255G and complete depletion with R255L, whereas WT is only able to convert 19 ± 6%.

Figure 4.24. Product analysis via GC-FID after conversion of dibenzofuran with lysate from EV (negative control), P450 BM3 WT, and from variants R255G and R255L. The data was obtained from 1 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. a—dibenzofuran, b—unknown product, i.s.—cyclododecanol. A conversion of 77 ± 0% and 85 ± 3% of dibenzofuran is visible for R255G and R255L, respectively, whereas WT is only able to convert 16 ± 4%.

59 Results and Discussion

Figure 4.25. Comparative conversions of 1 h reaction with the different substrates benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran (concentration 1.2 mM) by P450 BM3 WT and variants R255G and R255L. Numbers are presented in percentages. Error bars represent one SD of the mean from three replicates. Values are taken from GC-FID analysis.

60 Results and Discussion

4.1.7. Rationale behind the Activity Improvement of R255G and R255L Variants over the WT

Computational analysis was carried out using benzo-1,4-dioxane as a substrate. Molecular docking studies were performed to investigate the interactions of benzo-1,4-dioxane with P450 BM3’s active site. Figure 4.26 shows the most probable binding orientation of benzo-1,4-dioxane in the binding pocket of WT, R255G, and R255L. The docking simulations revealed that in all three cases, the substrate binds in a similar manner (close to the water molecule covalently bound to the central iron atom). Furthermore, there were no significant differences in the binding energies, although a slight change in the distance between the substrate-closest C atom (C5) and the iron-bound water molecule was observed in both R255G and R255L (≈4.90 Å) vs. WT (≈5.14 Å). This observation suggests a slow activation of the substrate by WT despite the binding of benzo-1,4-dioxane; however, the influence of the

A B C

L181 L181 L181

L437 L437 L437

F87 F87 F87 T438 R255 T438 T438 I263 E267 I263 G255 L255 E267 E267 I263

Heme Heme Heme

A B C

L181 L181 L181

L437 L437 L437

F87 F87 F87 T438 R255 T438 T438 I263 E267 I263 G255 L255 E267 E267 I263

Heme Heme Heme

Figure 4.26. Molecular docking pose of benzo-1,4-dioxane in the active site of WT, R255G, and R255L (A) WT (binding energy: −5.63 kcal/mol), (B) R255G variant (binding energy: −5.66 kcal/mol), and (C) R255L variant (binding energy: −5.66 kcal/mol). Reciprocal arrows indicate the closest distance between the iron-bound water ligand and C5 atom of benzo-1,4-dioxane (WT: 5.14 Å, R255G and R255L: 4.90 Å). Substrate benzo-1,4-dioxane is depicted as a ball and stick, whereas all the active site residues including heme are depicted as sticks. Hydrogen bond between substrate and T438 is represented as a black dotted line.

61 Results and Discussion residue R255 on the activity remained unresolved. This residue is located on the distal side of the I-helix and far away from the substrate-binding pocket. It is known that the I-helix is the most prominent structural component in P450, providing a backbone for heme arrangement and the remaining chain [55]. Therefore, to get a deeper molecular understanding of the influence of R255 residue on P450 BM3 activity, molecular dynamics (MD) simulations were carried out. The enzyme-substrate complex of benzo-1,4-dioxane for WT and variants were further subjected to MD simulations to analyze the stability and orientation as well as the nature and energetics of substrate binding. Root mean square deviation (RMSD) (Figure 4.27) analysis shows the stability of the substrate-enzyme complex throughout the MD simulations. Substitution of R255 by either glycine or leucine indeed introduced flexibility in the I-helix, as

A B

Figure 4.27. Calculated (A) root mean square deviation (RMSD) and (B) root mean square fluctuation (RMSF) per residue of P450 BM3 WT and P450 BM3 variants (R225L and R255L) throughout three independent MD simulation runs (I-helix: residues 245 to 283). evidenced by the root mean square fluctuation (RMSF) per residue analysis. From MD simulations, it was observed that in WT, benzo-1,4-dioxane initially stays in close contact with the heme but moves away afterward (Figure 4.28A). By contrast, in R255G and R255L variants, the C5-atom on which the actual hydroxylation takes place remains in close contact with the iron-bound water molecule, allowing for hydroxylation to occur. Indeed, a recent study on isophorone hydroxylation using P450-WAL [56] showed that the ideal distance and angle between isophorone and heme for catalytically competent hydroxylation should be approximately 3 Å and 109–149 degrees, respectively, which is well supported by our R255G and R255L variant simulations (Figure 4.28). Additionally, R255 is important for the structural rigidity of the I-helix due to the formation of a salt bridge with D217 in WT. Hence, substituting R255 with either G or L, this salt-bridge will not be formed, leading to increased flexibility in

62 Results and Discussion

A B

Figure 4.28. (A) Calculated distance between C5-atom of substrate and iron-bound oxygen species. (B) Calculated angle between benzo-1,4-dioxane substrate and heme required for the hydroxylation of benzo-1,4-dioxane in WT and variants R255G and R255L along three independent 50 ns MD simulation trajectories. the I-helix of R255G and R255L variants (Figure 4.27B). A structural rearrangement in the heme-binding domain was also observed, especially in residue F87 (Figure 4.29), which causes the substrate to adapt and maintain the catalytically competent orientation. Indeed, throughout 50 ns of MD simulations, benzo-1,4-dioxane persistently kept a distance ≈ 3 Å and an angle of 109–149 degrees required for hydroxylation, as shown in Figure 4.28. This rearrangement could thus lead to the improved performance of P450 BM3 R255G and R255L towards benzo-1,4- dioxane.

H-helix G-helix

I-helix

Figure 4.29. Cartoon representation of the structural alignment of P450 BM3 WT (grey) and P450 BM3 variants (R255G in green and R255L in magenta). Heme is depicted in red lines and residue F87 in sticks. A substantial rearrangement of the G-, H-, and I-helixes is observed. The models of the P450 BM3 variants R255G and R255L were constructed using the swap function in YASARA Structure Version 17.4.17 and optimized using the SCWRL rotamer library search for the designated substitutions. 63 Results and Discussion

4.1.8. Conclusion

In conclusion, protein engineering by directed evolution and site saturation mutagenesis of identified positions revealed the important role of position R255 in boosting the catalytic performance of P450 BM3 towards aromatic O-heterocyclic compounds. The increased performance was not limited to the evolution substrate (i.e., benzo-1,4-dioxane; R255G: ≈ 90%; R255L: ≈ 95%; WT: ≤ 7%), and indeed, similar improvements in conversions were achieved for phthalan (R255L and R255G: ≈ 90%; WT: ≤ 2%), isochroman (R255L and R255G: ≥ 99%; WT: ≤ 2%), 2,3-dihydrobenzofuran (R255L and R255G: ≥ 99%; WT: ≤ 2%), benzofuran (R255G: 90 ± 2%; R255L: ≥ 99%; WT: 19 ± 6%), and dibenzofuran (R255G: 77 ± 0%; R255L: 85 ± 3%; WT: 16 ± 4%). The P450 BM3 variant R255L is ca. 22 times more active than the WT in hydroxylating benzo-1,4-dioxane. This substitution has a drastic influence on the catalytic activity of P450 BM3 as compared to the WT, increasing the coupling efficiency (25.7 ± 1.0 % vs. 8.8 ± 0.1 %), NADPH oxidation rate (1168 ± 163 min−1 vs. 8.3 ± 1.3 min−1), and TTN (860 ± 15 vs. 40 ± 3). Computational analysis reveals that breaking a salt bridge (formed between R255 and D217 in WT) introduces flexibility in the I-helix and leads to a productive heme rearrangement, thus improving benzo-1,4-dioxane hydroxylation. The improvement observed in P450 BM3 R255G and R255L towards benzo-1,4-dioxane provides for the first time an enzymatic routes to produce pharmaceutical precursors in a selective and environmentally friendly way via late-stage hydroxylation. This improvement was not limited to benyo-1,4-dioxane but also to phtalan, isochroman, benzofuran, 2,3-dihydrobenzofuran, and dibenzofuran known for being present in macrocyclic drugs.

64 Results and Discussion

4.2. P450 Cand_1

4.2.1. Development of a Screening Systems for Product-Based Detection of Indigo and Indirubin

The hydroxylation of indole (10) in Figure 4.30 using P450 Cand_1 leads to a mixture of indoxyl keto (18) and enol (7) tautomers. Indigo is formed when two indoxyl groups combine spontaneously in the presence of oxygen to form, in a stage-wise manner first acid leucoindigo and then indigotin (8). During hydroxylation by P450, a side reaction can occur where indoxyl

Figure 4.30. Products of enzyme-catalyzed oxidation of indole. Based on [123,201,216]. is over-oxidised to isatin (19), condensing then with more indoxyl to produce the red pigment indirubin (20). Additional products may be produced from the hydroxylation of indole such as 1H-indol-6-ol and 1H-indol-7-ol that in the presence of oxygen react to form a hydrophilic red/brown pigment depending on oxygen concentrations and substrate orientation in the active site [201]. Indigo (λmax = 620 nm) and Indirubin (λmax = 540-560 nm) have distinctive absorption spectra with a slight overlapping between λ 550 nm and λ 600 nm (Figure 4.31) and different color formation which offers itself as a simple mean for high-throughput screening. We applied the standard nicotinamide adenine dinucleotide phosphate (NADPH) depletion assay, to assess NADPH depletion rates and absorption at λ 620 nm for total indigo formation, however, the rate of depletion was far too low

Figure 4.31. Absorbance spectra of indigo and indirubin from 400 to 700 nm. 65 Results and Discussion to measure over a short period of time which also led to ineffective product formation rates. To overcome this, we measured product formation after 16 h reaction (in the dark to avoid degradation of indole by light) in the presence of a NADPH regeneration solution containing glucose dehydrogenase (GDH) (3 U/mL), glucose (60 mM) and catalase (1200 U/mL). Under these new conditions, indigo could be detected but not indirubin (likely due to low amounts). A standard deviation of 23.9 % (true standard deviation of 33.2 %) was observed after 16 h of reaction using the P450 Cand_1 WT (Figure 4.32) making it a less reliable screening but still usable system for finding improved variants. The observed high standard variation is often

Figure 4.32. Standard deviation of the indigo MTP screening assay with P450 Cand_1. Detected absorbance at λ 610 nm in descending order of P450 Cand_1 clone catalyzed conversion of indole to indigo in a 96-well plate. The standard deviation of 23.9 % is represented in black rectangles. The true standard deviation (33.2 %) is represented with white triangles. linked to evaporation rates, usually higher in the edge and corner wells [202,203], but also to expression fluctuations when using the same plasmid co-expression systems under two strong promoters (Ptac and Ptrp in pBIDI231a) [204] as well as plasmid copy number [205,206]. However, when using clarified cell lysates from flask expression the formation of indigo was detectable at λ 620 nm just after 5 minutes of reaction in the presence of NADPH and indole (Figure 4.33). Furthermore, the absorbance steadily increased in a 30 min reaction.

Figure 4.33. A) Absorbance at λ 620 nm over time in min from conversion of indole cell lysates of P450 Cand_1 and Empty Vector in Terrific Broth.

66 Results and Discussion

4.2.2. Quantification of Indigo via High-Performance Liquid Chromatography

The final two major products of the biotransformation of indole with P450_Cand1 wild type (WT) are identified to be indirubin and indigo. Indigo (λmax = 620 nm) and

Indirubin (λmax = 540-560 nm) have distinctive absorption spectra (see Figure 4.31) with different color formation as well as structures. This feature allows for their separation using HPLC [128,181,207]. A high-performance liquid chromatographic separation and quantitative method was developed (Figure 4.34) using a C18 column (Nucleosil 100-5 C18, 250× 4.6 mm, CS Chromatographie, Germany) eluted isocratically with methanol and water (70:30 (v/v)) to analyze the isomeric active compounds indigo and indirubin. Using this method the indigoids present in the DMSO extract of centrifuged E. coli cultures can be analyzed and quantified. Moreover, under these conditions indigo quantification using the HPLC showed a linear response from 2 to 100 mg/mL at λ 620 nm (Figure 4.35).

Figure 4.34. Product separation via HPLC. i.p.—injection peak; a—indigo; b—indirubin; DMSO- Dimethyl sulfoxide. Indigo monitored at λ 620 nm, indirubin monitored at λ 540 nm.

Figure 4.35. The HPLC linear detection range of indigo measured at λ 620 nm. Samples were prepared in DMSO with concentrations ranging from 2 mg/L to 100 mg/L. 67 Results and Discussion

4.2.3. Analysis of P450 Cand_1 tunnels

The role of alternative network channels exiting at the distal surface of the protein have been documented and compiled in two recent reviews [208–210]. The main findind is that these network channels play a critical role in modulating P450 functions, where amino acid residues at the entrance of the channels influence substrate specificity (ligand recognition) and catalytic mechanism [208,209]. Computational analysis on P450 Cand_1 tunnels for improved activity on indole was done using the enzyme-substrate complex obtained from the crystal structure of cytochrome P450 Cand_1 heme domain (PDB ID: 6HQD) [78]. The structure was loaded into YASARA Structure Version 17.4.17 and then minimized using a water box with the steepest descent (timestep of 2 fs, atom velocities scaled down by 0.9 every 10 step) starting from 98K, 198K and 298K with a time-averaged Berendsen thermostat until convergence was reached. Afterward, the minimized structure was handled by Hollow script [211] to investigate the tunnels leading to the active site of P450 Cand_1 and the residues surrounding the tunnel (Figure 4.36). The Hollow script is a volume filling script for protein structures that probes the cavities/tunnels of a given protein for probable positions of water molecules. This means a colored surface can be created out of the combined and summed water positions as seen in Figure 4.36 in red. By analyzing the outcome of P450 Cand_1, the residues surrounding the active site tunnel exit appear to be constricting it. This is likely the cause for the observed limitation on the indole conversion [78] and also for the observed low speed of conversion. The residues were identified as being D89, R192, D262, L407, and V407 and were selected for site- saturation-mutagenesis to better understand their influence on indole hydroxylation. Additionally, a SeSaM library of the P450 Cand_1 gene was generated to investigate other key positions.

A B

Figure 4.36. The interior volume of P450 Cand_1. A) The volume-filling spheres surface (red) identified in P450 Cand_1 using a grid-spacing of 0.2 Å and the residues (green stick display) that define the interior volume by proximity to the volume- filling spheres and to the heme (orange stick display). B) The residues selected for site-saturation-mutagenesis. Images were prepared using Pymol. 68 Results and Discussion

4.2.4. P450 Cand_1 Library Generation and Screening

The P450 Cand_1 engineering can be briefly described as the SeSaM method [173] was performed on P450 Cand_1 and each step was confirmed via agarose gel electrophoresis whenever possible (Figure 4.37). The SeSaM method started by introducing, the “SeSaM”- sequences by PCR with gene-specific primers binding in front of and behind the P450 Cand_1 gene. The PCR was carried out using a pre-defined mixture of phosphorothioate and standard nucleotides to ensure an even distribution of inserted mutations over the full length of P450 Cand_1 gene (Figure 4.37A). The products of Step 1 were cleaved specifically at the phosphorothioate bonds using an Iodine solution, which generated a pool of single-stranded DNA fragments of different lengths (Figure 4.37B). Afterward, the DNA single strands were elongated by one universal base, catalyzed by terminal deoxynucleotidyl transferase (TdT). A PCR to recombine the single-stranded DNA fragments with the corresponding reverse template of P450 Cand_1, generating the full-length double-stranded gene including the universal base in its sequence. The replacement of the universal bases in the gene sequence by random standard nucleotides in the last step generated a diverse array of full-length gene sequences with substitution mutations (Figure 4.37C).

A

B C

Figure 4.37. GelDoc XR+ Automatic Band and Lane detection from a 1 % (w/v) agarose gel confirmation of SeSaM library products. A) Amplification with biotinilated primer confirmation. 1 & 12 – MWM 1 kb GeneRuler, 2 to 11 – amplification of P450 Cand_1 heme domain with biotinylated primers (1418 bp). B) Cleavage confirmation. 1 & 7 – MWM 1 kb GeneRuler 2 to 6 – cleavage of products from A. C) Final amplification 1 & 6 – MWM 1 kb GeneRuler 2 to 5 – final amplified products.

69 Results and Discussion

The P450 Cand_1 gene libraries were cloned into the vector backbone (pBIDI231a) via homology recombination [212] and subsequently transformed into chemically competent E. Q1 coli BL21-Gold (DE3) lacI cells (section 3.7.2). Afterward, 1000 clones were picked to be screened. Simultaneously, SSM’s were performed on five different positions (D89, R192, D262, L407, and V408) on the substrate access tunnel to evaluate the relevance of these residues over indole hydroxylation as mentioned in section 4.2.3. The clones picked from the SeSaM library and SSM library were prepared and expressed as described in section 3.7.7 and section 3.7.8 and screened as in section 3.9.3. When using clarified cell lysates of P450 Cand_1 WT from flask expression, hydroxylation of indole was observed due to indigo formation as seen in Figure 4.38. However, the expression of P450 Cand_1 WT in 2 mL 96 deep-well plates is poor and inconsistent (Figure 4.39) which turned inviable finding an improved variant in both

A B

EV

P450 Cand_1 (1/10)

P450 Cand_1

Figure 4.38. A) Absorbance at λ 620 nm over time in min from conversion of indole cell lysates of P450 Cand_1 and Empty Vector in Terrific Broth. B) MTP Picture taken at the end of the indole conversion in A) using cell lysates from P450 Cand_1 and Empty Vector. Blue colour indicates indigo formation.

A B

Figure 4.39. Comparison of flask and deep-well plate expression of P450 Cand_1. A) Pelleted cells after expression of P450 Cand_1 and negative control (empty vector). B) Pelleted cells after P450 Cand_1 expression in 2.2 mL deep-well plates. generated libraries (SeSaM and SSM). The observed inconsistent expression causes a high standard variation and is often related to sub-optimal growth conditions such as evaporation rates, usually higher in the edge and corner wells [202,203], but also influenced by the expression system in this case, a single plasmid co-expression under two strong promoters (Ptac and Ptrp) [204] as well as high plasmid copy number [205,206]. However, since the later conditions are also true for flask expression, the exact cause for not having consistent expression in deep-well plates is still not known.

70 Results and Discussion

4.2.5. Conclusion

The search for an improved variant of P450 Cand_1 for bioconversion of indole to indigo was performed in this thesis. Previously, a limitation was observed in P450 Cand_1, it could only convert indole to only about 50 % of the loaded substrate (reported by Fiorentini et al. 2018) [78]. Investigation of this observation followed. A product based screening system is always preferred in order to avoid false positives when compared to the standard NADPH depletion assay. Indigo (λmax = 620 nm) has a distinctive absorption spectrum that can be used for quantification using absorbance methodology. We used HPLC to quantify and separate indigoids using isocratic elution with methanol and water (70:30 (v/v)). This method was established an exhibited linear detection between 2 mg/L and 100 mg/L for indigo. Furthermore, to screen with a high-throughput the generated libraries, a screening method was developed in a 96-well MTP format for indigo detection. This method exhibited a standard deviation of 23.9 % (true standard deviation of 32.1 %) when using P450 Cand_1 WT. Afterward, two engineering strategies were followed, a random approach by sequence saturation method (SeSaM) and a semi-rational SSM at 5 non-conserved active site amino acids. Both libraries were generated and screened using the established method, however, a significantly improved variant was not found. P450 Cand_1 WT and exhibited inconsistent and poor expression in 2.2 mL deep-well plates, which caused the previously observed high standard deviation. However, the cause for the inconsistent expression in deep-well plates was not possible to unveil. An enzymatic or whole cell route to produce indigo has, as described in section 1.7, enormous advantages over the traditional chemical route by avoiding the usage of aniline, formaldehyde, hydrogen cyanide, sodium amide and strong bases to name a few. By contrast, enzymatic and whole cell approaches can start from the inexpensive and inert tryptophan that can be cleaved by tryptophanase, leaving the indole exposed for hydroxylation by P450s as shown in Figure 4.30, and thus establishing an inexpensive enzymatic route for a high demand compound.

71 Results and Discussion

4.3. P450 Cand_10

4.3.1. Analysis of P450 Cand_10 tunnels

Computational analysis on P450 Cand_10 tunnels for improved activity on the heterocycles benzo-1,4-dioxane and indole was done using the enzyme-substrate complex obtained from the crystal structure of cytochrome P450 Cand_10 heme domain (PDB ID: 6HQG) [76]. The structure was loaded into YASARA Structure Version 17.4.17 and then minimized using a water box with the steepest descent (timestep of 2 fs, atom velocities scaled down by 0.9 every 10 steps) starting from 98K, 198K and 298K with a time-averaged Berendsen thermostat until convergence was reached. Afterward the minimized structure was handled by Hollow script [186] to investigate the tunnels leading to the active site of P450 Cand_10 and the residues surrounding the tunnel (Figure 4.40). The Hollow script is a volume filling script for protein structures that probes the cavities/tunnels of a given protein for probable positions of water molecules. This means a colored surface can be created out of the combined and summed water positions as seen in Figure 4.40 in red. Afterward, an alignment using ClustalW was performed with the 2473 reviewed P450 structures in UniProt followed by an amino-acid variability analysis at the previously identified positions. Using the latter strategy, from the 12 amino acids responsible for the active site cavity (I88, V89, M101, I103, L258, V261, D265, T266, L309, M312, F410, and V411), only 6 (V89, I013, L258, V261, L309, M312) were not strictly conserved (Figure 4.41) and therefore, were selected for simultaneous site-saturation-mutagenesis and screened for improved activity towards benzo-1,4-dioxane and indole. A B

Figure 4.40. A) The crystal structure of P450 Cand_10 illustrating the residues lining the tunnel (red surface) and B) the identified positions V89, I013, L258, V261, L309, M312. The positions selected are represented in B as sticks. The heme cofactor is depicted in orange sticks

72 Results and Discussion

Figure 4.41. The occurrence variability analysis of the alignment using ClustalW performed with 2473 reviewed P450 structures in UniProt at the positions V89, I013, L258, V261, L309, M312 4.3.2. P450 Cand_10 Library Generation and Screening

The previously identified positions V89, I013, L258, V261, L309, M312 in 4.3.1 above, were subjected to site-saturation-mutagenesis using NNK degenerate primers to evaluate the relevance of these residues over heterocycle hydroxylation. The PCR was performed as mentioned in 3.8.2, confirmed via agarose gel electrophoresis (Figure 4.42) and the generated library was transformed as in 3.7.2. The generated clones (132 clones per position) were picked from the SSM library and prepared, expressed as described in section 3.7.7 and 3.7.8, and screened as in 3.9.2 and 3.9.3. P450 Cand_10 is easily expressed in both flask and 96-well plates (Figure 4.43), exhibits activity towards n-octane as Fiorentini et al described and displays

Figure 4.42. GelDoc XR+ Automatic Band and Lane detection from an 1% (w/v) agarose gel confirmation of SSM library PCR products. 1 & 8 – MWM 1 kb GeneRuler, 2 – SSM89, 3 – SSM103, 4 – SSM258, 5 – SSM261, 6 – SSM 309, 7 – SSM312. A B

Figure 4.43. Comparison of flask and deep-well plate expression of P450 Cand_10. A) Pelleted cells after expression of P450 Cand_10 and negative control (empty vector). B) Pelleted cells after P450 Cand_10 SSM258 expression in 2.2 mL deep-well plates. 73 Results and Discussion a typical CO absorbance spectrum as shown in Figure 4.44. The WT of P450 Cand_10 displays no activity over benzo-1,4-dioxane or and indole. Despite screened 132 clones per SSM a variant significantly improved was not found for any of the positions saturated (Figure 4.45).

B A

Figure 4.44. A) P450 Cand_10 NADPH depletion in the presence and absence of 1.2 mM of n-octane. 50 µL clarified cell lysate, 150 µL KPi 50 mM pH 7.5/1.2 mM n-octane, DMSO as co.solvent, 50 µL NADPH 1 mM. B) Absorption spectra for the expressed P450 Cand_10 in its ferrous carbon monoxide complex.

Figure 4.45. The relative NADPH consumption of the clones present in plate from P450 Cand_10 SSM312. Green bar represents P450 Cand_10 (positive control), EV – Empty Vector (negative control), green line represents the threshold for classifying the clones improved. The observed consumption rates of NADPH were far too small to consider any activity over benzo-1,4-dioxane and indole (<0.0002 min-1). The fact that no single variant displayed consumption rates significantly exceeding the <0.0002 min-1 rate suggests that the determinants for P450 Cand_10 activity over the tested substrates are likely to be present in the active site tunnel access or protein shell. Indeed, studies on the role of alternative network channels exiting at the distal surface of the protein have been recently compiled in two reviews [208–210]. The main findind is that these network channels play a critical role in modulating P450 functions, where amino acid residues at the entrance of the channels influence substrate specificity (ligand recognition) and catalytic mechanism [208–210]. Furthermore, the plasticity of P450 structures is also relevant when looking at how channels might play their role because, the exclusive function of the protein shell in promiscuous enzymes might be the stabilization and accessibility of their very reactive catalytic intermediates [78,208,209,213]. 74 Final Summary

4.3.3. Conclusion

The search for an improved variant of P450 Cand_10 for bioconversion of benzo-1,4- dioxane and indole to indigo was performed in this thesis. P450 Cand_10 displays no activity towards benzo-1,4-dioxane or indole. We investigated non-conserved amino acids within the active site of P450 Cand_10 and saturated them in search of the determinants that could lead to activity. Product-based screening systems had been already successfully developed for both compounds. Using the Hollow script (section 4.3.1) 12 amino acids were identified as responsible for the active site cavity (I88, V89, M101, I103, L258, V261, D265, T266, L309, M312, F410, and V411) and sequence alignment studies revealed that only 6 (V89, I103, L258, V261, L309, M312) were not strictly conserved. A semi-rational engineering approach followed by performing SSM at those 6 positions using NNK degenerate primers (section 4.3.2) and screened using the previously developed methods. Despite the successful generation of SSM libraries, no active variant on benzo-1,4-dioxane or indole was found. Recent reviews compiled research on P450 tunnel engineering and found that other structural determinants such as alternative network channels that lead to the active site and/or protein shell play a role in the accessibility and recognition of ligands as well as reactive catalytic intermediates. This is likely to be the case for P450 Cand_10 as well.

5. Final Summary

Cytochrome P450 monooxygenases are recognized as essential bio-bricks in synthetic biology approaches to enable the production of high-value complex molecules. Using P450 for enzymatic oxidations will significantly extend the synthetic toolbox, offering chemists an attractive alternative to conventional chemical strategies. P450 BM3 has been the gold standard for many years regarding oxidative biocatalysis for high-value compounds however, some undesirable features limit its productivity and prevent its broad industrial application to relevant substrates. Within the EU’s Horizon 2020 Programme Research and Innovation actions H2020- EU.1.3.1 project OXYtrain (grant agreement no. 722390) aimed to “Harnessing the power of enzymatic oxygen activation” through the study of the mechanism, engineering, and application of monooxygenases. Within the frame of this training network, the objective of my thesis at RWTH Aachen – Lehrstühl fur Biotechnology was the evolution of heme-dependent monooxygenases towards the conversion of heterocyclic compounds and its molecular understanding. In this thesis three heme-dependent monooxygenases were engineered, Cytochrome P450 BM3 monooxygenase from Bacillus megaterium, P450 Cand_1 from Pseudomonas sp. 19-rlim and P450 Cand_10 from Phenylobacterium zucineum. The

75 Final Summary determinants that enabled hydroxylation of benzo-1,4-dioxane and indole, O- and N- heterocycle respectively were investigated by subjecting all three monooxygenases to engineering campaigns. Although the NADPH cofactor depletion assay has been reported as a general screening method for engineering campaigns, a product based screening system is always preferred in order to efficiently screen the library and avoid false positives due to uncoupling reactions. Hence, we developed an indigo screening method adapted to 96-well MTP format and a quantification method via HPLC (section 4.2.1 and section 4.2.2). Furthermore, we used the 4-AAP method (section 4.1.2) as a screening system for product- based quantification of 2,3-dihydro-1,4-benzodioxin-5-ol and 2,3-dihydro-1,4-benzodioxin-6- ol (benzo-1,4-dioxane bioconverted products). A 96-multiplexed capillary electrophoresis screening system was also developed to separate and detect benzo-1,4-dioxane products hydroxylated by P450s at the benzene ring (section 4.1.3). Both screening systems had low standard deviation (9.6 % for the 4-AAP and 15.6 %) for the MP-CE. Furthermore, a working linear response was observed for the 4-AAP (16 µM to 500 µM). The MP-CE required a GDH/glucose based NADPH regeneration system to increase product formation, require less cofactor and boost linear response range (50 µM mM to 2 mM). Each P450 was subjected to different engineering strategies, P450 BM3 to random approach by 2 rounds epPCR followed by site-saturation-mutagenesis (SSM) at beneficial positions found (section 4.1.5), P450 Cand_1 was subjected to two different engineering strategies, a random approach by sequence saturation method (SeSaM) and a semi-rational SSM at 5 amino acids in the active site (section 4.2.4) and P450 Cand_10 was subjected to a semi-rational SSM at 6 non-conserved active site amino acids (section 4.3.2). The evolution of P450 Cand_1 and Cand_10 did not yield any significantly improved variant however, the evolution of P450 BM3 led to the identification of beneficial substitution R255L and R255G. These substitutions greatly increased bioconversion of benzo-1,4-dioxane with improved NADPH oxidation by approx. 140-fold (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), total turnover number (TTN) by approx. 21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency by approx. ≈ 2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%) (section 4.1.6). The substitution of R255 by either glycine or leucine increases flexibility at the I-helix, (a salt-bridge formed between R255 and D217 in WT is no longer present) and leads to a productive heme rearrangement as well of residue F87, thus improving not only benzo-1,4-dioxane hydroxylation (section 4.1.7) but also other O-heterocycles such as phtalan, isochroman, benzofuran, 2,3-dihydrobenzofuran, and dibenzofuran (4.1.6). To conclude, in this thesis, the evolution of three heme-dependent monooxygenases towards the conversion of benzo-1,4-dioxane and indole was reported, different screening and

76 References quantification methods were successfully established and applied using different platforms, 96- MP CE, MTP based screening systems for product-based quantification, HPLC and GC. Three protein engineering approaches were successfully applied on three different P450s, SeSaM method, SSM and epPCR., however only the campaign on P450 BM3 yielded variants significantly improved with increased coupling efficiency, NADPH oxidation rate and TTN’s.

The improvement observed in P450 BM3 R255G and R255L towards benzo-1,4-dioxane, phtalan, isochroman, benzofuran, 2,3-dihydrobenzofuran, and dibenzofuran provides new biosynthtetic routes that lead to advancements in biocatalysis by generating drug precursors of great pharmaceutical importance such as antimicrobial, antigrastic, spasmolytic, antipsychotic, anxiolytic, hepatoprotective or α-adreno blocking, to name a few. These P450 engineered variants, can be used to hydroxylate O- and N-heterocycles in a selective and environmentally friendly way via late-stage hydroxylation

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Residue size at position 87 of cytochrome P450 BM-3 determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation. FEBS Lett. 2001, 508, 249–52. 198. Muller, C.A.; Weingartner, A.M.; Dennig, A.; Ruff, A.J.; Groger, H.; Schwaneberg, U. A whole cell biocatalyst for double oxidation of cyclooctane. J Ind Microbiol Biotechnol 2016, 43, 1641–1646. 199. Müller, C.A.; Akkapurathu, B.; Winkler, T.; Staudt, S.; Hummel, W.; Gröger, H.; Schwaneberg, U. In Vitro Double Oxidation of n-Heptane with Direct Cofactor Regeneration. Adv. Synth. Catal. 2013, 355, 1787–1798. 200. Dennig, A.; Marienhagen, J.; Ruff, A.J.; Guddat, L.; Schwaneberg, U. Directed Evolution of P450 BM3 into a p-Xylene Hydroxylase. ChemCatChem 2012, 4, 771–773. 201. McClay, K.; Boss, C.; Keresztes, I.; Steffan, R.J. Mutations of toluene-4-monooxygenase that alter regiospecificity of indole oxidation and lead to production of novel indigoid pigments. Appl. Environ. Microbiol. 2005, 71, 5476–5483. 202. Martuza, R.L.; Proffitt, M.R.; Moore, M.B.; Dohan, C.F. Evaporation as a cause of positional differences in cell plating and growth in microtiter plates. Transplantation 1976, 21, 271–273. 84 Appendix

203. Deshpande, R.R.; Wittmann, C.; Heinzle, E. Microplates with integrated oxygen sensing for medium optimization in animal cell culture. Cytotechnology 2004, 46, 1–8. 204. Protein Expression - E.coli - Co-Expression - EMBL Available online: https://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/co-expression/ (accessed on Oct 30, 2019). 205. Bentley, W.E.; Mirjalili, N.; Andersen, D.C.; Davis, R.H.; Kompala, D.S. Plasmid-encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria. Biotechnol. Bioeng. 1990, 35, 668–681. 206. Birnbaum, S.; Bailey, J.E. Plasmid presence changes the relative levels of many host cell proteins and ribosome components in recombinantEscherichia coli. Biotechnol. Bioeng. 1991, 37, 736–745. 207. Berry, A.; Dodge, T.; Pepsin, M.; Weyler, W. Application of metabolic engineering to improve both the production and use of biotech indigo. J. Ind. Microbiol. Biotechnol. 2002, 28, 127–133. 208. Urban, P.; Lautier, T.; Pompon, D.; Truan, G. Ligand access channels in cytochrome P450 enzymes: A review. Int. J. Mol. Sci. 2018, 19. 209. Kingsley, L.J.; Lill, M.A. Substrate tunnels in enzymes: Structure-function relationships and computational methodology. Proteins Struct. Funct. Bioinforma. 2015, 83, 599–611. 210. Kokkonen, P.; Bednar, D.; Pinto, G.; Prokop, Z.; Damborsky, J. Engineering enzyme access tunnels. Biotechnol. Adv. 2019, 37, 107386. 211. Ho, B.K.; Gruswitz, F. HOLLOW: Generating Accurate Representations of Channel and Interior Surfaces in Molecular Structures. BMC Struct. Biol. 2008, 8, 49. 212. Jacobus, A.P.; Gross, J. Optimal cloning of PCR fragments by homologous recombination in Escherichia coli. PLoS One 2015, 10, 1–17. 213. Fürst, M.J.L.J.; Romero, E.; Gómez Castellanos, J.R.; Fraaije, M.W.; Mattevi, A. Side-Chain Pruning Has Limited Impact on Substrate Preference in a Promiscuous Enzyme. ACS Catal. 2018, 8, 11648–11656. 214. Buchan, D.W.A.; Jones, D.T. The PSIPRED Protein Analysis Workbench: 20 years on. Nucleic Acids Res. 2019, 47, W402–W407. 215. Bloom, J.D.; Arnold, F.H. In the light of directed evolution: pathways of adaptive protein evolution. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 Suppl, 9995–10000. 216. Rui, L.; Reardon, K.F.; Wood, T.K. Protein engineering of toluene ortho-monooxygenase of Burkholderia cepacia G4 for regiospecific hydroxylation of indole to form various indigoid compounds. Appl. Microbiol. Biotechnol. 2005, 66, 422–429.

7. Appendix

7.1. List of Tables

Table 1.1. Classification and application areas of enzymes. Based on Averill et al [12] ...... 3 Table 1.2. Examples of biocatalysis use in commercial processes for the production of fine chemicals...... 3 Table 1.3. Organization of different cytochrome P450 systems. Based on Hannemann et al. and Cook et al. [25,26] ...... 5 Table 1.4. A glimpse into P450 BM3 selectivity-directing and activity-enhancing mutations compiled. A table of positions substituted, improvements and references A more comprehensive overview of P450 BM3 substitutions is described by Whitehouse et al [70] and is also available online in the muteinDB [www.MuteinDB.org] [71]...... 9 Table 1.5. Best bacterial strains and best enzyme resources with the ability of producing indigo from indole...... 16 Table 3.1. List of substrates used in this study and their respective supplier...... 20 Table 3.2. Machines and equipment used during this work...... 21 Table 3.3. Strains employed in this study and their genotypes...... 23 Table 3.4. Vectors employed in this study for genetic manipulation and recombinant protein expression...... 23 Table 3.5. Genes employed in this study and their origins. Gene and protein sequences are shown in appendix...... 23 Table 3.6. PCR master mix composition...... 29 Table 3.7. PCR cycling condition...... 29 Table 3.8. Composition of the gels for the SDS-PAGE...... 37

85 Appendix

Table 3.9. Preparation of monooxygenases long-term substrate conversion...... 40 Table 4.1. The P450 BM3 variants used for the selection of the starting variant...... 44 Table 4.2. Catalytic performance of benzo-1,4-dioxane conversion with purified P450 BM3 WT and variants R255G and R255L...... 56 Table 7.1. List of oligonucleotides used for cloning, sequencing and mutagenesis during this work ...... 94 Table 7.2. Gas-chromatography programs and compound retention time ...... 96

7.2. List of Figures

Figure 1.1. Proposed catalytic cycle of P450 monooxygenases Based on Cook et al. 2016 [26]...... 6 Figure 1.2. Schematic of putative electron transfer pathways in flavocytochrome P450 BM3 in its dimeric form. CPR: FAD and FMN. Reductase domain represented in yellow and heme domain in red. Based on Girvan and Munro 2016 and Neeli et al. 2005 [40,52]...... 8 Figure 1.3. Overview of approaches for protein engineering by random, rational and combined methods...... 10 Figure 1.4. Schematic outline of a typical directed evolution experiment. Based on Bloom and Arnold (2009) [215]...... 11 Figure 1.5. Overview of the KnowVolution strategy which comprises four phases: (I) Identification of potentially beneficial amino acid positions, (II) determination of beneficial amino acid positions and substitutions, (III) computational analysis and a group of amino acid substitution which might interact with each other, and (IV) recombination of beneficial substitutions in a simultaneous or iterative manner. The KnowVolution strategy can also be performed in an iterative manner to further improve targeted enzyme properties...... 12 Figure 1.6. Under alkaline condition and catalyzed by potassium peroxodisulfate, phenols (1) react with 4-APP (2) forming a product (3) that displays strong absorbance maxima at λ 509 nm...... 14 Figure 1.7. Schematic outline of a typical capillary electrophoresis experiment ...... 14 Figure 1.8. 2D chemical structure of the tested aromatic heterocycles in this work...... 15 Figure 1.9. Examples of oxygen, nitrogen and sulphur-based heterocycles building blocks...... 15 Figure 1.10. Manufacturing process of indigo. (4) aniline, (5) chloroaceticacid, (6) sodium phenylglycinate, (7) indoxyl and (8) indigo. Based on Yamamoto, Inoue and Takaki et al. [120]...... 16 Figure 1.11. Tryptophan (9) is converted to indole (10) by the a tryptophanase which is then oxygenated to indoxyl (7) by a mono- or dioxygenase which spontaneously oxidizes to indigo (8). Based on Hsu et al. [116]...... 17 Figure 1.12. Available chemical routes for the production of (13) 3-dihydrobenzo-1,4-dioxin-5-ol, (15) 3-dihydrobenzo-1,4- dioxin-6-ol and, (18) 3-dihydrobenzo-1,4-dioxin-2-ol. (11) dibromomethane, (12) benzene-1,2,3-triol, (14) 2,3-dihydro-1,4- benzodioxine-6-carbaldehyde, (16) ...... 18 Figure 3.1. A schematic representation of the PLICing method. A) DNA backbone with a normal phosphodiester bond (top) and a phosphorothiodiester bond (bottom) in which one oxygen at the α-phosphate is replaced by a sulfur. B) Three step scheme of the PLICing method: amplification, cleavage, and hybridization. The figure was based on Blanusa et al. [171] ...... 31 Figure 3.2. Chemical cleavage of phosphothiolated nucleotides in the presence of iodine and ethanol (I2/EtOH) under alkaline conditions. In step one the sulfur atom is alkylated by iodoethanol leading to an instable intermediate which releases the phosphorothiolated nucleotide from the DNA. Based on Eckstein and Gish [174]...... 32 Figure 4.1. NADPH consumption rate comparison between P450 BM3 WT and the selected variants (bars). M1 (R47S/Y51W), M2 (R47S/Y51W/I401M), M3 (R47S/Y51W/A330F/I401M), CM1 (R255P/P329H). Error bars represent one SD of the mean from three replicates...... 44 Figure 4.2. The hydroxylation of benzo-1,4-dioxane by cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) leads to the formation of 2,3-dihydrobenzo-1,4-dioxin-5-ol and 2,3-dihydrobenzo-1,4-dioxin-6-ol at a 70/30 ratio...... 45 Figure 4.3. The 4-AAP linear detection range of 2,3-dihydrobenzo-1,4-dioxin-5-ol and 2,3-dihydrobenzo- 1,4-dioxin-5-ol in 96-well MTP format...... 46

86 Appendix

Figure 4.4. The standard deviation of the 4-AAP assay with P450 BM3 WT. Measured absorption values at λ 509 nm in descending order of P450 BM3 WT catalyzed conversion of benzo-1,4-dioxane in a 96-well plate. The apparent standard deviation (6.8%) is depicted with white triangles. The white hexagons show the true standard deviation (9.4%) after subtraction of the empty vector background...... 46 Figure 4.5. Substrate and product separation via CE. EOF—electro-osmotic flow; i.s.— internal standard (benzyl alcohol); a— 2,3-dihydrobenzo-1,4-dioxin-6-ol; b—2,3-dihydrobenzo-1,4- dioxin-5-ol; c—benzo-1,4-dioxane; Mix-mixture of 2,3- dihydrobenzo-1,4-dioxin-6-ol, 2,3-dihydrobenzo-1,4-dioxin-5-ol and benzo-1,4-dioxane...... 47 Figure 4.6. The CE linear detection range of 2,3-dihydrobenzo-1,4-dioxin-5-ol in a 96-well MTP format...... 48 Figure 4.7. The standard deviation of the CE with P450 BM3 WT. Detected signal amplitude (mAU) at λ 214 nm in descending order of P450 BM3 variant WT catalyzed conversion of benzo-1,4-dioxane in a 96-well plate. The standard deviation (15.6%) is depicted with black triangles...... 48 Figure 4.8. The GC-FID chromatograms of benzo-1,4-dioxane derivates separation. A thermal gradient (were separated using the following program: (100 °C for 1 min, heating 10 °C/min up to 200 °C, heating 20 °C/min up to 250 and hold for 10 min) was applied on a Optima 17 MS column which yielded in separation of benzo-1,4-dioxane (a), 2,3-dihydro-1,4-benzodioxin- 5-ol (b) and 2,3-dihydro-1,4-benzodioxin-6-ol (c). (a) MTBE – (methyl tert-Butyl ether)...... 49 Figure 4.9. The standard curve of benzo derivatives 2,3-dihydro-1,4-benzodioxin-5-ol and 2,3-dihydro-1,4-benzodioxin-6-ol. The samples were prepared in MTBE with concentrations ranging from 0.125 mM to 3 mM.s ...... 50 Figure 4.10. The standard curve of the internal standard cyclododecanol. The Samples were prepared in MTBE with concentrations ranging from 0.2 mM to 2 mM ...... 50 Figure 4.11. Summary of P450 BM3 engineering strategy. Starting from the top to the bottom, in-house libraries of P450 BM3 were screened yielding P450 BM3 CM1 (R255P/P329H), which was subjected to epPCR, and P450 BM3 GS2 (R255S/P329H/F331L) was generated. P450 BM3 GS2 was subjected to another epPCR round yielding P450 BM3 GS3 (I122V/R255S/P329H/F331L). P450 BM3 WT was subjected to single site saturation mutagenesis (SSM) in the four identified positions and double SSM at positions I122 and R255, which led to the most active variants, P450 BM3 R255G and R255L...... 51

Figure 4.12. The relative NADPH consumption of the clones present in the sample plate from 0.05 mM MnCl2 P450 BM3- epPCR library. Green bar represents P450 BM3 CM1 (positive control), EV – Empty Vector (negative control), red line represents the threshold for classifying the clones active and green line the threshold for classifying the clones improved.... 52 Figure 4.13. Comparison between P450 BM3 WT and resulting variants from the 2 rounds of epPCR of NADPH consumption rate (bars) and product formation (circles) using a 4-AAP assay. Error bars represent one SD of the mean from three replicates. CM1 (R255P/P329H), GS2 (R255S/P329H/F331L), and GS3 (I122V/R255S/P329H/F331L). Error bars represent one SD of the mean from three replicates...... 52 Figure 4.14. The crystal structure of P450 BM3 WT illustrating the 4 identified positions (I222, R255, P329, and F331) from the two rounds of epPCR. The positions selected are represented as ball and sticks. The heme cofactor is depicted in red sticks...... 53 Figure 4.15. Comparison of product formation by the colorimetric 4-aminoantipyrine (4-AAP) assay (λ 509 nm). Results show the best-performing P450 BM3 from SSM at the positions 122, 255, 329, and 331. Error bars represent one SD of the mean from seven replicates. CM1 (R255P/P329H), GS2 (R255S/P329H/F331L), and GS3 (I122V/R255S/P329H/F331L). EV— negative lysate control. Error bars represent one SD of the mean from three replicates...... 53 Figure 4.16.Product analysis via CE after conversion of benzo-1,4-dioxane with lysate from EV (negative control), P450 BM3 WT, and variants R255G and R255L. The data was obtained from 4 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. EOF—electro-osmotic flow; i.s.— internal standard (benzyl alcohol); a—2,3-dihydrobenzo- 1,4-dioxin-6-ol; b—2,3-dihydrobenzo-1,4- dioxin-5-ol; c—benzo-1,4-dioxane...... 54 Figure 4.17. A) Absorption spectra for the produced variants of P450 BM3 in their ferrous carbon monoxide complex. A typical absorption spectrum for the WT of P450 BM3 is shown in blue. EV—empty vector (negative control), WT—wild-type, GS2 (R255S/P329H/F331L), and GS3 (I122V/R255S/P329H/F331L). B) SDS-PAGE (10% Acrylamide) analysis of the purified

87 Appendix

P450 BM3 WT and variants R255G and R255L. The molecular weight of P450 BM3 WT and variant proteins are ca. 118 kDa...... 55 Figure 4.18. Product formation of different P450 BM3 variants measured with GC-FID. (a) 2,3-dihydrobenzo- 1,4-dioxin-5-ol detected by GC-FID after 1 h of benzo-1,4-dioxane conversion. (b) 2,3-dihydrobenzo- 1,4-dioxin-6-ol detected by GC-FID after 1 h of benzo-1,4-dioxane conversion. Error bars represent one SD of the mean from three replicates...... 55 Figure 4.19. Product analysis using GC-FID after conversion of benzo-1,4-dioxane with lysate from EV (negative control), P450 BM3 WT, and from variants R255G and R255L. The data was obtained from 1 h conversion reactions employing glucose dehydrogenase (GDH) for efficient NADPH cofactor regeneration. a—benzo-1,4-dioxane, d—2,3-dihydrobenzo-1,4-dioxin-5- ol, e—2,3-dihydrobenzo- 1,4-dioxin-6-ol, i.s.—cyclododecanol, b, c—unknown product. An improved 2,3-dihydrobenzo-1,4- dioxin-5-ol formation is visible with R255G and R255L compared to WT...... 56 Figure 4.20. Product analysis via GC-FID after conversion of 2,3-dihydrobenzofuran with EV, P450 BM3 WT, and variants R255G and R255L. The data was obtained from 1 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. a—2,3-dihydrobenzofuran, i.s.—cyclododecanol, b, c, d, e, f, g—unknown product. A total 2,3- dihydrobenzofuran depletion is visible with R255G and R255L, whereas the WT does not convert the substrate...... 57 Figure 4.21. Product analysis via GC-FID after conversion of isochroman with lysate from EV (negative control), P450 BM3 WT, and from variants R255G and R255L. The data was obtained from 1 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. a—isochroman, i.s.—cyclododecanol, b, c, d—unknown product. A complete isochroman depletion is visible for R255G and R255L, whereas WT is virtually unable to convert isochroman (≤1.5 ± 0.2% conversion)...... 58 Figure 4.22. Product analysis via GC-FID after conversion of phthalan with lysate from EV (negative control), P450 BM3 WT, and from variants R255G and R255L. The data was obtained from 1 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. a—phthalan, i.s.—cyclododecanol, b, c, d, e, f—unknown product. A nearly 90% phthalan depletion is visible for both R255G and R255L, whereas WT can only convert 6 ± 1%...... 58 Figure 4.23. Product analysis via GC-FID after conversion of benzofuran with lysate from EV (negative control), P450 BM3 WT, and from variants R255G and R255L. The data was obtained from 1 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. a—benzofuran, i.s.—cyclododecanol. A nearly 90 ± 2% benzofuran depletion is visible with R255G and complete depletion with R255L, whereas WT is only able to convert 19 ± 6%...... 59 Figure 4.24. Product analysis via GC-FID after conversion of dibenzofuran with lysate from EV (negative control), P450 BM3 WT, and from variants R255G and R255L. The data was obtained from 1 h conversion reactions employing GDH for efficient NADPH cofactor regeneration. a—dibenzofuran, b—unknown product, i.s.—cyclododecanol. A conversion of 77 ± 0% and 85 ± 3% of dibenzofuran is visible for R255G and R255L, respectively, whereas WT is only able to convert 16 ± 4%...... 59 Figure 4.25. Comparative conversions of 1 h reaction with the different substrates benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran (concentration 1.2 mM) by P450 BM3 WT and variants R255G and R255L. Numbers are presented in percentages. Error bars represent one SD of the mean from three replicates. Values are taken from GC-FID analysis...... 60 Figure 4.26. Molecular docking pose of benzo-1,4-dioxane in the active site of WT, R255G, and R255L (A) WT (binding energy: −5.63 kcal/mol), (B) R255G variant (binding energy: −5.66 kcal/mol), and (C) R255L variant (binding energy: −5.66 kcal/mol). Reciprocal arrows indicate the closest distance between the iron-bound water ligand and C5 atom of benzo-1,4- dioxane (WT: 5.14 Å, R255G and R255L: 4.90 Å). Substrate benzo-1,4-dioxane is depicted as a ball and stick, whereas all the active site residues including heme are depicted as sticks. Hydrogen bond between substrate and T438 is represented as a black dotted line...... 61 Figure 4.27. Calculated (A) root mean square deviation (RMSD) and (B) root mean square fluctuation (RMSF) per residue of P450 BM3 WT and P450 BM3 variants (R225L and R255L) throughout three independent MD simulation runs (I-helix: residues 245 to 283)...... 62

88 Appendix

Figure 4.28. (A) Calculated distance between C5-atom of substrate and iron-bound oxygen species. (B) Calculated angle between benzo-1,4-dioxane substrate and heme required for the hydroxylation of benzo-1,4-dioxane in WT and variants R255G and R255L along three independent 50 ns MD simulation trajectories...... 63 Figure 4.29. Cartoon representation of the structural alignment of P450 BM3 WT (grey) and P450 BM3 variants (R255G in green and R255L in magenta). Heme is depicted in red lines and residue F87 in sticks. A substantial rearrangement of the G-, H-, and I-helixes is observed. The models of the P450 BM3 variants R255G and R255L were constructed using the swap function in YASARA Structure Version 17.4.17 and optimized using the SCWRL rotamer library search for the designated substitutions...... 63 Figure 4.30. Products of enzyme-catalyzed oxidation of indole. Based on [123,201,216]...... 65 Figure 4.31. Absorbance spectra of indigo and indirubin from 400 to 700 nm...... 65 Figure 4.32. Standard deviation of the indigo MTP screening assay with P450 Cand_1. Detected absorbance at λ 610 nm in descending order of P450 Cand_1 clone catalyzed conversion of indole to indigo in a 96-well plate. The standard deviation of 23.9 % is represented in black rectangles. The true standard deviation (33.2 %) is represented with white triangles...... 66 Figure 4.33. A) Absorbance at λ 620 nm over time in min from conversion of indole cell lysates of P450 Cand_1 and Empty Vector in Terrific Broth...... 66 Figure 4.34. Product separation via HPLC. i.p.—injection peak; a—indigo; b—indirubin; DMSO- Dimethyl sulfoxide. Indigo monitored at λ 620 nm, indirubin monitored at λ 540 nm...... 67 Figure 4.35. The HPLC linear detection range of indigo measured at λ 620 nm. Samples were prepared in DMSO with concentrations ranging from 2 mg/L to 100 mg/L...... 67 Figure 4.36. The interior volume of P450 Cand_1. A) The volume-filling spheres surface (red) identified in P450 Cand_1 using a grid-spacing of 0.2 Å and the residues (green stick display) that define the interior volume by proximity to the volume-filling spheres and to the heme (orange stick display). B) The residues selected for site-saturation-mutagenesis. Images were prepared using Pymol...... 68 Figure 4.37. GelDoc XR+ Automatic Band and Lane detection from a 1 % (w/v) agarose gel confirmation of SeSaM library products. A) Amplification with biotinilated primer confirmation. 1 & 12 – MWM 1 kb GeneRuler, 2 to 11 – amplification of P450 Cand_1 heme domain with biotinylated primers (1418 bp). B) Cleavage confirmation. 1 & 7 – MWM 1 kb GeneRuler 2 to 6 – cleavage of products from A. C) Final amplification 1 & 6 – MWM 1 kb GeneRuler 2 to 5 – final amplified products...... 69 Figure 4.38. A) Absorbance at λ 620 nm over time in min from conversion of indole cell lysates of P450 Cand_1 and Empty Vector in Terrific Broth. B) MTP Picture taken at the end of the indole conversion in A) using cell lysates from P450 Cand_1 and Empty Vector. Blue colour indicates indigo formation...... 70 Figure 4.39. Comparison of flask and deep-well plate expression of P450 Cand_1. A) Pelleted cells after expression of P450 Cand_1 and negative control (empty vector). B) Pelleted cells after P450 Cand_1 expression in 2.2 mL deep-well plates. ... 70 Figure 4.40. A) The crystal structure of P450 Cand_10 illustrating the residues lining the tunnel (red surface) and B) the identified positions V89, I013, L258, V261, L309, M312. The positions selected are represented in B as sticks. The heme cofactor is depicted in orange sticks ...... 72 Figure 4.41. The occurrence variability analysis of the alignment using ClustalW performed with 2473 reviewed P450 structures in UniProt at the positions V89, I013, L258, V261, L309, M312 ...... 73 Figure 4.42. GelDoc XR+ Automatic Band and Lane detection from an 1% (w/v) agarose gel confirmation of SSM library PCR products. 1 & 8 – MWM 1 kb GeneRuler, 2 – SSM89, 3 – SSM103, 4 – SSM258, 5 – SSM261, 6 – SSM 309, 7 – SSM312...... 73 Figure 4.43. Comparison of flask and deep-well plate expression of P450 Cand_10. A) Pelleted cells after expression of P450 Cand_10 and negative control (empty vector). B) Pelleted cells after P450 Cand_10 SSM258 expression in 2.2 mL deep-well plates...... 73

89 Appendix

Figure 4.44. A) P450 Cand_10 NADPH depletion in the presence and absence of 1.2 mM of n-octane. 50 µL clarified cell lysate, 150 µL KPi 50 mM pH 7.5/1.2 mM n-octane, DMSO as co.solvent, 50 µL NADPH 1 mM. B) Absorption spectra for the expressed P450 Cand_10 in its ferrous carbon monoxide complex...... 74 7.3. List of Equations

Equation 1.1. The simplified reaction of a heme-dependent monooxygenase ...... 5 Equation 3.1. Condition to consider a variant improved for NADPH depletion...... 36 Equation 3.2. Condition to consider a variant improved for product formation...... 36 Equation 3.3. The equation for calculating the P450 sample concentration...... 38 Equation 3.4. The equation for determining the NADPH oxidation rate...... 39 Equation 3.5. The equation for determining coupling efficiency...... 39

90 Appendix

7.4. List of Abbreviations

°C Degrees Celsius KM Michaelis Menten Constant 4-AAP 4-Aminoantipyrine KPi Potassium Phosphate Buffer ALA Aminolevulinic Acid LB Lysogeni Broth APS Ammonium Persulfate MCS Multiple Clonning Site atm Atmospheric min Minute(s) bar Pressure mol Molecule bp base pair MOPS 3-(N-Morpholino)Propanesulfonic Acid BM Bacillus Megaterium mRNA Messenger Ribonuclei Acid BSA Bovine Serum Albumin mM mili Molar CBB Coomassie-Brilliant Blue MS Mass Spectrosmetry cfu Colony Formation Unit MTBE Methyl Tert-Butyl Ether CYP Cytochrome P450 Monoxygenase MTP Microtiter Plate CV Column Volume m/z Mass-To-Charge ratio Nicotine Amide Adenine Dinucleotide dH2O De-ionized Water NADP+ Phosphate (Oxidized Form) Nicotine Amide Adenine Dinucleotide DMSO Dimethyl sulfoxide NADPH Phosphate (Reduced Form) DEAE Diethylethanolamine nt Nucleotide

DNA Deoxyribonucleic Acid OD600 Optical Density At Wavelength Of 600 nm dsDNA Double Strand DNA P450 Cytochrome P450 Monooxygenase dNTP Deoxynucleotide Triphosphate PCR Polymerase Chain Reaction E. coli Escherichia coli PFR Product Formation Rate Decimal logarithm of the reciprocal of the e.g. exempli gratia pH hydrogen ion activity error-prone Polymerase Chain Logarithmic measure of the acid dissociation epPCR pKa Reaction constant Phosphorothioate-Based Ligase-Independent et al. et alli PLICing gene cloning EtOH Ethanol Ref. Reference EV Empty Vector Rev Reverse FAD Flavin Adeninde Dinucleotide rpm Revolutions per minute FDH Formate Dehydrogenase RT Room Temperature FID Flame Ionization Detector SDM Site Directed Mutagenesis Sodium Dodecyl Sulfate Polyacrylamide Gel FMN Flavin Mononucleotide SDS-PAGE Electrophoresis Fwd Forward s Second(s) g Gravitational Force (9.8 m/s2) ssDNA Single strand Deoxyribonucleic Acid GC Gas Chromatography SSM Site Saturation Mutagenesis GDH Glucose Dehydrogenase TB Terrific Broth h Hour(s) TEMED N,N,N’,N’-Tetramethylethylenediamine HTS High Throughput Screening TFA Trifluoroacetic acid High Performance Liquid HPLC TFB Transformation Buffer Chromatography IPTG Isopropyl TOF Turnover K Kelvin Tris Tris(Hydroxymethyl)Aminomethane Kan Kanamycin TTN Total Turnover Number kb Kilobase(s) U Units kcat Catalytic Activity UV Ultraviolet WT Wild-Type vs. Versus w/v weight per volume v/v volume per volume

7.5. Additional Experimental Information and Data

91 Appendix

7.5.1. Gene and Protein Sequences

Gene sequence of P450 BM3 (WT): atgacaattaaagaaatgcctcagccaaaaacgtttggagagcttaaaaatttaccgttattaaacacagataaaccggttcaagctttgatgaaaattgcggatgaatt aggagaaatctttaaattcgaggcgcctggtcgtgtaacgcgctacttatcaagtcagcgtctaattaaagaagcatgcgatgaatcacgctttgataaaaacttaagtc aagcgcttaaatttgtacgtgattttgcaggagacgggttatttacaagctggacgcatgaaaaaaattggaaaaaagcgcataatatcttacttccaagcttcagtcag caggcaatgaaaggctatcatgcgatgatggtcgatatcgccgtgcagcttgttcaaaagtgggagcgtctaaatgcagatgagcatattgaagtaccggaagacat gacacgtttaacgcttgatacaattggtctttgcggctttaactatcgctttaacagcttttaccgagatcagcctcatccatttattacaagtatggtccgtgcactggatg aagcaatgaacaagctgcagcgagcaaatccagacgacccagcttatgatgaaaacaagcgccagtttcaagaagatatcaaggtgatgaacgacctagtagata aaattattgcagatcgcaaagcaagcggtgaacaaagcgatgatttattaacgcatatgctaaacggaaaagatccagaaacgggtgagccgcttgatgacgagaa cattcgctatcaaattattacattcttaattgcgggacacgaaacaacaagtggtcttttatcatttgcgctgtatttcttagtgaaaaatccacatgtattacaaaaagcag cagaagaagcagcacgagttctagtagatcctgttccaagctacaaacaagtcaaacagcttaaatatgtcggcatggtcttaaacgaagcgctgcgcttatggcca actgctcctgcgttttccctatatgcaaaagaagatacggtgcttggaggagaatatcctttagaaaaaggcgacgaactaatggttctgattcctcagcttcaccgtga taaaacaatttggggagacgatgtggaagagttccgtccagagcgttttgaaaatccaagtgcgattccgcagcatgcgtttaaaccgtttggaaacggtcagcgtgc gtgtatcggtcagcagttcgctcttcatgaagcaacgctggtacttggtatgatgctaaaacactttgactttgaagatcatacaaactacgagctcgatattaaagaaa ctttaacgttaaaacctgaaggctttgtggtaaaagcaaaatcgaaaaaaattccgcttggcggtattccttcacctagcactgaacagtctgctaaaaaagttcgcaaa aaggcagaaaacgctcataatacgccgctgcttgtgctatacggttcaaatatgggaacagctgaaggaacggcgcgtgatttagcagatattgcaatgagcaaag gatttgcaccgcaggtcgcaacgcttgattcacacgccggaaatcttccgcgcgaaggagctgtattaattgtaacggcgtcttataacggtcatccgcctgataacg caaagcaatttgtcgactggttagaccaagcgtctgctgatgaagtaaaaggcgttcgctactccgtatttggatgcggcgataaaaactgggctactacgtatcaaa aagtgcctgcttttatcgatgaaacgcttgccgctaaaggggcagaaaacatcgctgaccgcggtgaagcagatgcaagcgacgactttgaaggcacatatgaag aatggcgtgaacatatgtggagtgacgtagcagcctactttaacctcgacattgaaaacagtgaagataataaatctactctttcacttcaatttgtcgacagcgccgcg gatatgccgcttgcgaaaatgcacggtgcgttttcaacgaacgtcgtagcaagcaaagaacttcaacagccaggcagtgcacgaagcacgcgacatcttgaaattg aacttccaaaagaagcttcttatcaagaaggagatcatttaggtgttattcctcgcaactatgaaggaatagtaaaccgtgtaacagcaaggttcggcctagatgcatc acagcaaatccgtctggaagcagaagaagaaaaattagctcatttgccactcgctaaaacagtatccgtagaagagcttctgcaatacgtggagcttcaagatcctgt tacgcgcacgcagcttcgcgcaatggctgctaaaacggtctgcccgccgcataaagtagagcttgaagccttgcttgaaaagcaagcctacaaagaacaagtgct ggcaaaacgtttaacaatgcttgaactgcttgaaaaatacccggcgtgtgaaatgaaattcagcgaatttatcgcccttctgccaagcatacgcccgcgctattactcg atttcttcatcacctcgtgtcgatgaaaaacaagcaagcatcacggtcagcgttgtctcaggagaagcgtggagcggatatggagaatataaaggaattgcgtcgaa ctatcttgccgagctgcaagaaggagatacgattacgtgctttatttccacaccgcagtcagaatttacgctgccaaaagaccctgaaacgccgcttatcatggtcgg accgggaacaggcgtcgcgccgtttagaggctttgtgcaggcgcgcaaacagctaaaagaacaaggacagtcacttggagaagcacatttatacttcggctgccg ttcacctcatgaagactatctgtatcaagaagagcttgaaaacgcccaaagcgaaggcatcattacgcttcataccgctttttctcgcatgccaaatcagccgaaaaca tacgttcagcacgtaatggaacaagacggcaagaaattgattgaacttcttgatcaaggagcgcacttctatatttgcggagacggaagccaaatggcacctgccgtt gaagcaacgcttatgaaaagctatgctgacgttcaccaagtgagtgaagcagacgctcgcttatggctgcagcagctagaagaaaaaggccgatacgcaaaagac gtgtgggctgggtaa Protein sequence of P450 BM3 (WT): RKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYN GHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEA DASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASK ELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPL AKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKY PACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTIT CFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEE LENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKS YADVHQVSEADARLWLQQLEEKGRYAKDVWAG*

92 Appendix

Gene sequence of P450 Cand_1 (WT): atgacaccgagtactcccatcgatgacgccgaaattgctcgtagcattgcgttggaagatatagatgtctccaagccggaactttttgaacgcgatggtctgcatcctt atttcgagcgcctacgccgcgaagatccagttcattattgtaaagcctccgaatatggaccgtactggagtattacgaaattctccgatattgttgccattgacactaacc acaaagtctttagctcagatcacacgaatgggtccttcgttctggatgatacgacgttgaacgccgttgacggtggtatctacctacccaattttttagggatggatcca cccaaacatgatgtgcatagaatggttgtttcacctattgtggccccgcaaaatcttctgcgttttgaggcgactattcgcgagcgtactaaacgtgtgctctcagagtta ccaatcggtgaggagtttaattgggttgaccgcgtgtcgatcgagctcaccacaatgatgttagcaacgctgttggattttccctttgatgatcgccgcaaactgacac ggtggagtgatataattacaacgcgccctggttatggcttagtggattcttgggaacaacgcgaaagcgaactgatggaatgcttagcgtatttccaacgactgtatgc cgagcgccaggcgatgcccccaaagccggatctgatctctatgctggcccactcgccagagatgcaggacttgacacctacagactttttaggtaccctggcacttc ttattgtgggtggtaacgataccactcggtcttctatgtctggttcggcgatggcgtgccatttgtatccacaggaatttgacaaagtgaggaataacagagcactgctg gctagtgtgatacctgaagttgttcgttggcagaccccgattgcacacatgcgccgcactgctttagaggacgtagaatttcgtgggaagcagattcgcaaaggcga caaggtcgttatgtggtatctgtccggtaacagggatgacgaggtgatagatcggccaatggattttattgcggatcgccctcgtgcgagacatcacctgtcatttggc tttggtattcaccgctgcctgggcaatagactggccgaacttcagcttaaaattctgtgggaggaaatgtgtgaacgctactcccggatagaggtatgcggcgagcc ggtacgtgtaccttccaacctagtacatggttatattgacattccagttcgtttacatgcgtaa Protein sequence of P450 Cand_1 (WT): MTPSTPIDDAEIARSIALEDIDVSKPELFERDGLHPYFERLRREDPVHYCKASEYGPYWSITKFSDIVAIDT NHKVFSSDHTNGSFVLDDTTLNAVDGGIYLPNFLGMDPPKHDVHRMVVSPIVAPQNLLRFEATIRERTK RVLSELPIGEEFNWVDRVSIELTTMMLATLLDFPFDDRRKLTRWSDIITTRPGYGLVDSWEQRESELMEC LAYFQRLYAERQAMPPKPDLISMLAHSPEMQDLTPTDFLGTLALLIVGGNDTTRSSMSGSAMACHLYP QEFDKVRNNRALLASVIPEVVRWQTPIAHMRRTALEDVEFRGKQIRKGDKVVMWYLSGNRDDEVIDR PMDFIADRPRARHHLSFGFGIHRCLGNRLAELQLKILWEEMCERYSRIEVCGEPVRVPSNLVHGYIDIPV RLHA* Gene sequence of P450 Cand_10 (WT): atggacgatgcgtccatagacctgcaacgcgcggcgcgggatgccgcttattcaatgcccattgaagaaataaaccctgcggatcccgaattattccgcaccgata ccatgtggccatattttgagcgcctgcgtaaagaagatccagttcactggggtgtttccccccacgaagacgtaggcggttattggagcgtgacaaaatataacgata tcatggcagtcgataccaatcatgaagtctttagtagtgaacctacaattgttctgccagacccagccgatgactttactctgccaatgttcattgctatggacccaccga aacacgatgtccaacggaaaaccgttcagccaattgtggcaccgaatcatttggcttatcttgaacctatcatccgtgagcgtgcagggaaaattttagatgatctgcct atcggagaggagattaactgggtagacaaagtatccatcgagctcaccactatgacattagccacccttttcgattttccatgggaagatcgcagaaaactgacatttt ggtcagatgttgcaacgtctgctccggagtcgggcattttaggcaccacagacccagaggagcacgaaaacctgcgtcgccagactttgttcgaatgcgtcgactat tttatgcgcctgtggaatgaacgtgttaatgcacccccgaagccagacctaattagtatgttagcacacggcgagagcaccaaaaatatggatcgtatggaatacctg ggaaatctgattttgcttattgtcggagggaacgataccacacgaaacaccattagcggttctgttttagcattgcaccaaaatcccgaccaggaccgcaaattgcga gagaatcctggtcttatcccagctatggtgagcgaaaccattcgctggcaaacacccttagcttatatgagacggcgtgcaaagcgggatttcgaacttggcgggaa aacgatccgtgaaggggacaaagttgctatgtggtatgtttcgggcaacagagatgaagaggtaattgaccgcccgaacgattattggatagaacgcccaagagta agacagcacctaagctttggtttcggcgttcatcgatgcgtgggaaaccgattggcagaactccagctgaaaatcatttgggaagagatcctggcgcgtttcccaag attagaggtggtggggccgcctagacgagtatattcctcgttcgtgaaagggtatgaagaattacccgtcgttattccaacacgcaattaa Protein sequence of P450 Cand_10 (WT): MDDASIDLQRAARDAAYSMPIEEINPADPELFRTDTMWPYFERLRKEDPVHWGVSPHEDVGGYWSVT KYNDIMAVDTNHEVFSSEPTIVLPDPADDFTLPMFIAMDPPKHDVQRKTVQPIVAPNHLAYLEPIIRERA GKILDDLPIGEEINWVDKVSIELTTMTLATLFDFPWEDRRKLTFWSDVATSAPESGILGTTDPEEHENLR RQTLFECVDYFMRLWNERVNAPPKPDLISMLAHGESTKNMDRMEYLGNLILLIVGGNDTTRNTISGSVL ALHQNPDQDRKLRENPGLIPAMVSETIRWQTPLAYMRRRAKRDFELGGKTIREGDKVAMWYVSGNRD EEVIDRPNDYWIERPRVRQHLSFGFGVHRCVGNRLAELQLKIIWEEILARFPRLEVVGPPRRVYSSFVKG YEELPVVIPTRN*

93 Appendix

7.5.2. Primers and Vectors map

Table 7.1. List of oligonucleotides used for cloning, sequencing and mutagenesis during this work Use Target Primer Name Primer Sequence (5’ - 3’) Vector pALX FWD ctcataatacGCCGCTGCTTGTGCTATACG P450 BM3 Vector pALX REV gcgtattatgAGCGTTTTCTGCCTTTTTGC cloning Gene P450 BM3 FWD catgggcatGACAATTAAAGAAATGCCTCA Gene P450 BM3 REV gcgtattatgaGCGTTTTCTGCCTTTTTGC - T7 Prom TAATACGACTCACTATAGGG - T7 Term. GTTATTGCTCAGCGGTGGCAGCAG - 1 CTTTAACAGCTTTTACCGAGATCAGC Sequencing - 2 CAGCTTAAATATGTCGGCATGGTC P450 BM3 - 3 GTGCTATACGGTTCAAATATGG - 4 GTTTTCAACGAACGTCGTAGC - 5 GTCGATGAAAAACAAGCAAGC SSM122.FWD ATGATGGTCGATNNKGCCGTGCAGCTT I122 SSM122.REV AAGCTGCACGGCMNNATCGACCATCAT SSM255.FWD GACGAGAACATTNNKTATCAAATTATT R255 P450 BM3 SSM255.REV AATAATTTGATAMNNAATGTTCTCGTC SSM SSM329.FWD TGGCCAACTGCTNNKGCGTTTTCCCTA P329 SSM329.REV TAGGGAAAACGCMNNAGCAGTTGGCCA SSM331.FWD ACTGCTCCTGCGNNKTCCCTATATGCA F331 SSM331.REV TGCATATAGGGAMNNCGCAGGAGCAGT Sequencing - Lac.FWD GGAATTGTGAGCGGATAACAATTCC P450 Cand_1 - Seq2.FWD CTTCTATGTCTGGTTCGGCG SSM89.FWD CGTTCTGNNKGATACGACGTTGAACG D89 SSM89.REV GTCGTATCMNNCAGAACGAAGGACCC SSM192.FWD TACAACGNNKCCTGGTTATGGCTTAGTG R192 SSM192.REV AACCAGGMNNCGTTGTAATTATATCACTCC P450 Cand_1 SSM262.FWD GGTGGTAACNNKACCACTCGGTCTTC D262 SSM SSM262.REV GAGTGGTMNNGTTACCACCCACAATAAG SSM407.FWD TTCCAACNNKGTACATGGTTATATTGACATTCC L407 SSM407.REV CCATGTACMNNGTTGGAAGGTACACGTACC SSM408.FWD AACCTANNKCATGGTTATATTGACATTCCAGTT V408 SSM408.REV ATAACCATGMNNTAGGTTGGAAGGTACACG Sequencing - Lac.FWD GGAATTGTGAGCGGATAACAATTCC Cand_10 - Seq3.FWD GAGAATCCTGGTCTTATCCCAGC SSM89.FWD ACCTACAATTNNKCTGCCAGACC

SSM89.REV GGTCTGGCAGMNNAATTGTAGGT SSM103.FWD CCAATGTTCNNKGCTATGGACC

SSM.103.REV GGTCCATAGCMNNGAACATTGG SSM258.FWD GAAATCTGATTNNKCTTATTGTCG P450 SSM258.REV CGACAATAAGMNNAATCAGATTTC Cand_10 SSM261.FWD ATTTTGCTTATTNNKGGAGGGAACG SSM SSM261.REV CGTTCCCTCCMNNAATAAGCAAAAT SSM309.FWD GGCAAACACCCNNKGCTTATATGAGAC

SSM309.REV GTCTCATATAAGCMNNGGGTGTTTGCC SSM312.FWD CTTAGCTTATNNKAGACGGCGTGC

SSM312.REV GCACGCCGTCTMNNATAAGCTAAG Capital letters: Standard nucleotides connected through phosphorodiester bonds Lower case letters: Nucleotides connected through a phosphorothioatediester bond N: A, C, G, T K: G, T.

94 Appendix

Figure A1. pALEXtreme-1a vector map for the expression of P450 BM3. The plasmid contains a kanamycin resistance (KanR) and ColE1 replicon (ori). The expression is under control of the IPTG inducible T7 promotor.

Figure A2. pBIDI231a vector map for the co-expression of P450 Cand_1 and its reductase partners FdR and FdX. The plasmid contains a ampicilin resistance (AmpR) and a ColE1 replicon (ori). P450 Cand_1, FdR and FdX expression is under control of a IPTG inducible tac promoter.

Figure A3. pBIDI231a vector map for the co-expression of P450 Cand_10 and its reductase partners FdR and FdX. The plasmid contains a ampicilin resistance (AmpR) and a ColE1 replicon (ori). P450 Cand_1, FdR and FdX expression is under control of a IPTG inducible tac promoter.

95 Appendix

7.5.3. Gas-chromatography programs and compound retention time

Table 7.2. Gas-chromatography programs and compound retention time Extraction Compound Column RT (min) Program Int. Standard Solvent MTBE Optima-17 MS 1.36 1 - Cyclododecanol Benzo-1,4-dioxane Optima-17 MS 5.26 1 MTBE Cyclododecanol 2,3-dihydrobenzo[b][1,4]dioxin-5-ol Optima-17 MS 8.50 1 MTBE Cyclododecanol 2,3-dihydrobenzo[b][1,4]dioxin-6-ol Optima-17 MS 9.96 1 MTBE Cyclododecanol Benzofuran Optima-17 MS 2.81 1 MTBE Cyclododecanol Dibenzofuran Optima-17 MS 9.91 1 MTBE Cyclododecanol Phthalan Optima-17 MS 3.87 1 MTBE Cyclododecanol Isochroman Optima-17-MS 5.23 1 MTBE Cyclododecanol 2,3-dihydrobenzofuran Optima-17-MS 3.92 1 MTBE Cyclododecanol Program 1: SPL:250 °C, 100 °C for 1 min, 10 °C/min until 200 °C, 20 °C/min until 250 °C hold 10 min, FID: 300°C 7.5.4. Additional figures and graphs

Figure A4. P450 BM3 WT sequence annotated with targeted SSM positions and secondary structure. Yellow boxes as β- strand, red boxes as α-helix, gray boxes as coil, and blue boxes as the targeted position for SSM. Image prepared with PSIPRED Server [214]

96 Appendix

1 2 3 4 5 6 7 8 9 10 11 12 13

1.5 kb

1 kb

Figure A5. Agarose gel (1% (w/v)) confirmation of epPCR products. Amplifications using in-house Taq DNA polymerase, 1- MWM 1 kb GeneRuler, 2 to 12–0.05 mM heme domain (1452 bp).

A B C

D D

Figure A6. Colonies obtained after transformation with (A) DpnI control of Heme-domain insert (0.05 mM MnCl2 epPCR) pool. (B) Cleaved vector control. (C) DpnI control of vector and (D) DNA hybridisation of heme-domain insert (0.05 mM MnCl2 PCR) and vector. Plated volume: 75 µL.

97 Appendix

Curriculum vitae

Personal Information

Name: Gustavo de Almeida Santos

Date of birth: 14th of December 1990

Place of birth: Oliveira de Azeméis, Portugal

Education

2017 – 2020 PhD Fellow RWTH Aachen University, Institute of Biotechnology

2013 – 2015 Master of Science Molecular Genetics University of Minho, Portugal 2008 – 2011 Bachelor of Science Biotechnology Polytechnic Institute of Coimbra - Coimbra College of Agriculture, Portugal

98 Appendix

Declaration

I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work.

______

Aachen, Gustavo de Almeida Santos

99 Appendix

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 722390. ITN European Training Network “OXYTRAIN” for a training network on mechanistic and applied enzymology.

100