Discovery and Protein Engineering of Baeyer-Villiger monooxygenases Inauguraldissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Andy Beier geboren am 11.10.1988 in Parchim Greifswald, den 02.08.2017 I Dekan: Prof. Dr. Werner Weitschies 1. Gutachter: Prof. Dr. Uwe T. Bornscheuer 2. Gutachter: Prof. Dr. Marko Mihovilovic Tag der Promotion: 24.10.2017 II We need to learn to want what we have, not to have what we want, in order to get stable and steady happiness. - The Dalai Lama - III List of abbreviations % Percent MPS Methyl phenyl sulfide % (v/v) % volume per volume MPSO Methyl phenyl sulfoxide % (w/v) % weight per volume MPSO2 Methyl phenyl sulfone °C Degrees Celsius MTS Methyl p-tolyl sulfide µM µmol/L MTSO Methyl p-tolyl sulfoxide aa Amino acids MTSO2 Methyl p-tolyl sulfone + AGE Agarose gel electrophoresis NAD Nicotinamide adenine dinucleotide, oxidized aq. dest. Distilled water NADH Nicotinamide adenine dinucleotide, reduced + BLAST Basic Local Alignment Search NADP Nicotinamide adenine dinucleotide Tool phosphate, oxidized bp Base pair(s) NADPH Nicotinamide adenine dinucleotide phosphate, reduced BVMO Baeyer-Villiger monooxyge- OD600 Optical density at 600 nm nase CHMO Cyclohexanone monooxyge- PAGE Polyacrylamide gel electrophoresis nase Da Dalton PAMO Phenylacetone monooxygenase DMF Dimethyl formamide PCR Polymerase chain reaction DMSO Dimethyl sulfoxide PDB Protein Data Bank DMSO2 Dimethyl sulfone rpm Revolutions per minute DNA Desoxyribonucleic acid rv Reverse dNTP Desoxynucleoside triphosphate SDS Sodium dodecyl sulfate E. coli Escherichia coli SOC Super Optimal broth with Catabolite repression ee Enantiomeric excess TAE TRIS-Acetate-EDTA FAD Flavin adenine dinucleotide TB Terrific broth Fig. Figure TCE 2,2,2-Trichloroethanol FMN Flavin adenine mononucleotide TCEP tris(2-carboxyethyl)phosphine FMO Flavoprotein monooxygenase TEMED Tetramethylethylenediamine fw Forward TRIS Tris(hydroxymethyl)aminomethane GC Gas chromatography UV Ultraviolet h Hours x g Times gravity of Earth HAPMO 4-Hydroxyacetophenone mo- nooxygenase His(6) hexahistidine tag IPTG Isopropyl β-D-1- thiogalactopyranoside Furthermore, SI units (base, derived and prefixes) and L Liter the common notation for amino acids and nucleic ac- LB Lysogenic broth ids are used. M mol/L min Minutes 1 U is defined as the amount of enzyme that catalyzes the depletion of 1 µmol NADPH per minute in the NADPH depletion assay. IV Table of contents 1 Introduction ....................................................................................................................... 1 1.1 White Biotechnology ............................................................................................................... 1 1.2 Protein Engineering ................................................................................................................. 2 1.3 Flavin-dependent monooxygenases......................................................................................... 6 1.3.1 Baeyer-Villiger-Monooxygenases ................................................................................. 10 2 Scope of this thesis .......................................................................................................... 34 3 Results .............................................................................................................................. 35 3.1 Baeyer-Villiger monooxygenases participating in the metabolism of ketones in yeasts ...... 35 3.1.1 Determination of metabolites from Candida maltosa and other yeasts from 2-dodecanone and 1-dodecene ...................................................................................... 35 3.1.2 Investigations of novel BVMOs from yeasts ................................................................ 37 3.2 Switch of the cofactor specificity of the cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871 .................................................................................................. 61 3.2.1 Mutations of the phosphate recognition site .................................................................. 62 3.2.2 Investigating residues in proximity of NAD(P)H .......................................................... 68 3.2.3 Determination of kinetic parameters ............................................................................. 71 3.2.4 Biocatalysis with variants of CHMOAcineto .................................................................... 72 4 Discussion ......................................................................................................................... 73 4.1 Baeyer-Villiger monooxygenases participating in the metabolism of ketones in yeasts ...... 73 4.1.1 Determination of metabolites from yeasts from 2-dodecanone and 1-dodecene........... 73 4.1.2 Investigations of novel BVMOs from yeasts ................................................................ 78 4.2 Switch of the cofactor specificity of CHMOAcineto ............................................................... 101 4.2.1 Mutation of the phosphate recognition site ................................................................. 101 4.2.2 Mutation of residues in proximity of NADPH ............................................................ 106 4.2.3 Kinetics and uncoupling of CHMOAcineto ..................................................................... 109 4.2.4 Biocatalysis with CHMOAcineto .................................................................................... 110 4.2.5 Structural investigation of S186_S208E_K326H ........................................................ 112 4.2.6 Outlook ........................................................................................................................ 114 5 Summary ........................................................................................................................ 116 V 6 Material and Methods .................................................................................................. 118 6.1 Equipment ........................................................................................................................... 118 6.2 Chemicals ............................................................................................................................ 119 6.3 Buffers, growth media and solutions ................................................................................... 119 6.4 Kits / markers / enzymes ..................................................................................................... 123 6.5 Strains, plasmids and primers .............................................................................................. 124 6.6 Microbiological methods ..................................................................................................... 131 6.6.1 Strain maintenance ...................................................................................................... 131 6.6.2 Cultivation and expression Pichia pastoris X-33 ........................................................ 131 6.6.3 Cultivation of Yarrowia lipolytica ............................................................................... 131 6.6.4 Cultivation and expression in E. coli BL21(DE3) ....................................................... 132 6.7 Molecular biological methods ............................................................................................. 135 6.7.1 Determination of DNA concentration ......................................................................... 135 6.7.2 Isolation of genomic DNA from yeasts ....................................................................... 135 6.7.3 Plasmid preparation ..................................................................................................... 135 6.7.4 Agarose gel electrophoresis ......................................................................................... 136 6.7.5 Sequencing .................................................................................................................. 136 6.7.6 Cloning ........................................................................................................................ 136 6.7.7 Transformation ............................................................................................................ 147 6.7.8 Colony PCR ................................................................................................................. 150 6.7.9 Site-directed mutagenesis ............................................................................................ 152 6.7.10 DpnI digestion ............................................................................................................. 153 6.8 Biochemical methods .......................................................................................................... 153 6.8.1 Cell disruption ............................................................................................................. 153 6.8.2 Enzyme purification .................................................................................................... 154 6.8.3 Determination of protein concentration ....................................................................... 155 6.8.4 SDS-PAGE .................................................................................................................