Flavoproteins in directed evolution [Elektronische Ressource] : iterative CASTing to evolve YqjM and P450BM3 / vorgelegt von Sabrina Kille

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Publié le : vendredi 1 janvier 2010
Lecture(s) : 19
Source : WWW-BRS.UB.RUHR-UNI-BOCHUM.DE/NETAHTML/HSS/DISS/KILLESABRINA/DISS.PDF
Nombre de pages : 145
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Flavoproteins in Directed Evolution



Iterative CASTing to evolve YqjM and P450 BM3









Dissertation
Zur Erlangung des Grades eines Doktors der Naturwissenschaften
der Fakultät für Chemie und Biochemie der Ruhr-Universität Bochum








vorgelegt von Diplom Chemikerin
Sabrina Kille
aus Cloppenburg



Max-Planck-Institut für Kohlenforschung, Mülheim an der Ruhr
2010


































Referent: Professor Dr. M. T. Reetz
Korreferent: Professor Dr. M. Feigel


Tag der mündlichen Prüfung: 06.08.2010





















‘Die Neugier steht immer an erster Stelle eines Problems, das gelöst werden will.’

Galileo Galilei










































Parts of this thesis were published:

Bougioukou, D. J.; Kille, S.; Taglieber, A.; Reetz, M. T., directed evolution of an Enantioselective Enoate-
Reductase: Testing the Utility of Iterative Saturation Mutagenesis. Adv. Synth. Catal. 2009, 351 (18), 3287-
3305.

Sanchis, J.; Fernandez, L.; Carballeira, J. D.; Drone, J.; Gumulya, Y.; Hobenreich, H.; Kahakeaw, D.; Kille, S.;
Lohmer, R.; Peyralans, J. J.; Podtetenieff, J.; Prasad, S.; Soni, P.; Taglieber, A.; Wu, S.; Zilly, F. E.; Reetz, M. T.,
Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution:
application to difficult-to-amplify templates. Appl. Microbiol. Biotechnol. 2008, 81 (2), 387-97.



Copyright of the figures belongs to the author of the thesis, if not mentioned otherwise.

Danksagung
Meinem Docktorvater danke ich für die faszinierenden und herausfordernden Projekte und die gewährte
Freiheit bei dessen Durchführung, sowie für die Unterstützung bei fachlichen und darüber
hinausgehenden Fragen und Problemen.

Ich danke Herrn Prof. Dr. M. Feigel für die Übernahme des Korreferats sowie Herrn Prof. Dr. M. Muhler für
die Abnahme der Nebenfachprüfung.

Ich danke Frau Rathofer, Frau Enk und Frau Lohmer für die administrative Unterstützung.

Danke Hermi für deine geduldigen Unterweisungen in alle möglichen technischen Geräte (GC, Tecan
Roboter usw.) und für deine Hilfsbereitschaft bei Computer- und sonstigen Problemen. Danke Petra, für
die Synthese von 1b-e und 3b. Und Danke an das Team der Kaffeerunde (Gerlinde, Piwi, Hermi, Frederik,
Horst, Andreas und Felipe).

Mein besonderer Dank geht an die beiden Chromatographie-Abteilungen im Hause, ohne deren Hilfe diese
Arbeit nicht möglich gewesen wäre. Frau Rosentreter (GC) danke ich für die akribische Bestimmung
unzähliger ee-Messungen. Herrn Deege (HPLC) für die geduldige Trennung meiner Progesterone Proben.
Mein besonderer Dank gebührt Heike (HPLC) für die schnelle Messung von weit über 20.000
Steroidproben und ihrer unendlichen Geduld, wenn ich mal wieder Sonderwünsche hatte.

I also would like to thank all the current and former group members, who I got to know during my three
years in Mülheim. It was a great experience to meet and to get known to all of you and your cultures.

I would like to thank A. Taglieber and J. Sanchis for their introduction to the YqjM project and into DE in
general. Thank you Despina for your input during the cooperation on the YqjM project and your endlessly
literature knowledge.

Besonderer Dank geht an Felipe, für das an Bord holen ins P450 Projekt. Vielen Dank Felipe für die
perfekte Zusammenarbeit, die ausführlichen Diskussionen und die vielen lustigen und langen Stunden an
der Laborbank bei dessen Umsetzung.




Zu guter Letzt danke ich all meinen Freunden, meiner Familie und besonders Horst für die moralische
Unterstützung und den Glauben an mich, besonders als ich ihn dringend benötigte. Ohne eure
Unterstützung hätte es diese Arbeit nicht gegeben.
1
Table of Contents
Table of Contents
TABLE OF CONTENTS ...................................................................................................................... 1
I INTRODUCTION .............................................................................................................................. 6
1 BIOCATALYSIS ............................................................................................................................ 6
2 ENZYME DESIGN ........................................................................................................................ 7
2.1 General ..................................................................................................................................................................... 7
2.2 Rational and random genetic diversity creating techniques ............................................................... 7
2.3 Directed evolution ............................................................................................................................................... 8
2.3.1 CASTing and ISM methodology ..................................................................................................................................... 9
2.3.2 The ‘Numbers Problem’ ................................................................................................................................................. 10
2.3.3 Protein fitness landscape .............................................................................................................................................. 11
3 THE OLD YELLOW ENZYME FAMILY ................................................................................ 12
3.1 General .................................................................................................................................................................. 12
3.2 Structure ............................................................................................................................................................... 13
3.3 Catalytic mechanism of OYEs ........................................................................................................................ 14
3.3.1 The reductive half reaction and cofactor preference ....................................................................................... 15
3.3.2 The oxidative half reaction .......................................................................................................................................... 16
3.3.3 Enzyme catalyzed H-tunneling reactions .............................................................................................................. 17
4 CYTOCHROME P450 MONOOXYGENASES ...................................................................... 18
4.1 General .................................................................................................................................................................. 18
4.2 The catalytic cycle of Cytochrome P450’s ................................................................................................ 18
4.3 Structure and Organization ........................................................................................................................... 19
4.4 The CYP102A - family ....................................................................................................................................... 23
4.4.1 CYP102A1 – P450 ...................................................................................................................................................... 24 BM32
Table of Contents
5 THESIS GOALS ......................................................................................................................... 25
II RESULTS AND DISCUSSION ..................................................................................................... 26
6 DIRECTED EVOLUTION OF YQJM ...................................................................................... 26
6.1 Directed evolution strategy ........................................................................................................................... 26
6.2 Directed evolution of YqjM ............................................................................................................................ 27
6.2.1 ‘Always the best’ ............................................................................................................................................................... 27
st nd6.2.2 ‘1 and 2 best hit’ .......................................................................................................................................................... 29
st6.2.3 Combination of 1 generation mutations .............................................................................................................. 31
6.3 Substrate scope of mutants ............................................................................................................................ 33
6.4 A discussion about the possible origin of enantioselectivity in OYEs ............................................ 35
6.5 Preparative scale bioreduction of α/β-unsaturated enones ............................................................. 37
7 DIRECTED EVOLUTION OF P450 ................................................................................ 40 BM3
7.1 Directed evolution strategy for P450 ................................................................................................... 40 BM3
7.2 Construction of pETM11-BM3 and mutant F87A ................................................................................... 42
7.3 Library creation ................................................................................................................................................. 43
7.3.1 Traditional PCR: QuikChange™, Kirsch+Joly, Zheng et al. .............................................................................. 44
7.3.2 Saturation mutagenesis for difficult-to-amplify templates ........................................................................... 45
7.3.3 Further optimization efforts ....................................................................................................................................... 48
7.3.4 Comparison of PCR generated and synthesized libraries .............................................................................. 54
7.4 Screening platforms for directed evolution of P450 ...................................................................... 62 BM3
7.4.1 Lysate based screening platform for ethyl 2-phenylacetate screening ................................................... 62
7.4.2 Resting-cells based screening platform for P450 libraries ..................................................................... 67 BM3
7.4.2.1 Detection of aromatic α-hydroxyketones by TTC-Assay ..................................................................... 68
7.4.2.2 Detection of Steroids by HPLC ......................................................................................................................... 71
7.5 Iterative CASTing applied to P450 ........................................................................................................ 72 BM3
7.5.1 Steroid hydroxylation by P450s ................................................................................................................................ 72
7.5.2 Saturation of position 87 in P450 ....................................................................................................................... 73 BM3
st7.5.3 1 generation of P450 evolution towards selective steroid hydroxylation ..................................... 74 BM3
7.5.4 The best of the best: a summary. ............................................................................................................................... 82
nd7.5.5 2 generation P450 evolution. ............................................................................................................................ 84 BM33
Table of Contents
7.5.6 Single point libraries ....................................................................................................................................................... 85
7.5.7 Hit characterization with progesterone and β-estradiol ................................................................................ 87
7.5.8 Docking of steroids into P450 -F87A: A possible clue for the source of regioselectivity ........... 90 BM3
7.5.9 A closing discussion ........................................................................................................................................................ 93
III SUMMARY AND OUTLOOK ..................................................................................................... 95
IV MATERIALS AND METHODS .................................................................................................. 98
8 MICROORGANISMS, VECTORS, PLASMIDS, AND PRIMERS ....................................... 98
8.1 Microorganisms ................................................................................................................................................. 98
8.2 Vectors and Plasmids ....................................................................................................................................... 98
8.3 Primers .................................................................................................................................................................. 98
9 NUTRITION MEDIA, BUFFERS AND SOLUTIONS ........................................................ 100
9.1 Nutrition Medias ..............................................................................................................................................100
9.2 Media Additives................................................................................................................................................100
9.3 Buffers and Solutions .....................................................................................................................................101
10 GENERAL METHODS ........................................................................................................ 101
10.1 Isolation of plasmid DNA from E. coli.......................................................................................................101
10.2 Agarose gel electrophoresis ........................................................................................................................101
10.3 DNA Purification ..............................................................................................................................................102
10.4 Preparation of electrocompetent cells ....................................................................................................102
10.5 Transformation of E. coli Cells ....................................................................................................................102
10.6 Storage of E. coli strains ................................................................................................................................102
10.7 Library Quality Control .................................................................................................................................103
11 YQJM PROTOCOLS ............................................................................................................ 103
11.1 YqjM mutagenesis ...........................................................................................................................................103 4
Table of Contents
11.2 Library screening ............................................................................................................................................103
11.3 Low-load cell mass biotransformation ....................................................................................................104
11.4 Conditions for YqjM Trees ............................................................................................................................104
11.5 Protein Isolation of YqjM wild-type and mutants ...............................................................................105
11.5.1 Affinity chromatography ...................................................................................................................................... 105
11.5.2 Ion exchange chromatography .......................................................................................................................... 105
11.6 Scale-Up Bioreductions .................................................................................................................................106
11.7 GC analysis .........................................................................................................................................................107
12 P450 PROTOCOLS ...................................................................................................... 108 BM3
12.1 Isolation of genomic DNA from B. megaterium ....................................................................................108
12.2 BM3 amplification from genomic DNA ....................................................................................................108
12.3 Cloning of bm3 and pETM11-BM3 construction ..................................................................................108
12.3.1 Vector preparation .................................................................................................................................................. 108
12.3.2 Insert preparation ................................................................................................................................................... 109
12.3.3 Ligation and Transformation .............................................................................................................................. 109
12.3.4 Introducing F87A into pETM11-BM3 ............................................................................................................. 110
12.4 Standardized PCR protocols: comparison of QC, Kirsch-Joly, Zheng et al. and ‘improved
method’ for chapter 7.3.1and 7.3.2. ........................................................................................................................110
12.5 Improved PCR for difficult templates, optimized protocol ..............................................................112
12.5.1 cyp102A1 optimized library creation: one pot PCR ................................................................................. 112
12.5.2 cyp102A1 megaprimer approach: two pot PCR ......................................................................................... 112
12.5.3 Primer combinations and annealing temperatures.................................................................................. 113
12.5.4 QC with pETM11-BM3 template ....................................................................................................................... 114
12.6 Sloning library creation ................................................................................................................................114
12.7 Library screening ............................................................................................................................................115
12.7.1 Resting-cells process for steroid screening ................................................................................................. 115
12.7.1.1 Expression and enzymatic reaction ...................................................................................................... 115
12.7.1.2 Plate preparation for HPLC analysis ..................................................................................................... 115
12.7.1.3 Screening of Lib C-NNC with Deoxybenzoin ..................................................................................... 115
12.7.2 Lysate based screening platform ...................................................................................................................... 116
12.7.2.1 Screening of Lib C-NNC with ethyl 2-phenylacetate ..................................................................... 116 5
Table of Contents
12.8 10-pNCA Assay ..................................................................................................................................................116
12.9 Indigo formation ..............................................................................................................................................117
12.10 High-load cell mass biotransformation ..............................................................................................117
12.11 Scale – Up Biooxidation ............................................................................................................................117
12.12 TLC analysis of hydroxylated steroid products ...............................................................................118
12.13 HPLC analysis of hydroxylated steroids .............................................................................................118
12.14 HPLC/MS analysis .......................................................................................................................................119
12.15 HPLC purification of hydroxylated progesterones ........................................................................120
12.16 NMR analysis of hydroxylated progesterons....................................................................................120
V ABBREVIATIONS AND ACRONYMS ..................................................................................... 126
VII SUPPLEMENTARY ................................................................................................................. 127
VI REFERENCES ............................................................................................................................. 132

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