Directed Evolution of DNA Polymerases for Advancement of the SeSaM Mutagenesis Method and Biotransformations with P450 BM3 Monooxygenase 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 Master of Science (MSc) Molecular Life Science Tsvetan Dinkov Kardashliev aus Stara Zagora, Bulgarien Berichter: Universitätsprofessor Dr. rer. nat. Ulrich Schwaneberg Universitätsprofessor Dr. rer. nat. Lothar Elling Tag der mündlichen Prüfung: 29. Januar 2015 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar Abstract ABSTRACT Directed evolution is a powerful algorithm to tailor proteins to needs and requirements in industry. Error-prone PCR (epPCR) based methods are the “golden standard” in random mutagenesis due to their robustness and simplicity. Despite their wide use in protein engineering experiments, epPCR methods are limited in their ability to generate highly diverse mutant libraries. This is due to 4 reasons – 1) the redundancy of the genetic code, 2) the low mutagenic frequency and lack of subsequent nucleotide exchanges in a standard epPCR library, 3) the tendency of polymerases to introduce mutations preferentially in certain DNA sequence contexts and 4) the innate transition bias of DNA polymerases leading to conservative amino acid exchanges. Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method that has been developed to overcome the aforementioned limitations. SeSaM complements the mutagenic spectrum of epPCR by introducing transversions and consecutive nucleotide substitutions. Indeed, SeSaM libraries enriched in consecutive nucleotide subsitutions and transversion mutations were reported; however the frequency of occurrence of such mutations remains low. In particular, the fraction of consecutive transversion mutations accounted for a meager 4.6 %. The increase of the fraction of consecutive transversion mutations is critical in order to advance the SeSaM technology and access previously unattainable sequence space. This can be achieved by the use of DNA polymerases adapted to the requirements of the SeSaM method. The biggest challenge is posed in SeSaM step 3 in which the employed DNA polymerase must be able to elongate consecutive transversion mismatches formed between degenerate base analogs in the primer strand and standard nucleobases in the template strand. The most feasible solution to this problem is to engineer a DNA polymerase capable of efficient consecutive transversion mismatch elongation. The latter has been the main objective pursued in CHAPTER I of this doctoral thesis. The work towards fulfilling this goal included identification from genetic databases of 4 potential candidates from the Y-family of DNA polymerases (exclusively involved in translesion DNA synthesis), followed by expression and preliminary biochemical characterization of the putative polymerases, and, finally, selection of one candidate (Dpo4 from Sulfolobus solfataricus) for directed evolution. The protein engineering work comprised development of a novel high-throughput screening system for non-processive DNA polymerases, screening and identification of Dpo4 variants capable of 1 Abstract consecutive mismatch elongation. Finally, the most promising polymerase mutant was used in the preparation of a model SeSaM library. Direct comparison to data generated using an earlier version of the SeSaM protocol indicated a marked improvement in frequency of consecutive transversion mutations (relative increase of 40 %) in the libraries prepared with the identified Dpo4 polymerase mutant. The identified polymerase variant enabled a significant advancement in consecutive transversion generation and, consequently, of the SeSaM random mutagenesis method. In CHAPTER II of this dissertation, P450 BM3 monooxygenase was studied in the context of regioselective biotransformations of benzenes with isolated enzyme (P450 Project I) and as a whole cell catalyst (P450 Project II). P450 BM3 is a promising biocatalyst which enables challenging chemical reactions, e.g., insertion of an oxygen atom into a non-activated C-H bond. In P450 Project I, P450 BM3 in purified form was employed in the synthesis of mono- and di-hydroxylated products from six monosubstituted benzene substrates. A P450 BM3 mutant (P450 BM3 M2 (R47S/Y51W/A330F) proved to be promiscuous and highly regioselective (95 % - 99 %) hydroxylation catalyst achieving good overall yields (up to 50 % with benzenes and ≥90 % with phenols). The developed process showed promising potential for sustainable synthesis on a semi-preparative scale of valuable chemical precursors, i.e., phenols and hydroquinones, even prior to bioprocess optimizations. Nevertheless, productivity, especially with non-halogenated substrates, would need to be further improved. Especially, whole cell cofactor regeneration systems have to be developed in order to bring this attractive synthesis route closer to industrial exploitation. The issue of cofactor regeneration served as a stimulus to initiate follow-up project dealing with whole cell catalysis with P450 BM3. The requirement for expensive reduced cofactor (NAD(P)H) in equimolar amounts is a major disadvantage of P450 BM3 preventing its wider use in organic synthesis. The NAD(P)H dependency of P450 BM3 necessitates the use of whole cells in preparative synthesis in order to achieve cost-efficient cofactor regeneration. However, the semi-permeable nature of the outer membrane of industrially relevant bacteria such as E. coli limits their use as whole cell systems. The issue of substrate permeability in whole-cell biocatalysts has been addressed in this thesis by co-expressing a large passive diffusion channel, FhuA Δ1-160, in the outer membrane of E. coli. The influence of the channel protein on P450 BM3-catalyzed conversions of 2 monosubstituted benzenes has been investigated in P450 Project II. Preliminary experiments indicated that the co-expression in the outer E. coli 2 Abstract membrane of FhuA Δ1-160 had a positive effect, possibly of global nature, on the mass transfer in whole cell biotransformations (up to 10-fold relative increase of product titers). The presented drawback of whole-cell biocatalysis has not been extensively addressed to date, and with these preliminary and promising first results, we hope to entice further interest in the topic. In summary, this doctoral thesis addresses three questions from the related fields of protein engineering and biocatalysis: 1) Advancement of diversity generation methods in directed protein evolution; 2) Establishing biocatalytic process for oxy-functionalization of generally unreactive aromatic C-H bonds; 3) Developing a strategy to improve the mass transfer across the outer membrane of E. coli in whole cell oxy-functionalization of aromatic compounds. 3 Publications PUBLICATIONS Published: 1) Kardashliev T., Ruff A. J., Zhao J., Schwaneberg U., “A High-Throughput Screening Method to Reengineer DNA Polymerases for Random Mutagenesis”, 2014, Molecular Biotechnology, 56(3):274-83 2) Ruff A. J., Kardashliev T., Dennig A., Schwaneberg U. “The Sequence Saturation Mutagenesis (SeSaM) method”, 2014, Methods in Molecular Biology Vol. 1179: Directed Evolution Library Creation 2nd ed: 45-68 3) Zhao J., Kardashliev T., Ruff A. J., Bocola M., Schwaneberg U., “Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations”, 2014, Biotechnology and Bioengineering, 111: 2380-2389 4) Cheng F., Kardashliev T., Pitzler C., Shehzad A., Lue H, Bernhagen J., Zhu L., Schwaneberg U., “A competitive flow cytometry screening system for arginine-metabolizing enzyme in cancer treatment”, 2015, ACS Synthetic Biology, DOI: 10.1021/sb500343g Presented as posters and oral presentations: 5) Kardashliev T., Ruff A. J., Schwaneberg U., “Engineering of DNA polymerases for applications in random mutagenesis”, Biotrans 2013 International Conference, Manchester, UK, poster presentation. 6) Kardashliev T., Ruff A. J., Schwaneberg U., “A high-throughput screening system for engineering of DNA polymerases”, IBN 2013 International Conference, Hamburg, Germany, poster presentation. 7) Kardashliev T., “Advancement of Sequence Saturation Mutagenesis (SeSaM) method for genetic diversity generation”, Biokatalyse2021 Scientific Cluster Conference, Hamburg, Germany, oral presentation. Planned for publication: 8) Kardashliev T., Dennig A., Halmschlag B., Ruff AJ., Schwaneberg U. “One step synthesis of hydroquinones from benzenes with a single cytochrome P450 BM3 variant”, 2015, submitted to Advanced Synthesis & Catalysis (in Feb 2015). 9) Kardashliev T., Dennig A., Arlt M., Ruff AJ., Schwaneberg U. “Improving the mass transfer in whole-cell biocatalysis by expression of a passive diffusion channel in the outer membrane of E. coli”, 2015 4 Table of Contents TABLE OF CONTENTS ABSTRACT ................................................................................................................................................ 1 PUBLICATIONS ......................................................................................................................................... 4 TABLE OF CONTENTS ............................................................................................................................... 5 CHAPTER I. Engineering of DNA Polymerases for Application in Directed Protein Evolution ................. 8 1. Introduction