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Engineering of Hyaluronic Acid Synthases from Streptococcus Equi Subsp

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Engineering of Hyaluronic Acid Synthases from Streptococcus Equi Subsp Engineering of Hyaluronic Acid Synthases from Streptococcus equi subsp. zooepidemicus and Pasteurella multocida Towards Improved HA Chain Length and Titer 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 John Cyrus Baltazar Mandawe, MSc aus Paraῆaque, Philippinen Berichter: Universitätsprofessor Dr. rer. nat. Ulrich Schwaneberg Universitätsprofessor Dr.-Ing. Lars M. Blank Tag der mündlichen Prüfung: 31.Oktober 2018 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek verfügbar. I "It is not the critic who counts; not the man who points out how the strong man stumbles, or where the doer of deeds could have done them better. The credit belongs to the man who is actually in the arena, whose face is marred by dust and sweat and blood; who strives valiantly; who errs, who comes short again and again, because there is no effort without error and shortcoming; but who does actually strive to do the deeds; who knows great enthusiasms, the great devotions; who spends himself in a worthy cause; who at the best knows in the end the triumph of high achievement, and who at the worst, if he fails, at least fails while daring greatly, so that his place shall never be with those cold and timid souls who neither know victory nor defeat." -Theodore Roosevelt II Acknowledgements “I've learned that people will forget what you said, people will forget what you did, but people will never forget how you made them feel” - Maya Angelou My deepest gratitude to Prof. Dr. Ulrich Schwaneberg for allowing me the opportunity to pursue my PhD studies. I have learned and grown so much because of you. My sincere appreciation to Prof. Dr. Lars Blank and Prof. Dr. Lothar Elling as supportive project collaborators and as members of my dissertation committee. I also extend my gratitude to Prof. Dr. Uwe Conrath as the chairperson of my dissertation committee. I acknowledge the Deutsche Bundesstiftung Umwelt (DBU) for the financial support from 2013-2016. Very special thanks to SeSaM-Biotech for giving me the opportunity to sustain myself for the last several months of my doctoral studies so that I could bring all these efforts to fruition. I acknowledge those who contributed to the pmHAS KnowVolution publication: Dr. Belen Infanzon, Anna Eisele, Henning Zaun, Dr. Jürgen Kuballa, Dr. Mehdi D. Davari, Dr. Felix Jakob, Prof. Dr. Lothar Elling and Prof. Dr Ulrich Schwaneberg. Also, special thanks to Shohana Islam for the PLICing primers and JARA-HPC from RWTH Aachen University under projects JARA0169 for granting computer simulations resources. Thank you as well to Dr. Gaurao Dhoke for providing assistance with the homology model manipulations for the ChemBioChem cover page. I thank Dr. Felix Jakob for the supervision and assistance and the members of the Biohybrid subgroup for the scientific discussions. I would like to thank every single member of the Schwaneberg group, not only for their technical and administrative support, but also for being amazing colleagues. Thank you to Dr. Kristin Rübsam for the help with confocal microscopy. I also extend my special appreciation to Shohana Islam, Patrizia Pazdzior and Dr. Juliana Kurniadi for their support and kindness during the stormy days. I ingrain this indebtedness to my memory. My sincerest thanks to my brothers Paul, George and Ringo (“The Beatles”), and my families and friends in Canada and Germany for their support, inspiration and encouragement throughout the years. Tim, Swanny, Mom Astrid and Anne, thank you for welcoming me into your respective families and for your kindness, care and thoughtfulness over the years. I highly appreciate you. Finally, I dedicate this PhD to my parents, Maria Lucila and Alvin, who gave up their education to raise their “Beatles”. Thank you for all the sacrifices you have made for us and for your endless love and support. You have instilled in me the values of hard work, diligence, perseverance, independence, responsibility, integrity and respect. I offer you this doctoral degree. I keep the life lessons. Todo para la familia! III Publications Parts of this thesis have been published: Mandawe J, Infanzon B, Eisele A, Zaun H, Kuballa J, Davari MD, et al. Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida Towards High Molecular Weight Hyaluronic Acid. ChemBioChem. 2018;19:1414-23. Parts of this thesis will be published: Mandawe J*, Anand D*, Jakob F, Zaun H, Kuballa H, Schwaneberg U. A facile and inexpensive toolbox for differential hyaluronic acid synthesis: From kilodalton to megadalton scale (manuscript in preparation). *shared first authorship Other publications: Vargas WA, Mandawe J and Kenerley CM. Plant-derived sucrose is a key element in the symbiotic association between Trichoderma virens and maize plants. Plant Physiol. 2009;151:792-808. Jakob F, Martinez R, Mandawe J, Hellmuth H, Siegert P, Maurer KH, Schwaneberg U. Surface charge engineering of a Bacillus gibsonii subtilisin protease. Appl Microbiol Biotechnol. 2012;97:6793-6802. IV Abstract Hyaluronan (hyaluronic acid; HA) is the non-sulfated glycosaminoglycan product of HA synthases that can vary in length from 103-107 Daltons. Depending on polymer length, concentration and localization, HA possesses variable physicochemical properties that serve useful in pharmaceutical, biomedical and cosmetic applications worth billions ($) in commercial valuation worldwide. A deeper molecular understanding of the control of HA polymerization by the synthase machinery can, therefore, facilitate the tuned production of HA, according to the intended application. The general objective of this doctoral investigation, therefore, is to implement protein engineering principles to Class I and Class II HA synthases, with supplication of computational modeling, to improve HA production by means of polymer length and quantity. The Class I HAS from Streptococcus equi subsp. zooepidemicus (seHAS) was first subjected to protein engineering. seHAS was recombinantly expressed in three microbial hosts (E. coli, B. subtilis and S. cerevisiae) and could direct HA synthesis in all three hosts. E. coli was selected for enzyme engineering due to easy handling and quick doubling time. Fluorescent epitope tagging confirmed the presence of seHAS and its localization in the outer membrane of the Gram-negative host, contrary to the inner membrane reported in literature. Of the many screening systems attempted to be established, the agarose gel electrophoresis screening platform was the most reliable and could discriminate between empty vector controls, wild type and seHAS variants. Screening of the site-saturation mutagenesis libraries (of the conserved Cys226, Cys262, and Cys281 and polar membrane residues Lys48 and Glu327) and one random mutagenesis library (1392 error-prone PCR variants) failed to identify one seHAS variant with improved chain length specificity. However, alternative positive results were discovered. Site-saturation mutagenesis variants (K48L and K48E) produced consistently monodispersed low molecular weight (LMW; < 0.5 MDa) HA products, while epPCR variants H2 (N345S/F403L) and A6 (R347S/F362S) produced high molecular weight (HMW; >1 MDa) HA with polydispersity lower than that of seHAS- WT. Homology model analysis hinted at the potential role of HA-HAS interaction in the control of HA polymerization. The discovery of these new positions bifurcates into another dimension of HA, which is chain polydispersity. A better understanding of these product-enzyme interactions can provide clues for production of monodispersed HA. The second protein engineering campaign involved the Class II HAS from Pasteurella multocida (pmHAS). The knowledge-gaining directed evolution (KnowVolution) approach successfully improved the enzymatic activity of the membrane-associated pmHAS. Two screening systems were simultaneously employed to detect improvements in enzymatic output: agarose gel electrophoresis for chain length and the CTAB turbidimetric assay for HA titer. With CTAB, absorbance values of HA synthesized by pmHAS-expressing E. coli BL21 GOLD (DE3) cells were at least 5-fold higher than that V of the baseline (empty vector control). Through KnowVolution, seven improved epPCR variants out of 1392 were identified, eight prospective beneficial positions from these variants were saturated and the most beneficial amino acid substitutions (T40L, V59M and T104A) were recombined to generate the final variant (pmHAS-VF). Production of HA up to 4.7 MDa and with a two-fold improvement in mass-based total turnover number over wild type was achieved. This is the first case of a Class II HA synthase directed evolution and an example of a simultaneous dual property improvement resulting from protein engineering. The most complete and validated model to date of pmHAS32-703 was also generated to gain molecular insight into the improved properties. The substitutions in pmHAS-VF are located at the N-terminal domain, away from either glycosyltransferase active sites of pmHAS, suggesting their non-catalytic role. Molecular dynamics simulations reveal the improved flexibility of the N-terminal region allowing it to swing from the GlcNAc-transferase domain to the GlcA-transferase domain. This suggests a newly found importance of the N-terminal domain in HA synthesis. Overall, the ability to synthesize longer HA polymers at higher output brings promise to improved HA production.
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