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An Enzyme-Based Process for the Production of Long-Chain Α,Ω-Dicarboxylic Acids

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A CYP solution: an enzyme-based process for the production of long-chain α,ω-dicarboxylic acids Ir. Delphine Devriese Supervisor: Prof. Dr. Bart Devreese Academic year 2020–2021 A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Doctor of Sciences: Biochemistry and Biotechnology Supervisor Prof. Dr. Bart Devreese Laboratory of Microbiology – Protein Research Unit, Ghent University Examination committee Prof. Dr. Savvas Savvides (Chair) VIB-UGent Center for Inflammation Research Dr. Kenneth Verstraete (Secretary) VIB-UGent Center for Inflammation Research Prof. Dr. ir. Inge Van Bogaert Centre for Synthetic Biology (CSB), Ghent University Prof. Dr. Serge Tavernier Intelligence in Processes, Advanced Catalysts & Solvents (iPRACS), University of Antwerp Dr. Lina De Smet Laboratory of Molecular Entomology and Honeybee Pathology (L-MEB), Ghent University This dissertation was part of the project Enzymes for Added Sustainability and Efficiency (EnzymASE). The project was funded by The Flemish Agency for Innovation and Entrepreneurship (VLAIO) and supported by Catalisti. A CYP solution: an enzyme-based process for the production of long-chain α,ω-dicarboxylic acids Ir. Delphine Devriese Supervisor: Prof. Dr. Bart Devreese Academic year 2020–2021 A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Doctor of Sciences: Biochemistry and Biotechnology The author and the supervisors give the authorization to consult and copy parts of this manuscript for personal use only. Any other use is limited by the laws of copyright, especially the obligation to refer to the source whenever results from this manuscript are cited. Ghent Author Supervisor Ir. Delphine Devriese Prof. Dr. Bart Devreese II Dankwoord De afgelopen vier jaar kreeg ik de kans om een doctoraatsonderzoek uit te voeren in de PRU onderzoeksgroep van het labo microbiologie, voormalig het L-PROBE labo. Ik heb mezelf hier kunnen ontwikkelen als onderzoeker maar ook daarbuiten leerde ik veel bij. Dit alles was niet mogelijk geweest zonder de volgende mensen en bij deze wil ik hen ook bedanken. Allereerst wil ik mijn promotor professor Bart Devreese bedanken. Iets meer dan vier jaar geleden botste ik op een vacature bij het toenmalige L-PROBE labo betreffende het onderzoek van enzymen voor de chemische industrie. Vanwege mijn interesse in de eiwitchemie, solliciteerde ik voor dit doctoraat en werd ik tot mijn vreugde aangenomen. Niet alleen wil ik mijn promotor bedanken om mij de kans te geven voor het uitvoeren van dit doctoraat, ook kon ik afgelopen vier jaar rekenen op een aangename samenwerking waarbij de deur ook vaak letterlijk openstond voor ons. In het labo kon ik altijd terugvallen op mijn collega’s. Stijn wil ik bedanken om mij bij de start van mijn doctoraat te begeleiden en mij te introduceren in het labo alsook in het EnzymASE project. Isabel verdient hier ook zeker een dankjewel. Als lab manager kon ik steeds op haar rekenen voor haar raad en daad met betrekking tot veiligheid, producten, toestellen en meer. Daarnaast was ze mee betrokken in EnzymASE en kon ik bij haar terecht voor het bediscussiëren van dit project. Steeds als ik iets meer wilde weten of hulp nodig had bij massa spectrometrie, kon ik aankloppen bij Sören. Voor extra informatie in verband met de membraanvesikels kon ik dan weer rekenen op Jolien Vitse. Ook mijn andere collega’s wil ik graag bedanken, namelijk Ingrid, Zhoujian en Gonzalez, alsook ex-collega’s Jolien Claeys, Sofie en Kasia en nieuwe collega’s Amir, Amaury en Eva. Iedereen droeg en draagt bij aan een aangename sfeer in het labo, van een babbeltje tussendoor en tijdens de lunch, tot de jaarlijkse teambuilding, waardoor ik steeds met plezier naar het labo kwam. Doorheen mijn doctoraat kon ik ook rekenen op mensen buiten het PRU labo. Zo wil ik Jordy Bauwelinck en Pieter Surmont bedanken voor het uitvoeren van de GC-MS analyses. Om mijn membraanvesikels te analyseren, kon ik gebruik maken van de NanoSight toestellen, beschikbaar in het labo experimenteel kankeronderzoek in het UZ Gent en in het labo algemene biochemie en fysische farmacie aan de faculteit farmaceutische wetenschappen. Daarbij wil ik Lien Lippens en Herlinde De Keersmaecker, respectievelijk, in het bijzonder bedanken voor de introductie van het NanoSight toestel. Ik wil van de gelegenheid gebruik maken om mijn examen commissie te bedanken voor de kritische analyse van mijn doctoraat en de behulpzame suggesties die gegeven werden om deze dissertatie te maken tot wat ik hier nu mag afleveren. III Afgelopen vier jaar kon ik rekenen op mijn familie en vrienden en kon ik er ook steeds terecht voor een ontspannend momentje. Ik zou hierbij graag mijn ouders bedanken voor hun steun doorheen de jaren. Daarnaast verdient mijn zus Joke hier zeker een grote dankjewel. Ook zij legde een doctoraat af. Ze wist me zo steeds bij te staan met raad en daad en als er nood was aan ontspanning, stond ze alvast klaar met haar gezelschapsspelen. Als laatste had ik mijn vriend Toon willen bedanken. Doorheen de afgelopen jaren heeft hij altijd een luisterend oor geboden en klaargestaan voor mij. Hij wist me steeds te motiveren en kon mij ook tot rust brengen bij stress momenten. Dankjewel iedereen! Delphine IV Summary α,ω-Dicarboxylic acids (DCAs) are valuable chemical compounds and are used in a wide variety of applications. They serve as monomers for polyamides such as nylons and polyesters. Moreover, these compounds are used as precursors for pharmaceuticals, perfumes, lubricants, adhesives, plasticizers, powder coatings and corrosion inhibitors. Today, mainly short- and medium-chain DCAs are used. The application of DCAs with longer chain lengths (C > 14) is less widespread due their lower availability and elevated costs. This is the result of their challenging production process. On the one hand, long-chain α,ω-DCAs are produced through chemical conversion of fatty acids by means of metathesis. On the other hand, a fermentation process with the industrial DCA producer Candida viswanathii is established. Unfortunately, both processes come with a number of disadvantages and there is a need for an alternative long- chain α,ω-DCA production process for increased availability and decreased prices. Cytochrome P450 monooxygenases (CYP) constitute a large superfamily of enzymes, performing a wide variety of reactions and accepting a diverse range of substrates. These enzymes have the ability to introduce one oxygen atom from molecular oxygen in a non- activated C-H bond, while reducing the other oxygen atom to water. This is a reaction difficult to achieve by conventional chemical routes. Therefore, CYPs are considered of great value in synthetic biology applications. Unfortunately, disadvantages such as low specific activity, the need for a redox partner and the cofactor NAD(P)H limits their use, and current application is restricted to whole-cell bioconversions. A number of CYP families have been reported to hydroxylate fatty acid substrates at the ω-position to their corresponding ω-hydroxy fatty acids. In some cases, overoxidation to the α,ω-DCAs was observed. One of these enzymes was selected for the development of an in vitro biocatalytic process, producing α,ω-DCAs from fatty acid substrates, i.e. CYP52A13 from the industrial DCA producer C. viswanathii. An in vitro biocatalytic process was pursued due to its many advantages over the currently used processes. No rare metal catalysts are needed, the enzyme performs its conversion at ambient temperature in mild reaction conditions, no side reactions occur due to absence of other enzymes and no cell membrane needs to be crossed, especially important with respect to the desired long-chain DCAs. Moreover, a biocatalytic process is easier to implement in the chemical industry as fermentation requires specific know-how and specialized equipment. In the first experimental chapter (chapter 3) it was investigated whether it would be feasible to create a soluble self-sufficient enzyme for the implementation in an enzyme reactor. CYP52A13 belongs to the class II CYP enzymes, meaning it is a membrane-bound protein, requiring the redox partner CYP reductase (CPR), which is also membrane-bound. As a soluble system was of interest due to its ease of implementation in a reactor system, both enzymes were truncated in order to remove their membrane anchor. Pichia pastoris was chosen as this fungal host offers many advantages towards industrial protein production. For example, little endogenous protein is secreted by this recombinant host. Producing CYP and CPR in a secreted V form was therefore pursued as this should greatly facilitate downstream processing. Furthermore, it was of interest to create a chimeric construct, where both redox partners were present in the same polypeptide chain. In this way, only one enzyme instead of two needs to be produced and fusion of the redox partners was reported to enhance coupling efficiency. For the optimization of this construct, a nontruncated variant was produced in the same recombinant host. Both truncated CYP and CPR were successfully produced using an artificial N-terminal tag, replacing the N-terminal membrane anchor. Even more, this N-tag enabled secretion to the medium for both enzymes. Unfortunately, activity could only be proven for CPR. A chimer was successfully produced but not secreted in P. pastoris and the chimer was retained in the microsomal membrane. However, the artificial N-tag proved to be valuable still. The chimer could be isolated in a soluble form in absence of detergents by washing the microsome with a high salt buffer. Furthermore, the N-tag improved the production yield of this class II CYP compared to the nontruncated form, which was only produced to a low level. In the following chapter (chapter 4), an in vivo immobilization strategy was considered in order to enhance stability of the CYP by introduction in a membrane environment, while still retaining the advantages of an in vitro approach.
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  • Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites

    Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites

    Int. J. Mol. Sci. 2012, 13, 15901-15924; doi:10.3390/ijms131215901 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites Giovanna Di Nardo and Gianfranco Gilardi * Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +39-011-670-4593; Fax: +39-011-670-4643. Received: 27 September 2012; in revised form: 1 November 2012 / Accepted: 13 November 2012 / Published: 28 November 2012 Abstract: Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may be oxidized by more than one enzyme. As a result, multiple products may be obtained from the same drug, and as the metabolites can be biologically active and may cause adverse drug reactions (ADRs), the metabolic profile of a new drug has to be known before this can be commercialized. Therefore, the metabolites of a certain drug must be identified, synthesized and tested for toxicity. Their synthesis must be in sufficient quantities to be used for metabolic tests. This review focuses on the progresses done in the field of the optimization of a bacterial self-sufficient and efficient cytochrome P450, P450 BM3 from Bacillus megaterium, used for the production of metabolites of human enzymes.