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Biosynthesis of New Alpha-Bisabolol Derivatives Through a Synthetic Biology Approach Arthur Sarrade-Loucheur

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Biosynthesis of New Alpha-Bisabolol Derivatives Through a Synthetic Biology Approach Arthur Sarrade-Loucheur Biosynthesis of new alpha-bisabolol derivatives through a synthetic biology approach Arthur Sarrade-Loucheur To cite this version: Arthur Sarrade-Loucheur. Biosynthesis of new alpha-bisabolol derivatives through a synthetic biology approach. Biochemistry, Molecular Biology. INSA de Toulouse, 2020. English. NNT : 2020ISAT0003. tel-02976811 HAL Id: tel-02976811 https://tel.archives-ouvertes.fr/tel-02976811 Submitted on 23 Oct 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE En vue de l’obtention du DOCTORAT DE L’UNIVERSITÉ DE TOULOUSE Délivré par l'Institut National des Sciences Appliquées de Toulouse Présentée et soutenue par Arthur SARRADE-LOUCHEUR Le 30 juin 2020 Biosynthèse de nouveaux dérivés de l'α-bisabolol par une approche de biologie synthèse Ecole doctorale : SEVAB - Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingenieries Spécialité : Ingénieries microbienne et enzymatique Unité de recherche : TBI - Toulouse Biotechnology Institute, Bio & Chemical Engineering Thèse dirigée par Gilles TRUAN et Magali REMAUD-SIMEON Jury Mme Véronique DE BERARDINIS, Rapporteure, Chercheur CEA M. Jean-Etienne BASSARD, Rapporteur, Chargé de Recherche, CNRS Mme Danièle WERCK-REICHHART, Examinatrice, Directeur de Recherche Emérite, CNRS M. Gilles TRUAN, Directeur de thèse, Directeur de Recherche, CNRS Mme Magali REMAUD-SIMEON, Co-directrice de thèse, Professeur des Universités, INSA Mme Fayza DABOUSSI, Présidente, Directeur de Recherche INRAE 2 Author: Arthur Sarrade-Loucheur Year: 2020 Biosynthesis of new α-bisabolol derivatives through a synthetic biology approach The rise of synthetic biology now enables the production of new to nature molecules. In the frame of this thesis we focused on the diversification of the (+)-epi-α-bisabolol scaffold. This molecule coming from the plant Lippia dulcis belongs to the vast family of sesquiterpenes. While sesquiterpenes possess diverse biological activities, (+)-epi-α- bisabolol is the precursor of hernandulcin, an intense sweetener. However, the last oxidative step(s) of the hernandulcin biosynthetic pathway remain elusive. Rather than seeking the native oxidase responsible for hernandulcin synthesis among L. dulcis enzymes we turned our choice towards oxidative enzymes known to be promiscuous and that could functionalize (+)-epi-α-bisabolol in order to i) generate diversity from (+)-epi-α-bisabolol; ii) hopefully identify an oxidative enzyme catalyzing hernandulcin synthesis. First, a yeast chassis strain enabling the in vivo screening of cytochromes P450 (CYPs) was constructed by coexpressing two key enzymes: the (+)-epi-α-bisabolol synthase (BBS) and the NADPH cytochrome P450 reductase. Then, the in vivo screening assays revealed that 5 CYPs out of our library of 25 animal CYPs involved in xenobiotic metabolism oxidized (+)-epi-α-bisabolol and produced new hydroxylated regioisomers. Of the oxidized products, the structure of one compound, 14-hydroxy-(+)-epi-α-bisabolol, was fully elucidated by NMR while the probable structure of a second one was determined, 9-hydroxy-(+)-epi-α-bisabolol. In parallel, the production of (+)-epi-α-bisabolol derivatives was enhanced through addition of a supplementary genomic copy of BBS that augmented the final titer of hydroxylated product to 64 mg/L. We thus demonstrate that drug metabolism CYPs can be used to produce novel compounds from a sesquiterpene scaffold. Furthermore, different products were obtained with two homologous enzymes i.e. CYP2B6 and CYP2B11. This prompted us to study the molecular determinants putatively responsible for enzyme regiospecificity. From chimeric enzymes composed of secondary structure elements originating from CYP2B6 and CYP2B11 we were not able to identify specific motifs that could explain the CYPs regiospecificity. This approach suggests that the molecular determinants cannot be attributed to specific structural elements of the two enzymes but are rather widespread in the protein sequences. In order to generate a wider molecular diversity from α-bisabolol or hernandulcin, and synthesize molecules with different physico-chemical and biological properties (i.e. solubility, sweetening power etc.), we attempted the glycosylation of these compounds using a plant glucosyltransferase (UGT93B16) or human glucuronosyltransferases. UGT93B16 was found to glucosylate both (-)-α-bisabolol and hernandulcin. In addition, we carried out whole cell catalysis using E. coli and S. cerevisiae as recombinant producers of UGT93B16. Comparison of the two microbial hosts showed that glycosylation using yeast cells was more efficient. In parallel, we investigated α-bisabolol glucosylation by human UDP-glucuronosyltransferases involved in the xenobiotic metabolism. In vitro enzymatic assays demonstrated a weak activity of UGT1A9, UGT2B4 and UGT2B7 towards α-bisabolol and hernandulcin. However, their introduction in yeast failed to produce detectable amounts of glucuronide products. In summary, we proved the feasibility of producing new to nature sesquiterpene glucosides using either enzyme-based assay or bioconversion in two different hosts that are widely used in biotechnology. To conclude, the approaches used in this thesis highlight the assets of screening promiscuous enzymes for the production of new molecules from α-bisabolol. We also explored the limits of our chassis strain and a tighter regulation of S. cerevisiae metabolism could improve the production of (+)-epi-α-bisabolol oxidized products. Finally, the coupling in yeast of cytochrome P450 and glucosyltransferase steps can now be envisioned. Keywords: Synthetic biology, sesquiterpene, metabolic engineering, cytochrome P450, Saccharomyces cerevisiae, enzymatic promiscuity 3 4 Auteur : Arthur Sarrade-Loucheur Année : 2020 Biosynthèse de nouveaux dérivés de l’α-bisabolol par une approche de biologie de synthèse La biologie de synthèse permet désormais la production de nouvelles molécules n’existant pas dans la nature. Au cours de cette thèse, nous nous sommes focalisés sur la diversification de produits issus du (+)-epi-α-bisabolol. Cette molécule issue de la plante Lippia dulcis appartient à la vaste famille des sesquiterpènes qui recèle de nombreuses activités biologiques. En particulier, le (+)-epi-α-bisabolol est le précurseur de l’hernandulcine, un édulcorant intense. Cependant, la (les) dernière(s) étape(s) d’oxydation conduisant à ce composé demeure(nt) inconnue(s). Plutôt que de rechercher la (ou les enzymes) d’oxydation intervenant dans la voie de biosynthèse de l’hernandulcine chez Lippia dulcis, nous avons choisi de mettre à profit la promiscuité connue de cytochromes P450 pour diversifier les molécules issues du (+)-epi-α-bisabolol ou reproduire la voie de synthèse naturelle. Tout d’abord, une souche châssis adaptée au criblage in vivo a été construite en exprimant la (+)-epi-α-bisabolol synthase (BBS) et une NADPH cytochrome P450 réductase. Puis, le criblage in vivo chez la levure a montré que 5 des cytochromes P450 (CYPs) parmi notre banque de 25 enzymes impliquées dans le métabolisme des xénobiotiques (CYPs animaux) oxydaient le (+)- epi-α-bisabolol. Parmi ces produits, le 14-hydroxy-(+)-epi-α-bisabolol a été purifié et caractérisé par RMN tandis que la structure probable d’un second produit a été obtenue (9-hydroxy-(+)-epi-α-bisabolol). En parallèle, la production de produits hydroxylés (+)-epi-α-bisabolol a été optimisée notamment par l’addition d’une copie génomique supplémentaire de BBS, avec un titre de de produit hydroxylé de 64 mg/L. Ainsi, nous avons démontré le potentiel des CYPs du métabolisme des xénobiotiques dans la synthèse de nouvelles molécules issues des sesquiterpènes. Parmi les enzymes identifiées, deux enzymes homologues, CYP2B6 et CYP2B11 ont catalysé la formation de produits différents à partir du (-)-α-bisabolol et du trans,trans-farnésol. Pour identifier les possibles déterminants moléculaires responsables de la spécificité de chacune de ces deux enzymes, des chimères échangeant divers éléments de structure secondaire de CYP2B6 dans CYP2B11 ont été comparées. Ainsi, la spécificité ne peut pas être expliquée par un seul élément de la structure secondaire mais plus probablement par des déterminants disséminés en divers endroits des séquences protéiques des deux enzymes. Afin d’étendre la diversité de molécules produites à partir de l’α-bisabolol et de l’hernandulcine et produire des composés aux propriétés physico-chimiques améliorées (solubilité, pouvoir sucrant…) des essais de glycosylation par une glucosyltransférase de plante (UGT93B16) et par des glucuronosyltransférases humaines ont été menés. UGT93B16 a démontré son potentiel en glucosylant l’α-bisabolol et l’hernandulcine. La bioconversion de ces molécules en utilisant E. coli et S. cerevisiae a également montré une meilleure conversion chez la levure. Concernant les UDP- glucuronosyltransférases, des essais enzymatiques ont mis en évidence une activité d’UGT1A9, d’UGT2B4 et d’UGT2B7 pour l’α-bisabolol et l’hernandulcine. Toutefois, l’introduction de ces enzymes dans la levure n’a pas
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