Team:Bordeaux/Design

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Design

Recent studies have shown that multiple organisms have the ability of metabolizing 5-hydroxymethylfurfural (HMF). For instance, enzyme able to oxidize HMF into Furan-2,5-dicarboxylic acid (FDCA) has been discovered in fungal species such as Caldariomyces fumago [1] or in prokaryotic species Cupriavidus basilensis (C. basilensis) [2]. and Methylovorus sp. [4].

As we intended to work with Escherichia coli (E. coli), we focused on the enzymes discovered in prokaryotes. Due to glycosylations on the enzymes that has been found in Caldaryomyces fumago and Eukaryotes/Prokaryotes gene compatibility issue, fungal enzymes have been dismissed. The following section describes the enzymes selected to achieve an efficient transformation of HMF into FDCA.

HmfH is an enzyme that has been discovered in C. basilensis. This enzyme is part of a gene cluster implied in furanic compound degradation. HmfH homologues as also been found in several other bacterial species [6]. In a study, HmfH gene has been cloned into Pseudomonas putida in order to enable this bacteria to synthetize FDCA from HMF. HmfH catalyse two successive oxidations that transform HMF into 5-(hydroxymethyl)furoic acid ( HMF acid) and then into FDCA [5] ( fig 2. ). The modified P. putida with HmfH was able to produce FDCA without excessive amount of other furan derivatives. One issue with this enzyme is that the second oxidation of HMF acid into FDCA is slower than the first one. It results in the accumulation of the intermediate product, HMF acid. In fed batch culture the accumulation of intermediate product could lead to efficiency issue to the production of high purity FDCA.

One way to compensate for that is to add other enzymes to the artificial metabolic pathway, in order to speed up the second oxidation of HMF acid into FDCA.

An enzyme of interest, HMF dehydrogenase (Aldh1), is found to catalyse identical reaction as HmfH. This enzyme oxidizes HMF into FDCA with HMF acid as a reaction intermediate. The Kcat of this enzyme on 5-HMF has been estimated to 5.1 mM-1.min-1 [8]. By acting synergically with HmfH, Aldh1 could limit the accumulation of HmfAcid.

An other enzyme that may turn worthwhile for the biosynthesis of FDCA is HmfO (5-(hydroxymethyl)furfural Oxydase). This enzyme is present in Methylovorus sp. and in C. basilensis. HmfO belongs to a C. basilensis HMF14 gene cluster involved in Hmf degradation pathway. HmfO and HmfH are homologous, they both belongs to the GMC (glucose-methanol-choline) oxidoreductase proteins family. The N-ter GMC domain bind to FAD, and release H2O2 as a byproduct [4]. However, unlike HmfH , HmfO needs three successive oxidations of HMF to reach FDCA (fig 1.). Thereby two intermediary compounds are formed, 2,5-Furandicarboxaldehyde (furfural) (DFF) and 5-formyl-2-furancarboxylic acid (FFA) [4].

In order to obtain an efficient whole cell biocatalyst genes coding for the three previously described enzymes have to be under control of a strong promoter. To maximize the expression of these proteins it was planned to put them under control of promoter improved by freiburg 2011 iGem team and characterized by Slovenia HS 2015 iGem team (BBa_K608002).

Genes overexpression could be a drag on bacterial growth. In order to able bacterial culture to reach stationary phase quicker it was planned to use an inducible promoter.The part pBAD strong (BBa_K206000) , registered by iGEM09_British_Columbia, offer a high expression level.

In order to study each proteins activity individually, it was planned to cloned each one of them in pSB1-C3 under control of BBa_K608002 (“strong promoter strong RBS”). Moreover , so as to study their joint activity, an operon structure of HmfH, HmfO and Aldh1 under control of an constitutive and inducible promoter has been designed (fig 3).

References :

1. 5-hydroxymethylfurfural conversion by fungal aryl-alcohol oxidase and unspecific peroxygenase Juan Carro, Patricia Ferreira, Leonor Rodrıguez, Alicia Prieto, Ana Serrano, Beatriz Balcells, Ana Arda, Jesus Jim enez-Barbero, Ana Gutierrez, Rene Ullrich, Martin Hofrichter and Angel T. Martınez
2. Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of Cupriavidus basilensis HMF14. Koopman F, Wierckx N, de Winde JH, Ruijssenaars HJ.
3. "Enzyme-catalyzed oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid." Dijkman W.P., Groothuis D.E., Fraaije M.W.
4. "Discovery and characterization of a 5-hydroxymethylfurfural oxidase from Methylovorus sp. strain MP688." Dijkman W.P., Fraaije M.W.
5. 2010 Aug;101(16):6291-6. doi: 10.1016/j.biortech.2010.03.050. Epub 2010 Apr 3. Efficient whole-cell biotransformation of 5-(hydroxymethyl)furfural into FDCA, 2,5-furandicarboxylic acid. Koopman F1, Wierckx N, de Winde JH, Ruijssenaars HJ.
6. Microbial degradation of furanic compounds: biochemistry, genetics, and impact ; Nick Wierckx, Frank Koopman, Harald J. Ruijssenaars, and Johannes H. de Winde
7. Inhibition performance of lignocellulose degradation products on industrial cellulase enzymes during cellulose hydrolysis. Jing X, Zhang X, Bao J.
8. Metabolic engineering of Raoultella ornithinolytica BF60 for the production of 2, 5- 2 furandicarboxylic acid from 5-hydroxymethylfurfural Gazi Sakir Hossain, Haibo Yuan, Jianghua Li, Hyun-dong Shin, Miao Wang, Guocheng Du, Jian Chen, Long Liu

Lab Work

Team

Yasmine Amrani, supervised the lab work. Arthur Grimaud and Sandro Papaïs designed the experiments and the parts used for the lab work. Sandro Papaïs, François Hebrard and Maëlle Allender, accomplished the lab work by themselves. They also wrote their own experiment protocols with the help of David Mauboules. Finally, Maëlle Allender did the experiments regarding the chemistry part.

External Help

Thanks to the European Institute of Chemistry and Biology (IECB) and the Laboratory of Chemistry of Organic Polymers (LCPO) of Bordeaux for lending us their premises. Denis Dupuy and Delphine Daguerre who supervised our trainees for the lab work in the IECB and Stéphane Grelier and Eric Virol in the LCPO. Florian Alonso (CRTCB - U1045) and Thierry Dakhli for their help and advices to understand our mistakes and use them to carry out our progress.

Sponsoring

Thomas Favraud, Marguerite Batsale, Ines Baghou, were responsible for finding sponsors and building application files. Lorine Debande has set up our crowdfunding campaign.

Human Practices

Team

Our human practice work has been rich and diversified. Thus, we were all involved in some aspect of it.

Each conference has succeeded thanks to the coordination of all of us. Marguerite Batsale, Thomas Favraud, Yasmine Amrani and Jean-Clément Gallardo were greatly active for finding and inviting our speakers. Frederic Jung was responsible to find premises for hosting each event. They were diffused with posters and flyers designed by Pierre Jacquet, Marguerite Batsale, Thomas Favraud, and Francois Hebrard .

The interviews to the Landes forest actors were carried out by Marguerite Batsale . The scientific workshops for children were designed by Anaïs Labecot and took place with the help of Pierre Jacquet, Marguerite Batsale, David Mauboules, and Maëlle Allender. The survey we have held came from an idea of Yasmine Amrani and Lorine Debande. It was thus designed and analyzed by Ines Baghou.

External Help

• Raphael Devillard, University lecturer at the University of Bordeaux
• Etienne Roux, Associate professor of Physiology at the University of Bordeaux
• The Antony Mainguene Foundation
• Patrice Binder, Biosecurity Specialist and Expert
• Ijsbrand Kramer, Professor at the University of Bordeaux
• Regis Pommier, researcher in Civil and Environmental Engineering and passionate about the Landes forest
• Stephane Grelier, University lecturer at the University of Bordeaux.

They all took part to our events aiming to diffuse, talk, and exchange about Science. They shared their knowledge and brought reflexions among their public.

Collaborations

Our collaborative projects have involved the organization and the lodging of our collaborators. Beside the academic work to perform we had at heart to welcome them properly and make them a good time so they keep of this collab a good memory. To do this, thank you to Jean-Clément Gallardo, Anaïs Labecot, Arthur Grimaud, Sandro Papaïs , Ines Baghou and David Mauboules. They have been good for both working IGEM and having fun.

Meetups

The meetups are important events for the team. It permits to build a network, exchange with other igemers, training for presentations and finally increase our visibility. Most of them were far from our hometown but some of us took the time to travel to it. Yasmine Amrani, Lorine Debande, Jean-Clément Gallardo, Anaïs Labecot, François Hebrard , Estelles Lesbros and David Mauboules did their best to proudly represent our team abroad.

Communication

Communication is essential for the visibility and the credibility of the team. Ines Baghou was in charge to manage our Facebook page and Twitter account, helped along with Estelle Lebros. Lorine Debande designed our booklet used for finding sponsors by presenting us in a good form. To complete this task she also designed business cards to spread all around.

Wiki

Each of us had a specific chapter to write, therefore we were all involved in the writing of the Wiki. The video clip presentation of our supervisor was filmed by Sandro Papais and animated by Yasmine Amrani. The computer building part was managed by Pierre Jacquet and Sebastien Goncalves Claro. Pierre Jacquet designed a new logo used to represents the team.

Giant Jamboree Organization

Marguerite Batsale set the organization of the traveling to Boston which includes flight estimates and reservations, and housing. Helped by Arthur Grimaud she also took care of the registration of the team, and of each member of the team.

IGEM teams ❤

• IGEM Munich
• IGEM Aix-Marseille
• IGEM Pasteur: For organizing and inviting us to their meetup, during which we met who became later our friends and collaborators
• IGEM Groningen who came all its way from the Netherlands to bring us their skills in chemistry. It has resulted in a very rich IGEM collaboration.
• IGEM Sorbonne who invited us to take part to their conference in Sustainable Development.
• IGEM Toulouse who brought us in Bordeaux some lab materials to enrich our experiments.
• IGEM Le Caire who took part of our first conference by presenting their project in videoconference.

Fundings

• The Excellence Initiative of the University of Bordeaux (IDEX)
• The Solidarity and Development Fund of Student Initiatives (FSDIE)
• The French embassy to the United-States
• The Synthetic Biology community in Bordeaux
• All the donors to our crowdfunding campaign: They all trusted in our project and our ambitions, their financial help permitted us to cover all the expenses generate by the competition. There is no way we could make it to Boston without them.

Personal Acknowledgements

We thank Denis Dupuy who is on the initiative of the IGEM project at the University of Bordeaux.

We thank the University of Bordeaux who lend us their premises to organize our events.

Again, we thank to the IECB where all the members of the team could regularly meet to exchange about the conduct of our project.

Last, we would like to warmly thanks all the people who trusted in our project and encouraged us to pursue. They are friends, family, colleagues, teachers, or even unknown, and they are all precious to us !p>