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     <!-- Intro Team IGEM-->
 
     <!-- Intro Team IGEM-->
 
     <div class="container text_intro">
 
     <div class="container text_intro">
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         <h2>Design</h2>
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         <h1 class="text-longshadow">Design</h1>
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         <div class="text_intro"> The IGEM Bordeaux team is responsible for all the aspects of running the project. From the reflection of the subject,
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         <p> Recent studies have shown that multiple organisms have the ability of metabolizing 5-hydroxymethylfurfural (HMF).
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             to the writing of the protocols, setting them up, finding sponsors and promote our subject among a large and
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            For instance, enzyme able to oxidize HMF into Furan-2,5-dicarboxylic acid (FDCA) has been discovered in fungal
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             diversified public. We also carried out all the writing and and all the computer part of the development of the
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            species such as
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             Wiki. The tasks were split as below: </div>
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            <i>Caldariomyces fumago</i> [1] or in prokaryotic species
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    </div>
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            <i>Cupriavidus basilensis (C. basilensis)</i> [2]. and
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    <div class="bg-color2 section-padding separator">
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            <i>Methylovorus sp.</i> [4].</p>
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         <div class="container">
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        <p>As we intended to work with
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             <div class="row">
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            <i>Escherichia coli (E. coli)</i>, we focused on the enzymes discovered in prokaryotes. Due to glycosylations on
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                <div class="col-12">
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            the enzymes that has been found in
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                    <h2 class="section-title">Lab Work</h2>
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            <i>Caldaryomyces fumago</i> and Eukaryotes/Prokaryotes gene compatibility issue, fungal enzymes have been dismissed.
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                </div>
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             The following section describes the enzymes selected to achieve an efficient transformation of HMF into FDCA.
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                <div class="col-md-6 col-12">
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        </p>
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                    <h4>Team</h4>
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        <p>HmfH is an enzyme that has been discovered in
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                    <p>
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            <i>C. basilensis</i>. This enzyme is part of a gene cluster implied in furanic compound degradation. HmfH homologues
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                        <b>Yasmine Amrani</b>, supervised the lab work.
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            has also been found in several other bacterial species [6]. In a study, HmfH gene has been cloned into
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                        <b>Arthur Grimaud</b> and
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            <i>Pseudomonas putida</i> in order to enable this bacteria to synthetize FDCA from HMF. HmfH catalyse two successive
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                        <b>Sandro Papaïs</b> designed the experiments and the parts used for the lab work.
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            oxidations that transform HMF into 5-(hydroxymethyl)furoic acid ( HMF acid) and then into FDCA [5] ( fig 2. ).
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                        <b>Sandro Papaïs</b>,
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             The modified
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                        <b>François Hebrard</b> and
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            <i>P. putida</i> with HmfH was able to produce FDCA without excessive amount of other furan derivatives. One issue
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                        <b>Maëlle Allender</b>, accomplished the lab work by themselves. They also wrote their own experiment
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            with this enzyme is that the second oxidation of HMF acid into FDCA is slower than the first one. It results
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                        protocols with the help of
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            in the accumulation of the intermediate product, HMF acid. In fed batch culture the accumulation of intermediate
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                        <b>David Mauboules</b>. Finally,
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             product could lead to an efficient production of high pure FDCA.
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                        <b>Maëlle Allender </b> did the experiments regarding the chemistry part.</p>
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        </p>
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                </div>
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        <p>One way to compensate for that is to add other enzymes to the artificial metabolic pathway, in order to speed up
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                <div class="col-md-6 col-12">
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            the second oxidation of HMF acid into FDCA.</p>
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                    <h4>External Help</h4>
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        <p>An enzyme of interest, HMF dehydrogenase (Aldh1), is found to catalyse identical reaction as HmfH. This enzyme oxidizes
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                    <p>Thanks to the European Institute of Chemistry and Biology (IECB) and the Laboratory of Chemistry of Organic
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            HMF into FDCA with HMF acid as a reaction intermediate. The Kcat of this enzyme on 5-HMF has been estimated to
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                        Polymers (LCPO) of Bordeaux for lending us their premises.
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            5.1 mM-1.min-1 [8]. By acting synergically with HmfH, Aldh1 could limit the accumulation of HmfAcid.</p>
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                        <b>Denis Dupuy</b> and
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         <figure class="figure">
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                        <b>Delphine Daguerre</b> who supervised our trainees for the lab work in the IECB and
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             <img src="https://static.igem.org/mediawiki/2018/1/1f/T--Bordeaux--HmfO.png" class="figure-img img-fluid rounded" alt="HmfO protein structure">
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                        <b>Stéphane Grelier</b> and
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            <figcaption class="figure-caption">HmfO protein structure</figcaption>
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                        <b>Eric Virol</b> in the LCPO.
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        </figure>
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                        <b>Florian Alonso (CRTCB - U1045)</b> and
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        <p>An other enzyme that may turn worthwhile for the biosynthesis of FDCA is HmfO (5-(hydroxymethyl)furfural Oxydase).
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                        <b>Thierry Dakhli </b> for their help and advices to understand our mistakes and use them to carry out
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            This enzyme is present in
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                        our progress.</p>
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            <i>Methylovorus sp.</i> and in
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                 </div>
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            <i>C. basilensis</i>. HmfO belongs to a
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             </div>
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            <i> C. basilensis</i> 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].</p>
 +
        <figure class="figure">
 +
            <img src=" https://static.igem.org/mediawiki/2018/4/46/T--Bordeaux--HMF-FDCA.png" class="figure-img img-fluid rounded" alt="Fig 2. Artificial metabolic pathway of oxidation of HMF into FDCA, using HmfH,HmfO and Aldh1 enzyms. Oxydation reactions
 +
            imply the use of a FAD co-factor and the release of hydrogen peroxyde.">
 +
            <figcaption class="figure-caption">Artificial metabolic pathway of oxidation of HMF into FDCA, using HmfH,HmfO and Aldh1 enzyms. Oxydation reactions
 +
                imply the use of a FAD co-factor and the release of hydrogen peroxyde.</figcaption>
 +
        </figure>
 +
        <p>In order to obtain an efficient whole cell biocatalyst genes coding for the three previously described enzymes have
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            to be under control of a strong promoter. To maximize the expression of these proteins it was planned to put
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            them under control of promoter improved by freiburg 2011 iGem team and characterized by Slovenia HS 2015 iGem
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            team (BBa_K608002).</p>
 +
        <p>Genes overexpression could be a drag on bacterial growth. In order to able bacterial culture to reach stationary
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            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.</p>
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        <p>In order to study each proteins activity individually, it was planned to clone each one of them in pSB1-C3 under
 +
            control of BBa_K608002 (“strong promoter strong RBS”). Moreover, to study their joint activity, an operon structure
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            of HmfH, HmfO and Aldh1 under control of an constitutive and inducible promoter has been designed (fig 3).
 +
        </p>
 +
        <img src="https://static.igem.org/mediawiki/2018/0/0a/T--Bordeaux--mainplasmid.png" class="col-12 img-fluid">
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        <p>See standards biobrick constructions below (click to view it fullscreen)</p>
 +
        <div class="row col-12" style="margin-bottom: 50px; margin-top: 50px; text-align: center!important">
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            <a href="https://static.igem.org/mediawiki/2018/6/6d/T--Bordeaux--stdbiobrick1.png" class="col-4" target="_blank">
 +
                <img src="https://static.igem.org/mediawiki/2018/6/6d/T--Bordeaux--stdbiobrick1.png" class="img-fluid">
 +
                <p style="text-align: center">pSB1C3 + Aldh1 (Standard BioBrick)</p>
 +
            </a>
 +
            <a href="https://static.igem.org/mediawiki/2018/a/af/T--Bordeaux--stdbiobrick2.png" class="col-4" target="_blank">
 +
                <img src="https://static.igem.org/mediawiki/2018/a/af/T--Bordeaux--stdbiobrick2.png" class="img-fluid">
 +
                <p style="text-align: center">pSB1C3 + HmfH (Standard BioBrick)</p>
 +
            </a>
 +
            <a href="https://static.igem.org/mediawiki/2018/a/a3/T--Bordeaux--stdbiobrick3.png" class="col-4" target="_blank">
 +
                <img src="https://static.igem.org/mediawiki/2018/a/a3/T--Bordeaux--stdbiobrick3.png" class="img-fluid">
 +
                 <p style="text-align: center">pSB1C3 + HmfO (Standard BioBrick)</p>
 +
             </a>
 
         </div>
 
         </div>
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    </div>
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         <div id="ref">
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    <div class="section-padding separator">
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             <h4>References :</h4>
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         <div class="container">
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             <ol type="1">
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             <div class="row">
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                 <li>5-hydroxymethylfurfural conversion by fungal aryl-alcohol oxidase and unspecific peroxygenase Juan Carro,
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                <div class="col-12">
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                    Patricia Ferreira, Leonor Rodrıguez, Alicia Prieto, Ana Serrano, Beatriz Balcells, Ana Arda, Jesus Jim
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                    <h2 class="section-title">Sponsoring</h2>
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                    enez-Barbero, Ana Gutierrez, Rene Ullrich, Martin Hofrichter and Angel T. Martınez</li>
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                </div>
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                 <li>Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of
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                <div class="col-md-12 col-12">
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                     Cupriavidus basilensis HMF14. Koopman F, Wierckx N, de Winde JH, Ruijssenaars HJ.</li>
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                    <p>
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                 <li>"Enzyme-catalyzed oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid." Dijkman W.P., Groothuis
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                        <b>Thomas Favraud</b>,
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                    D.E., Fraaije M.W.
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                        <b>Marguerite Batsale</b>,
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                 </li>
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                        <b>Ines Baghou</b>, were responsible for finding sponsors and building application files.
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                 <li>"Discovery and characterization of a 5-hydroxymethylfurfural oxidase from
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                        <b>Lorine Debande</b> has set up our crowdfunding campaign.</p>
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                     <i>Methylovorus sp.</i> strain MP688." Dijkman W.P., Fraaije M.W.
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                </div>
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                 </li>
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             </div>
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                 <li>2010 Aug;101(16):6291-6. doi: 10.1016/j.biortech.2010.03.050. Epub 2010 Apr 3. Efficient whole-cell biotransformation
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        </div>
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                     of 5-(hydroxymethyl)furfural into FDCA, 2,5-furandicarboxylic acid. Koopman F1, Wierckx N, de Winde JH,
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    </div>
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                     Ruijssenaars HJ.</li>
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    <div class="bg-color1 section-padding separator">
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                <li>Microbial degradation of furanic compounds: biochemistry, genetics, and impact ; Nick Wierckx, Frank Koopman,
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        <div class="container">
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                    Harald J. Ruijssenaars, and Johannes H. de Winde
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            <div class="row">
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                </li>
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                 <div class="col-12">
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                 <li> Inhibition performance of lignocellulose degradation products on industrial cellulase enzymes during cellulose
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                    <h2 class="section-title">Human Practices</h2>
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                     hydrolysis. Jing X, Zhang X, Bao J.</li>
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                </div>
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                 <li>Metabolic engineering of Raoultella ornithinolytica BF60 for the production of 2, 5- 2 furandicarboxylic
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                <div class="col-md-6 col-12">
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                    acid from 5-hydroxymethylfurfural Gazi Sakir Hossain, Haibo Yuan, Jianghua Li, Hyun-dong Shin, Miao Wang,
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                    <h4>Team</h4>
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                    Guocheng Du, Jian Chen, Long Liu</li>
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                    <p>Our human practice work has been rich and diversified. Thus, we were all involved in some aspect of it.
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             </ol>
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                        <br>
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                        <br> Each conference has succeeded thanks to the coordination of all of us.
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                        <b>Marguerite Batsale</b>,
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                        <b>Thomas Favraud</b>,
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                        <b>Yasmine Amrani</b> and
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                        <b>Jean-Clément Gallardo</b> were greatly active for finding and inviting our speakers.
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                        <b>Frederic Jung</b> was responsible to find premises for hosting each event. They were diffused with
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                        posters and flyers designed by
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                        <b>Pierre Jacquet</b>,
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                        <b>Marguerite Batsale</b>,
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                        <b>Thomas Favraud</b>, and
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                        <b>Francois Hebrard </b>.
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                        <br>
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                        <br>The interviews to the Landes forest actors were carried out by
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                        <b>Marguerite Batsale </b>. The scientific workshops for children were designed by
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                        <b>Anaïs Labecot</b> and took place with the help of
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                        <b>Pierre Jacquet</b>,
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                        <b>Marguerite Batsale</b>,
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                        <b>David Mauboules</b>, and
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                        <b>Maëlle Allender</b>. The survey we have held came from an idea of
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                        <b>Yasmine Amrani</b> and
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                        <b>Lorine Debande.</b> It was thus designed and analyzed by
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                        <b>Ines Baghou</b>.</p>
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                 </div>
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                <div class="col-md-6 col-12">
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                    <h4>External Help</h4>
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                    <p>
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                        <ul class="pointylist">
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                            <li>
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                                <b>Raphael Devillard</b>, University lecturer at the University of Bordeaux</li>
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                            <li>
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                                <b>Etienne Roux</b>, Associate professor of Physiology at the University of Bordeaux</li>
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                            <li>The Antony Mainguene Foundation</li>
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                            <li>
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                                <b>Patrice Binder</b>, Biosecurity Specialist and Expert</li>
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                            <li>
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                                <b>Ijsbrand Kramer</b>, Professor at the University of Bordeaux</li>
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                            <li>
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                                <b>Regis Pommier</b>, researcher in Civil and Environmental Engineering and passionate about
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                                the Landes forest</li>
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                            <li>
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                                <b>Stephane Grelier</b>, University lecturer at the University of Bordeaux.</li>
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                        </ul>
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                    </p>
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                    <p>
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                        <br>
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                        <b>
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                            <i>They all took part to our events aiming to diffuse, talk, and exchange about Science. They shared
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                                their knowledge and brought reflexions among their public.</i>
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                        </b>
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                    </p>
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    <div class="section-padding separator">
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                     <h2 class="section-title">Collaborations</h2>
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                </div>
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                <div class="col-12">
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                    <p>Our collaborative projects have involved the organization and the lodging of our collaborators. Beside
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                        the academic work to perform we had at heart to welcome them properly and make them a good time so
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                        they keep of this collab a good memory. To do this, thank you to
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                        <b>Jean-Clément Gallardo</b>,
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                        <b>Anaïs Labecot</b>,
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                        <b>Arthur Grimaud</b>,
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                        <b>Sandro Papaïs </b>,
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                        <b>Ines Baghou</b> and
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                        <b>David Mauboules</b>. They have been good for both working IGEM and having fun.</p>
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                    <p>The meetups are important events for the team. It permits to build a network, exchange with other igemers,
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                        training for presentations and finally increase our visibility. Most of them were far from our hometown
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                        but some of us took the time to travel to it.
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                        <b>Yasmine Amrani</b>,
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                        <b>Lorine Debande</b>,
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                        <b>Jean-Clément Gallardo</b>,
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                        <b>Anaïs Labecot</b>,
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                        <b>François Hebrard </b>,
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                        <b>Estelles Lesbros</b> and
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                        <b>David Mauboules</b> did their best to proudly represent our team abroad.</p>
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                    <p>Communication is essential for the visibility and the credibility of the team.
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                        <b>Ines Baghou</b> was in charge to manage our Facebook page and Twitter account, helped along with
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                        <b>Estelle Lebros</b>.
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                        <b>Lorine Debande</b> designed our booklet used for finding sponsors by presenting us in a good form.
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                        To complete this task she also designed business cards to spread all around.</p>
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                     <h2 class="section-title">Wiki</h2>
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                    <p>Each of us had a specific chapter to write, therefore we were all involved in the writing of the Wiki.
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                        The video clip presentation of our supervisor was filmed by
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                        <b>Sandro Papais</b> and animated by
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                        <b>Yasmine Amrani</b>. The computer building part was managed by
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                        <b>Pierre Jacquet</b> and
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                        <b>Sebastien Goncalves</b>
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                        Claro.
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                        <b>Pierre Jacquet</b> designed a new logo used to represents the team.</p>
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                    <h2 class="section-title">Giant Jamboree Organization</h2>
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                    <p>
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                        <b>Marguerite Batsale</b> set the organization of the traveling to Boston which includes flight estimates
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                        and reservations, and housing. Helped by
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                        <b>Arthur Grimaud</b> she also took care of the registration of the team, and of each member of the
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                        team. </p>
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                    <h2 class="section-title">IGEM teams ❤</h2>
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                        <ul class="pointylist">
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                            <li>IGEM Munich</li>
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                            <li>IGEM Aix-Marseille</li>
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                            <li>IGEM Pasteur:
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                                <i>For organizing and inviting us to their meetup, during which we met who became later our
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                                    friends and collaborators</i>
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                            </li>
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                            <li>IGEM Groningen who came all its way from the Netherlands to bring us their skills in chemistry.
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                                It has resulted in a very rich IGEM collaboration.</li>
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                            <li>IGEM Sorbonne who invited us to take part to their conference in Sustainable Development. </li>
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                            <li>IGEM Toulouse who brought us in Bordeaux some lab materials to enrich our experiments.</li>
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                            <li>IGEM Le Caire who took part of our first conference by presenting their project in videoconference.
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                     <h2 class="section-title">Fundings</h2>
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                            <li>The Excellence Initiative of the University of Bordeaux (IDEX)</li>
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                            <li>The Solidarity and Development Fund of Student Initiatives (FSDIE) </li>
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                            <li>The French embassy to the United-States</li>
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                            <li>The Synthetic Biology community in Bordeaux</li>
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                            <li>
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                                <b>All the donors to our crowdfunding campaign:
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                                    <i>They all trusted in our project and our ambitions, their financial help permitted us
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                                        to cover all the expenses generate by the competition. There is no way we could make
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                                        it to Boston without them. </i>
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                                </b>
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                     <h2 class="section-title">Personal Acknowledgements</h2>
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                    <p>We thank
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                        <b>Denis Dupuy</b> who is on the initiative of the IGEM project at the University of Bordeaux.
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                        <br>
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                        <br>We thank the University of Bordeaux who lend us their premises to organize our events.
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                        <br>Again, we thank to the IECB where all the members of the team could regularly meet to exchange about
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                        the conduct of our project.
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                        <br>
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                        <br>Last, we would like to warmly thanks all the people who trusted in our project and encouraged us
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Latest revision as of 09:59, 16 October 2018

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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 has 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 an efficient production of high pure 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.

HmfO protein structure
HmfO protein structure

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].

Fig 2. Artificial metabolic pathway of oxidation of HMF into FDCA, using HmfH,HmfO and Aldh1 enzyms. Oxydation reactions imply the use of a FAD co-factor and the release of hydrogen peroxyde.
Artificial metabolic pathway of oxidation of HMF into FDCA, using HmfH,HmfO and Aldh1 enzyms. Oxydation reactions imply the use of a FAD co-factor and the release of hydrogen peroxyde.

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 clone each one of them in pSB1-C3 under control of BBa_K608002 (“strong promoter strong RBS”). Moreover, 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).

See standards biobrick constructions below (click to view it fullscreen)

pSB1C3 + Aldh1 (Standard BioBrick)

pSB1C3 + HmfH (Standard BioBrick)

pSB1C3 + HmfO (Standard BioBrick)

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