Team:Tokyo-NoKoGen/g3dh

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<center><img src="https://static.igem.org/mediawiki/2014/8/8d/Noko14_G3dhreact5.png" width="70%"></center><br><br>
<h2>About G3DH</h2>
<h2>About G3DH</h2>
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<p> The glucose-3-dehydrogenase (G3DH) operon from <i>Rhizobium tumefaciens</i> EHA101 encodes the heterotrimer G3DH, which converts glucose to 3-ketoglucose.  
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<p> The enzyme, glucoside 3-dehydrogenase (E.C.1.1.99.13) or glucose-3-dehydrogenase (G3DH) catalyzes the oxidation of the C-3 hydroxyl group of the glucosides and converting them to corresponding 3-ketoglucosides (1). Because G3DH has wide substrate specificity, it can convert not only monosaccharides but disaccharides, including trehalose.</p>  
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The deduced primary structure and secondary structure of G3DH are similar to sorbitol dehydrogenase from <i>Gluconobacter oxidans</i> and 2-keto-D-gluconate dehydrogenases from <i>Erwinia herbicola</i>. These enzymes are composed of three subuints; catalytic subunit, cytochrome c subunit, and small subunit. Catalytic domain has flavin adenine dinucleotide (FAD) as a cofactor. And cytochrome c subunit bounds to cytoplasmic membrane. The function of small subunit is unknown.</p><br>
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<img src="https://static.igem.org/mediawiki/2014/c/c4/Noko14_G3DH_reaction.PNG" width="80%"><br>
<img src="https://static.igem.org/mediawiki/2014/c/c4/Noko14_G3DH_reaction.PNG" width="80%"><br>
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<p> This G3DH we use also has cytochrome c subunit. Cytochrome c subunit needs to be matured by cytochrome c maturation enzymes (CCM). Therefore, we use the plasmid, pEC86 which has the gene of CCM.</p><br>
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<p> G3DH is composed of three subunits: catalytic subunit, cytochrome <i>c</i> subunit, and small subunit. The catalytic subunit has a flavin adenine dinucleotide (FAD) as cofactor, and the cytochrome<i> c</i> subunit is bound to the cytoplasmic membrane in the periplasm.</p>  
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   <p> And this G3DH is a homolog of G3DH from a marine bacterium <i>Halomonas </i>sp. α-15 which shows wide substrate specificity. It is already reported that G3DH from <i> Halomonas </i> sp. α-15 can also convert trehalose (a disaccharide made up of two glucose moieties) into 3,3'-diketotrehalose, which is trehalase inhibitor. Therefore, the G3DH we use can also convert trehalose to 3,3'-diketotrehalose.</p><br><br>
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   <p> In our project, we used G3DH derived from <i>Rhizobium tumefaciens</i> EHA101, formerly known as <i>Agrobacterium tumefaciens</i>. The enzymatic activity of G3DH in this microorganism was first reported in 1967 (2). The gene encoding this enzyme was found from a putative proteins, which is homologous to G3DH derived from <i>Halomonas </i>sp. α-15 (3), which was reported to convert trehalose to 3,3’dkT (see below). The functional expression of <i>R.tumefaciens</i> derived putative enzyme confirmed that the gene encodes the G3DH complex (unpublished data from Sode laboratory, Tokyo univ. of Agric. and Technol.).</p><br><br>
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 +
<h2>About 3,3'-diketotrehalose (3,3'-dkT) </h2>
 +
<p> 3,3’-dkT is a novel trehalose derivative in which the third hydroxyl group of both glucose moieties are oxidized. It was already reported that 3,3-dkT showes an inhibitory effect toward the trehalase from pig-kidney and <i>Bombyx mori</i> (silkworm)(3).</p><br><br>
    
    
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<p><b>Reference</b></p>
 
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<p>(1) Cloning and Expression of Glucose 3-Dehydrogenase from <i>Halomonas</i> sp. α-15 in <i>Escherichia coli</i>.,(2001),K Kojima<i> et al.,</i></p>
 
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<p>(2) Overproduction of the <i>Bradyrhizobium japonicum</i> c-Type Cytochrome Subunits of the cbb3 Oxidase in <i>Escherichia coli</i>.,(1998),E Arslan <i>et al.,</i><p><br><br>
 
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<h2><b>Construction of Biobrick</b></h2>
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<h2>1.Construction of Biobrick</h2>
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<p>  We amplified G3DH gene from <i>Rhizobium tumefacience</i> by PCR. PCR products were inserted in pSB1C3 using standard cloning techniques. Original G3DH gene has two <i>Eco</i>RI restriction sites, which is incompatible with BioBrick parts. These restriction sites was removed without altering the codon by overlap extension PCR using designed primers(Fig.1).
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<p> G3DH gene were cloned from pTrc99a-G3DH which originated from <i>Agrobacterium tumefaciens</i>. PCR products were inserted pSB1C3. Original G3DH gene has two illegal restriction sites. In order to remove these restriction sites, the G3DH gene were amplified by overlap extension PCR. We designed two primer sets for overlap extension PCR.</p><br>
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</p><br>
<img src="https://static.igem.org/mediawiki/2014/7/72/Noko14_G3dh-rest.png" width="80%"><br>
<img src="https://static.igem.org/mediawiki/2014/7/72/Noko14_G3dh-rest.png" width="80%"><br>
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<!--<img src="https://static.igem.org/mediawiki/2014/b/be/Noko14_G3dh-remrest.png" width=80%"><br>-->
<!--<img src="https://static.igem.org/mediawiki/2014/b/be/Noko14_G3dh-remrest.png" width=80%"><br>-->
<img src="https://static.igem.org/mediawiki/2014/5/54/G3dh-remrest2.png" width="80%"><br>
<img src="https://static.igem.org/mediawiki/2014/5/54/G3dh-remrest2.png" width="80%"><br>
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<p>Fig.1. Remove illegal restriction sites</p><br><br>
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<p>Fig.1. The schematic image to remove internal <i>Eco</i>RI sites in G3DH.</p><br><br>
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<p> G3DH gene fragments were amplified and three PCR products were connected.</p>
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<p>  G3DH gene fragments were amplified and three PCR products were ligated. Then, G3DH (removed illegal restriction sites) were ligated with four different promoters and double terminator (BBa_B0015)(Fig.2).</p>
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<p> G3DH (removed illegal restriction sites) were ligated with four promoters and double terminator (BBa_B0010 and BBa_B0012).</p>
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<p> One of those promoters is arabinose inducible, and the others are constitutive promoter.</p><br>
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<p>Table 1. Promoter</p>
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<img src="https://static.igem.org/mediawiki/2014/e/e1/Noko14_Promoter.PNG" width="80%"><br>
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<p>Fig 2. Constructed plasmid</p><br><br>
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<h2>2. Evaluation</h2><br>
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<img src="https://static.igem.org/mediawiki/2014/8/85/Noko14_G3dhvecconst1.png" width="50%"><img src="https://static.igem.org/mediawiki/2014/6/68/Noko14_G3dhveccon2.png" width="50%"><br>
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<p> This G3DH needs the maturation of cytochrome c subunit to have catalytic activity. Therefore, we transformed G3DH with the plasmid, pEC86; which expresses cytochrome c maturation (CCM) enzymes (1).</p>
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<p>Fig 2. BioBrick parts containing G3DH.</p><br><br>
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<p>This is the method of culturing and extraction of production (Fig. 1).</p>
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<p>We cultured <i>E. coli</i> TOP10 transformed with two plasmids; G3DH expression vector, and cytochrome c maturation enzymes expression vector (pEC86) in LB medium containing 20 mM Trehalose. For expression of G3DH, we used four different promoters.</p>
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<p>When OD<sub>660</sub> achieves 0.6, 0.2 % arabinose was added to the medium for induction in the case we used P<sub>BAD</sub> as a promoter.</p>
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<p>After culturing for 20 hours at 37 ℃, we extracted products by boiling and centrifugation.</p>
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<p>0.007U trehalase and 20 mM trehalose mixed sample and incubated 30h.</p>
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<p>First, we investigated expression of G3DH by SDS-PAGE analysis. Second, we measured the glucose dehydrogenase activity of G3DH. Finally we tried to detect 3,3’-dkT by thin-layer chromatography (TLC).</p><br>
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<img src=”” alt=”scheme”><br><br>
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<p> One promoter is arabinose inducible (P<sub>BAD</sub>), and the others are constitutive promoters (Table.1).</p><br>
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<p>Fig. 3 evaluation of G3DH</p>
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<p>Table 1. Promoter connected with G3DH genes.</p>
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<img src="https://static.igem.org/mediawiki/2014/e/e1/Noko14_Promoter.PNG" width="80%"><br><br><br>
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<p> By SDS-PAGE analysis, there was the band which showed about 68 kDa on G3DH under the constitutive promoter P106 and P100, pBAD. Therefore, we confirmed the expression of G3DH (Fig. 2).</p><br>
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<h2><b>Evaluation</b></h2><br>
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<p>  The G3DH requires the maturation of cytochrome <i>c</i> subunit for the catalytic activity. Therefore, we transformed <i>E. coli</i> TOP 10 with the constructed plasmids and pEC86, which expresses cytochrome <i>c</i> maturation (CCM) enzymes (3).</p>
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<p>This is the method of culturing and extraction of production (Fig. 3).</p>
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<p> We cultured TOP10 transformed with below plasmids in LB medium containing 20 mM Trehalose.
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P<sub>high</sub>, <sub>medium</sub>-RBS-G3DH-DT and P<sub>BAD</sub>-RBS-G3DH-DT were cultured at 80 rpm, 37 ℃, for 24 hours and at 150 rpm, 37 ℃ respectively.</p>
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<p> When OD<sub>660</sub> achieves 0.6, 0.2 % arabinose was added to the medium for induction (PEC86,P<sub>BAD</sub>-RBS-G3DH-DT).
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The transformants were harvested, suspended in 10 mM sodium phosphate buffer (SPB) and pellet was obtained by centrifugation.</p>
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<!-- <p> We cultured <i>E. coli</i> TOP10 transformed with two plasmids; G3DH expression vector, and cytochrome c maturation enzymes expression vector (pEC86) in LB medium containing 20 mM Trehalose. For expression of G3DH, we used four different promoters.</p>
 +
<p> When OD<sub>660</sub> achieves 0.6, 0.2 % arabinose was added to the medium for induction in the case we used P<sub>BAD</sub> as a promoter.</p>
 +
<p> After culturing for 20 hours at 30 ℃, we extracted products by boiling and centrifugation. To investigate the inhibition effect of products, 0.007U trehalase and 20 mM trehalose mixed sample and incubated 30h. </p>-->
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<br>
 +
 
 +
<p> First, we investigated expression of G3DH by SDS-PAGE analysis. Second, we measured the glucose dehydrogenase activity of G3DH. Finally we tried to detect 3,3’-dkT by thin-layer chromatography (TLC).</p><br>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/2/28/Noko14_Flow-g3dh.PNG" alt="sheme" width="80%">
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<br><br>
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<p>Fig. 3 Evaluation of G3DH</p><br><br>
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 +
 
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<p><b>SDS-PAGE</b></p>
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<p> By SDS-PAGE analysis, there was the band which showed about 68 kDa on G3DH under the constitutive promoter P<sub>medium</sub> and P<sub>high</sub>, P<sub>BAD</sub>. Therefore, we confirmed the expression of G3DH (Fig. 4).</p><br>
<img src="https://static.igem.org/mediawiki/2014/9/97/Noko14_Sdsg3dh3.PNG" width="80%"><br>
<img src="https://static.igem.org/mediawiki/2014/9/97/Noko14_Sdsg3dh3.PNG" width="80%"><br>
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<p>Fig. 4 SDS-PAGE analysis</p><br><br>
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<p>Fig. 4 SDS-PAGE analysis of G3DH expression. </p><br><br>
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<p><b> Measurement of G3DH productivity</b> </p>
 +
<p> We measured the oxidase activity of G3DH by PMS-DCIP assay (Fig. 5). On this assay, we measured the decrease in absorbance of DCIP at 600 nm.  Substrates were glucose and trehalose.  PMS stands for phenazine methosulfate, and DCIP stands for 2,6-dichlorophenol-indophenol.</p><br>
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<img src="https://static.igem.org/mediawiki/2014/a/a8/Noko14_PMS-DCIP.PNG" width="50%"><br>
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<p>Fig.5 The scheme of PMS-DCIP assay.</p><br>
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<p>Trehalase inhibition measurement (Fig. 3) </p><br>
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<img src="https://static.igem.org/mediawiki/2014/3/39/Noko14_Pms-dcip1.png" "width="500" height="500">
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<p>Fig.6 Measurement of the G3DH productivity<br>
 +
<p>Productivity is the enzyme activity(U) from 1L culture. P<sub>high</sub> had the highest productivity. It is reason that  we confirmed expression of P<sub>high</sub>-G3DH by SDS-PAGE.
 +
Both of P<sub>BAD</sub>-G3DH(+) and (-) had comparatively high productivity. Increase of P<sub>BAD</sub>(-) productivity results that regulation by non-arabinose was not enough. P<sub>BAD</sub> (+) was also seemed to express at insoluble fraction (misfolded), therefore we could not confirm activity improvement.</p><br><br>
 +
 
 +
<br>
 +
 
 +
<p><b>Trehalase inhibition measurement</b> </p>
 +
<p> This is the result of trehalase inhibition activity assay(Fig. 7). Each value of activity was normalized at the value of activity of empty vector which were not induced by arabinose.
 +
Samples of G3DH which induced by P<sub>high</sub> and P<sub>medium</sub> activity was lower than that of empty vector.  </p><br>
 +
<p> We concluded that G3DH which induced by P<sub>high</sub> and P<sub>medium</sub> expressed G3DH. And G3DH converted trehalose to 3,3’-dkT.</p><br><br>
<img src="https://static.igem.org/mediawiki/2014/8/83/Noko14_33dktinh.png" width="80%"><br>
<img src="https://static.igem.org/mediawiki/2014/8/83/Noko14_33dktinh.png" width="80%"><br>
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<p> Fig. 3 Trehalose inhibition activity assay </p><br><br>
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<p> Fig. 7 Trehalase inhibition activity assay </p><br><br>
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<p> This is the result of trehalase inhibition activity assay. Each value of activity was normalized at the value of activity of empty vector which were not induced by arabinose.
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Samples of G3DH which induced by P100 and P106 activity was lower than that of empty vector. </p><br>  
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<p> We concluded that G3DH which induced by P100 and P106 expressed G3DH. And G3DH converted trehalose to 3,3'-diketotorehalose  .</p>
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<h2>Reference</h2><br>
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 +
<p>(1) K Kojima<i> et al</i>., (2001) Cloning and Expression of Glucose 3-Dehydrogenase from <i>Halomonas</i> sp. α-15 in <i>Escherichia coli</i>. <i>Biochem Biophys Res Commun</i>, <b>282</b>, 21-27</p>
 +
<p>(2) K Hayano <i>et al</i>., (1967) Purification und properties of 3-ketosucrose-forming enzyme from the cells of <i>Agrobacterium tumefaciens</i>. <i>J. Biol. Chem.</i>, <b>242</b>, 3665-3672</p>
 +
<p>(3) K Sode <i>et al</i>., (2001) Enzymatic synthesis of a novel trehalose derivative, 3,3’-diketotrehalose, and its potential application as the trehalase enzyme inhibitor. <i>FEBS Letters</i>, <b>489</b>, 42-45</p>
 +
<p>(4) E Arslan <i>et al</i>., (1998) Overproduction of the <i>Bradyrhizobium japonicum</i> c-Type Cytochrome Subunits of the cbb3 Oxidase in <i>Escherichia coli</i>. <i>Biochem Biophys Res Commun</i>, <b>251</b>, 744-747</p>
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<p>>><a href="http://www.brenda-enzymes.org/enzyme.php?ecno=1.1.99.13" target="_blank">BRENDA</a></p><br>
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 +
<h2>Future work</h2>
 +
 
 +
<p> We confirmed the production of trehalose and 3,3’-dkT respectively.  
 +
To produce 3,3’-dkT from glucose directly in <i>E. coli</i>, we are going to cotransform <i>E. coli</i> with 3 different plasmids which harbor OtsA+OtsB, G3DH and cytochrome c maturation enzymes, respectively. </p>
 +
 
 +
<p> In this study, we confirmed low productivity of 3,3’-dkT in <I>E. coli</I>. It might be caused by low amount of precursor (trehalose).
 +
In our project, we used OtsA and OtsB derived from <i>E. coli</i> to produce trehalose, but <i>E. coli</i> and other microorganisms have different kind of pathway to produce trehalose. To increase the production of trehalose, we are planning to use other kind of enzyme to produce trehalose. </p>
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<br>
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<p>Further improvement</p>
 +
 <p> To improve the specificity of <i>Exterminator coli</i>, we are going to introduce cockroach pheromone expression system to it. If we want to create an <i>Exterminator coli</i> for other insects, we can engineer it by introducing specific pheromone for each insect.</p>

Latest revision as of 03:56, 18 October 2014



About G3DH

 The enzyme, glucoside 3-dehydrogenase (E.C.1.1.99.13) or glucose-3-dehydrogenase (G3DH) catalyzes the oxidation of the C-3 hydroxyl group of the glucosides and converting them to corresponding 3-ketoglucosides (1). Because G3DH has wide substrate specificity, it can convert not only monosaccharides but disaccharides, including trehalose.


 G3DH is composed of three subunits: catalytic subunit, cytochrome c subunit, and small subunit. The catalytic subunit has a flavin adenine dinucleotide (FAD) as cofactor, and the cytochrome c subunit is bound to the cytoplasmic membrane in the periplasm.

 In our project, we used G3DH derived from Rhizobium tumefaciens EHA101, formerly known as Agrobacterium tumefaciens. The enzymatic activity of G3DH in this microorganism was first reported in 1967 (2). The gene encoding this enzyme was found from a putative proteins, which is homologous to G3DH derived from Halomonas sp. α-15 (3), which was reported to convert trehalose to 3,3’dkT (see below). The functional expression of R.tumefaciens derived putative enzyme confirmed that the gene encodes the G3DH complex (unpublished data from Sode laboratory, Tokyo univ. of Agric. and Technol.).



About 3,3'-diketotrehalose (3,3'-dkT)

 3,3’-dkT is a novel trehalose derivative in which the third hydroxyl group of both glucose moieties are oxidized. It was already reported that 3,3-dkT showes an inhibitory effect toward the trehalase from pig-kidney and Bombyx mori (silkworm)(3).



Construction of Biobrick

 We amplified G3DH gene from Rhizobium tumefacience by PCR. PCR products were inserted in pSB1C3 using standard cloning techniques. Original G3DH gene has two EcoRI restriction sites, which is incompatible with BioBrick parts. These restriction sites was removed without altering the codon by overlap extension PCR using designed primers(Fig.1).









Fig.1. The schematic image to remove internal EcoRI sites in G3DH.



  G3DH gene fragments were amplified and three PCR products were ligated. Then, G3DH (removed illegal restriction sites) were ligated with four different promoters and double terminator (BBa_B0015)(Fig.2).


Fig 2. BioBrick parts containing G3DH.



 One promoter is arabinose inducible (PBAD), and the others are constitutive promoters (Table.1).


Table 1. Promoter connected with G3DH genes.




Evaluation


 The G3DH requires the maturation of cytochrome c subunit for the catalytic activity. Therefore, we transformed E. coli TOP 10 with the constructed plasmids and pEC86, which expresses cytochrome c maturation (CCM) enzymes (3).

This is the method of culturing and extraction of production (Fig. 3).

 We cultured TOP10 transformed with below plasmids in LB medium containing 20 mM Trehalose. Phigh, medium-RBS-G3DH-DT and PBAD-RBS-G3DH-DT were cultured at 80 rpm, 37 ℃, for 24 hours and at 150 rpm, 37 ℃ respectively.

 When OD660 achieves 0.6, 0.2 % arabinose was added to the medium for induction (PEC86,PBAD-RBS-G3DH-DT). The transformants were harvested, suspended in 10 mM sodium phosphate buffer (SPB) and pellet was obtained by centrifugation.


 First, we investigated expression of G3DH by SDS-PAGE analysis. Second, we measured the glucose dehydrogenase activity of G3DH. Finally we tried to detect 3,3’-dkT by thin-layer chromatography (TLC).


sheme

Fig. 3 Evaluation of G3DH



SDS-PAGE

 By SDS-PAGE analysis, there was the band which showed about 68 kDa on G3DH under the constitutive promoter Pmedium and Phigh, PBAD. Therefore, we confirmed the expression of G3DH (Fig. 4).



Fig. 4 SDS-PAGE analysis of G3DH expression.



Measurement of G3DH productivity

 We measured the oxidase activity of G3DH by PMS-DCIP assay (Fig. 5). On this assay, we measured the decrease in absorbance of DCIP at 600 nm. Substrates were glucose and trehalose. PMS stands for phenazine methosulfate, and DCIP stands for 2,6-dichlorophenol-indophenol.



Fig.5 The scheme of PMS-DCIP assay.


Fig.6 Measurement of the G3DH productivity

Productivity is the enzyme activity(U) from 1L culture. Phigh had the highest productivity. It is reason that we confirmed expression of Phigh-G3DH by SDS-PAGE. Both of PBAD-G3DH(+) and (-) had comparatively high productivity. Increase of PBAD(-) productivity results that regulation by non-arabinose was not enough. PBAD (+) was also seemed to express at insoluble fraction (misfolded), therefore we could not confirm activity improvement.




Trehalase inhibition measurement

 This is the result of trehalase inhibition activity assay(Fig. 7). Each value of activity was normalized at the value of activity of empty vector which were not induced by arabinose. Samples of G3DH which induced by Phigh and Pmedium activity was lower than that of empty vector.


 We concluded that G3DH which induced by Phigh and Pmedium expressed G3DH. And G3DH converted trehalose to 3,3’-dkT.




Fig. 7 Trehalase inhibition activity assay



Reference


(1) K Kojima et al., (2001) Cloning and Expression of Glucose 3-Dehydrogenase from Halomonas sp. α-15 in Escherichia coli. Biochem Biophys Res Commun, 282, 21-27

(2) K Hayano et al., (1967) Purification und properties of 3-ketosucrose-forming enzyme from the cells of Agrobacterium tumefaciens. J. Biol. Chem., 242, 3665-3672

(3) K Sode et al., (2001) Enzymatic synthesis of a novel trehalose derivative, 3,3’-diketotrehalose, and its potential application as the trehalase enzyme inhibitor. FEBS Letters, 489, 42-45

(4) E Arslan et al., (1998) Overproduction of the Bradyrhizobium japonicum c-Type Cytochrome Subunits of the cbb3 Oxidase in Escherichia coli. Biochem Biophys Res Commun, 251, 744-747

>>BRENDA


Future work

 We confirmed the production of trehalose and 3,3’-dkT respectively. To produce 3,3’-dkT from glucose directly in E. coli, we are going to cotransform E. coli with 3 different plasmids which harbor OtsA+OtsB, G3DH and cytochrome c maturation enzymes, respectively.

 In this study, we confirmed low productivity of 3,3’-dkT in E. coli. It might be caused by low amount of precursor (trehalose). In our project, we used OtsA and OtsB derived from E. coli to produce trehalose, but E. coli and other microorganisms have different kind of pathway to produce trehalose. To increase the production of trehalose, we are planning to use other kind of enzyme to produce trehalose.


Further improvement

 

 To improve the specificity of Exterminator coli, we are going to introduce cockroach pheromone expression system to it. If we want to create an Exterminator coli for other insects, we can engineer it by introducing specific pheromone for each insect.