Team:Wageningen UR/Why Aspergillus nigem

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<img src="https://static.igem.org/mediawiki/2013/1/13/Aspergillusheader.png">
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<div class="content aspergillus">
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<h1>Why <i>Aspergillus nigem</i>?</h1>
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<h2>Cause there ain't no party like an <i>Aspergillus</i> party!</h2>
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            <li id="li_tab1" onclick="tab('tab1')" class="active"><a>Introduction</a></li>
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            <li id="li_tab2" onclick="tab('tab2')"><a>Secondary metabolites</a></li>
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            <li id="li_tab3" onclick="tab('tab3')"><a>Toolbox</a></li>
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            <li id="li_tab4" onclick="tab('tab4')"><a>Host engineering</a></li>
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            <li id="li_tab5" onclick="tab('tab5')"><a>Summary</a></li>
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         <li class="currentfirst">Why <i>Aspergillus nigem</i>?</li>
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         <li class="first pad">Why <i>Aspergillus nigem</i>?</li>
         <li><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
         <li><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
         <li><a href="https://2013.igem.org/Team:Wageningen_UR/Lovastatin">Lovastatin</a></li>
         <li><a href="https://2013.igem.org/Team:Wageningen_UR/Lovastatin">Lovastatin</a></li>
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         <li><a href="https://2013.igem.org/Team:Wageningen_UR/ATP_biosensor">ATP Biosensor</a></li>
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         <li><a href="https://2013.igem.org/Team:Wageningen_UR/Modeling">Modeling</a></li>
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         <li><a href="https://2013.igem.org/Team:Wageningen_UR/Biosensors">Biosensors</a></li>
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         <li><a href="https://2013.igem.org/Team:Wageningen_UR/Cytoskeleton_and_septa">Cytoskeleton and Septa</a></li>
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         <li><a href="https://2013.igem.org/Team:Wageningen_UR/Host_engineering">Host engineering</a></li>
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         <li><a href="https://2013.igem.org/Team:Wageningen_UR/Summary">Summary</a></li>
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         <li class="last"><a href="https://2013.igem.org/Team:Wageningen_UR/Summary">Summary</a></li>
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        <li class="first pad">Why <i>Aspergillus nigem</i>?</li>
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        <li class="smbg"><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
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        <li class="bgbg"><a href="https://2013.igem.org/Team:Wageningen_UR/Lovastatin">Lovastatin</a></li>
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        <li class="bgsm"><a href="https://2013.igem.org/Team:Wageningen_UR/Modeling">Modeling</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Biosensors">Biosensors</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Chromoproteins">Chromoproteins</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Host_engineering">Host engineering</a></li>
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        <li class="last"><a href="https://2013.igem.org/Team:Wageningen_UR/Summary">Summary</a></li>
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    <ul>
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        <li class="first pad">Why <i>Aspergillus nigem</i>?</li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
 +
        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Lovastatin">Lovastatin</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Modeling">Modeling</a></li>
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        <li class="smbg"><a href="https://2013.igem.org/Team:Wageningen_UR/Biosensor">Biosensors</a></li>
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        <li class="bgbg"><a href="https://2013.igem.org/Team:Wageningen_UR/Infrastructure">Infrastructure</a></li>
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        <li class="bgsm"><a href="https://2013.igem.org/Team:Wageningen_UR/Chromoproteins">Chromoproteins</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Host_engineering">Host engineering</a></li>
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        <li class="last"><a href="https://2013.igem.org/Team:Wageningen_UR/Summary">Summary</a></li>
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        <li class="first pad">Why <i>Aspergillus nigem</i>?</li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
 +
        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Lovastatin">Lovastatin</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Modeling">Modeling</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Biosensors">Biosensors</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Infrastructure">Infrastructure</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Chromoproteins">Chromoproteins</a></li>
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        <li class="smbgsm"><a href="https://2013.igem.org/Team:Wageningen_UR/Host_engineering">Host engineering</a></li>
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        <li class="last"><a href="https://2013.igem.org/Team:Wageningen_UR/Summary">Summary</a></li>
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    </ul>
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<div id="tab5">
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<div class="tournav">
 +
    <ul>
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        <li class="first pad">Why <i>Aspergillus nigem</i>?</li>
 +
        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
 +
        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Lovastatin">Lovastatin</a></li>
 +
        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Modeling">Modeling</a></li>
 +
        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Biosensors">Biosensors</a></li>
 +
        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Infrastructure">Infrastructure</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Chromoproteins">Chromoproteins</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Host_engineering">Host engineering</a></li>
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        <li  class="last bg"><a href="https://2013.igem.org/Team:Wageningen_UR/Summary">Summary</a></li>
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    </ul>
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<div class="pagina-titel">
 
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    <h1>Why <i>Aspergillus nigem</i>?</h1>
 
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    <h2>Cause there ain't no party like an <i>Aspergillus</i> party!</h2>
 
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    <img src="http://beauvillemedia.nl/igem/dna.png"/>
 
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== A Candidate Host ==
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== A Versatile and Powerful Synbio Host ==
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<p>Biotechnology has brought about a revolution in drug manufacturing since its inception. Many life-saving drugs have spun out of biotech companies over the past few decades. However, there is still a vast body of unexplored compounds such as the fungal secondary metabolites that have the potential to prolong human lifespan. </p>
+
<p>Biotechnology has brought about a revolution in drug manufacturing since its inception. Many life-saving drugs have spun out of biotech companies over the past few decades. However, there is still a vast body of unexplored compounds, such as fungal secondary metabolites, that have the potential to prolong human lifespan and increase the quality of life. </p>
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<p>The production of lovastatin, a drug used in lowering LDL cholesterol for patients suffering from cardiovascular disease, has been chosen as a proof of principle. Currently, it is produced in the fungi <i>Aspergillus terreus</i>, which also produces less desirable toxins. The aim is to transfer the entire lovastatin metabolic pathway from <i>A. terreus</i> into a GRAS organism like <i>Aspergillus niger</i>, which is closely related. <i>Aspergillus niger</i> is amenable to genetic engineering, has a high protein secretion capacity. <i>A. niger</i> has already represented the most efficient, highest yielding bioprocess in practice for organic acids production.</p>
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<p>The production of lovastatin, a drug used in lowering LDL cholesterol for patients suffering from cardiovascular diseases, has been chosen as the Herculean task for our team. Currently, this secondary metabolite is produced in the fungus <i>Aspergillus terreus</i>, which also produces some less undesirable toxins. We aim to transfer the entire lovastatin metabolic pathway from <i>A. terreus</i> into the Generally Regarded as Safe organism <i>Aspergillus niger</i>. <i>A. niger</i> is much more amenable to genetic engineering, which already had profound effects in citric acid production.</p>
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== Modularity Approach ==
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== Modular Structure of DNA Sequence==
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  <p> The production of these secondary metabolites in most cases involves a large backbone enzyme that contains multiple catalytic domains. One of our goals is to establish a modular system of domain shuffling to generate a plethora of novel enzymes with new and improved functionalities. The possibilities are endless as there is a myriad of different domains from many fungi that can be added, removed, reordered or exchanged in this synthetic biology approach.</p>
+
  <p> The production of secondary metabolites in most cases involves very big genes that code for large backbone enzymes that contain multiple, apparently independent, catalytic domains. One of our goals is to establish a modular system of domain shuffling to be able to generate a plethora of novel enzymes with new functions. The possibilities are endless as there are various different domains from fungi that can be added, removed, reordered, or even customized by this modularity approach, which is shown in Figure 1.</p>
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<img src="https://static.igem.org/mediawiki/2013/a/a4/Lov_domain.png" style="width:75%;height:75%;"/>
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<p class="caption">Figure 1. Modularity approach of lovB. Since secondary metabolite producing enzymes consist of large modular proteins, it could be possible to brick the individual modules and use our new frame-shift-less assembly strategy to generate multidomain proteins in all kinds of configurations. </p>
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    <p>For instance, one of the main enzymes involved in the production of lovastatin is the 277kDa LovB enzyme, which contains 7 different catalytic domains. Prior studies on this pathway provide a good insight into the catalytic mechanisms of these individual domains. The individual domains have been mapped by homology modeling and are found to be well defined as well, which makes the modularity approach more feasible.</p>
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    <p>For instance, one of the main enzymes involved in the production of lovastatin is the 277kDa LovB enzyme, which contains 7 different catalytic domains. Prior studies on this pathway provide a good insight into the catalytic mechanisms of these individual domains. The individual domains have been mapped by homology modeling and are found to be well defined as well as often in a sequential order, which makes the modular approach more feasible.</p>
 
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== A Toolkit for <i>A. niger</i> ==
== A Toolkit for <i>A. niger</i> ==
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     <p>In addition to the modularity approach, a toolkit for <i>A. niger</i> is also being created, consisting of an ATP and a pH biosensor that can be targeted to specific compartments with the use of signal peptides, actin and septa GFP fusions to visualize the cytoskeleton and the junctions between adjacent cells, and chromoproteins that can serve as secondary selection markers. These tools allow one to obtain insight in cellular economy, physiology and architecture, which can be used to optimize the production processes in <i>A. niger</i>.</p>
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     <p>In addition to the modularity approach, a toolkit for <i>A. niger</i> is also being created, consisting of an ATP and a pH biosensor that can be targeted to specific compartments with the use of signal peptides, actin and septa GFP fusions to visualize the cytoskeleton and the junctions between adjacent cells, and chromoproteins that can serve as simple bioreporters such as secondary or neutral selection markers. These tools allow one to obtain insight in the cellular economy, physiology and architecture and logistics, which can be used to optimize production processes in <i>A. niger</i>.</p>
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== Host Engineering==
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<p> <i>A. niger</i> is a ‘workhorse’ commonly used in industry. However in this multicellular host only the hyphal tips are actively secreting whereas most of the biomass is vegetative. This is why we chose to expand the scope of our project by including host engineering. The goal is to create a single cell from the mycelium of <i>A. niger</i> that represents the active hyphal tip. Two different approaches are taken into consideration to do so.</p>
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Latest revision as of 03:58, 5 October 2013

Why Aspergillus nigem?

Cause there ain't no party like an Aspergillus party!


A Versatile and Powerful Synbio Host

Biotechnology has brought about a revolution in drug manufacturing since its inception. Many life-saving drugs have spun out of biotech companies over the past few decades. However, there is still a vast body of unexplored compounds, such as fungal secondary metabolites, that have the potential to prolong human lifespan and increase the quality of life.

The production of lovastatin, a drug used in lowering LDL cholesterol for patients suffering from cardiovascular diseases, has been chosen as the Herculean task for our team. Currently, this secondary metabolite is produced in the fungus Aspergillus terreus, which also produces some less undesirable toxins. We aim to transfer the entire lovastatin metabolic pathway from A. terreus into the Generally Regarded as Safe organism Aspergillus niger. A. niger is much more amenable to genetic engineering, which already had profound effects in citric acid production.

Modular Structure of DNA Sequence

The production of secondary metabolites in most cases involves very big genes that code for large backbone enzymes that contain multiple, apparently independent, catalytic domains. One of our goals is to establish a modular system of domain shuffling to be able to generate a plethora of novel enzymes with new functions. The possibilities are endless as there are various different domains from fungi that can be added, removed, reordered, or even customized by this modularity approach, which is shown in Figure 1.

Figure 1. Modularity approach of lovB. Since secondary metabolite producing enzymes consist of large modular proteins, it could be possible to brick the individual modules and use our new frame-shift-less assembly strategy to generate multidomain proteins in all kinds of configurations.

For instance, one of the main enzymes involved in the production of lovastatin is the 277kDa LovB enzyme, which contains 7 different catalytic domains. Prior studies on this pathway provide a good insight into the catalytic mechanisms of these individual domains. The individual domains have been mapped by homology modeling and are found to be well defined as well, which makes the modularity approach more feasible.

A Toolkit for A. niger

In addition to the modularity approach, a toolkit for A. niger is also being created, consisting of an ATP and a pH biosensor that can be targeted to specific compartments with the use of signal peptides, actin and septa GFP fusions to visualize the cytoskeleton and the junctions between adjacent cells, and chromoproteins that can serve as simple bioreporters such as secondary or neutral selection markers. These tools allow one to obtain insight in the cellular economy, physiology and architecture and logistics, which can be used to optimize production processes in A. niger.

Host Engineering

A. niger is a ‘workhorse’ commonly used in industry. However in this multicellular host only the hyphal tips are actively secreting whereas most of the biomass is vegetative. This is why we chose to expand the scope of our project by including host engineering. The goal is to create a single cell from the mycelium of A. niger that represents the active hyphal tip. Two different approaches are taken into consideration to do so.

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