Team:Wageningen UR/Why Aspergillus nigem

From 2013.igem.org

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== Synthetic biology approach ==
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== A Candidate Host ==
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     <p>Citric acid production by the filamentous ascomycete fungus, Aspergillus niger represents the most efficient, highest yielding bioprocess in practice.</p>
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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. The production of these secondary metabolites in most cases involves a large backbone enzyme that contains multiple catalytic domains. One of the 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. 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 Aspergillus terreus, which also produces less desirable toxins. The aim is to transfer the entire lovastatin metabolic pathway from A. terreus into a GRAS organism like Aspergillus niger, which is closely related. A. niger is amenable to genetic engineering, has a high protein secretion capacity and produces high amounts of organic acids. 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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<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. Citric acid production by the filamentous ascomycete fungus, Aspergillus niger represents the most efficient, highest yielding bioprocess in practice.</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 Aspergillus terreus, which also produces less desirable toxins. The aim is to transfer the entire lovastatin metabolic pathway from A. terreus into a GRAS organism like Aspergillus niger, which is closely related. A. niger is amenable to genetic engineering, has a high protein secretion capacity and produces high amounts of organic acids.</p>
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== Modularity Approach ==
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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>
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<p>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 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 A. niger.</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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== Abstract ==
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== A Toolkit for A. niger ==
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     <p>Economics is a vital aspect of any system, be it the world, a country, a person or even a microbe. Fungi are being used extensively by the biotechnology industry to produce useful compounds like drugs, organic acids, enzymes, fuels, etc. Thus efficient use of cellular resources is of prime importance in these industrial workhorses. Now the energy currency of any cell is Adenosine tri-phosphate (ATP) molecule and visualizing the dynamics of ATP levels in living cells has been a challenge. To this end we aim to produce a Fluorescence resonance energy transfer (FRET) based sensor for live cell ATP measurements in Aspergillus niger. With this we hope to measure ATP levels between compartments in growing Aspergillus niger during major metabolic shifts to understand the energy management processes in the cell.</p>
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     <p>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 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 A. niger.</p>
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Revision as of 08:53, 16 September 2013

Why Aspergillus nigem?

When IGEM encountered with Aspergillus niger

A Candidate 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 the fungal secondary metabolites that have the potential to prolong human lifespan. Citric acid production by the filamentous ascomycete fungus, Aspergillus niger represents the most efficient, highest yielding bioprocess in practice.

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 Aspergillus terreus, which also produces less desirable toxins. The aim is to transfer the entire lovastatin metabolic pathway from A. terreus into a GRAS organism like Aspergillus niger, which is closely related. A. niger is amenable to genetic engineering, has a high protein secretion capacity and produces high amounts of organic acids.

Modularity Approach

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.

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.

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 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 A. niger.


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