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| − | <h3>Cohesins and Dockerins</h3> | + | <h3 style="font-size:24px; font-family: Tahoma">The Alcohol-to-Carboxylic-Acid Pathway</h3> |
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| − | <p>The main gene products of interest in this work are known as Cohesins and Dockerins.</p>
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| − | <p>The Cohesins are structural proteins that form part of the outlay of the scaffold. These elements act as anchors for other proteins and are highly specific, which makes them reliable for the purpose of ordered display of proteins.</p> | + | <p>We believe that one of the strongest points about the scaffold is its ability to harbor proteins in an ordered fashion. This feature alone will have a huge impact on the engineering of processes, since artificial metabolic pathways can be tailored for specific needs, and then displayed on the scaffold for diverse industrial and research purposes. To affirm this presumption, we decided to build a metabolic pathway that could be easily measurable and in which the scaffold’s contribution to its efficiency could be readily measurable.</p> |
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| − | <p>The Dockerins are also proteins and are usually fused with some other enzyme. These elements act as adaptors for the enzyme they are attached to, and allow them to anchor themselves onto the Cohesin elements of the scaffold.</p> | + | <p>We prototyped the alcohol-to-carboxylic-acid pathway, which was thought to transform ethanol into acetic acid through a two-step mechanism. We displayed alcohol dehydrogenase and aldehyde dehydrogenase sequentially onto the scaffold which would, in principle, yield the desired compound in an efficient manner. The efficiency of the pathway would be readily measurable by assaying the production of the pathway’s final product, acetic acid. </p> |
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| − | <p>There are different types of Cohesins, with their respective complementary Dockerins, which make the scaffold a very powerful tool in the biotechnology landscape.</p>
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| − | <h3>Implications</h3>
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| − | <h4>Increased efficiency</h4>
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| − | <p>For many metabolic processes happening in the cell, the enzymatic substrate must be imported into the cytoplasm for processing. This usually requires energy from the cell, which diminishes the efficiency of the process. In the same way, if the metabolic product of such a reaction would be of interest, the organism would require a way to excrete it, which is, again, energetically consuming. With an external scaffold holding these enzymes in place, the import of substrate and export of product would be bypassed, and therefore the efficiency of a certain enzymatic process could be increased or facilitated.</p>
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| − | <h4>Ordered multi-step processes</h4>
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| − | <p>By ordering the correct pairs of cohesins with their respective dockerin-enzyme fusion products on the scaffold, it is possible to recreate or engineer diverse multi-step processes with high efficiency. Imitating the naturally occurring process of enzyme channeling, emulating multi-enzyme complexes and programming seemingly complex metabolic processes are only some of the many possible advantages of the scaffold.</p>
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| − | <h4>Fusion of non-prokaryotic proteins to scaffold</h4>
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| − | <p>Many proteins that have potential scientific or industrial interest are difficult or impossible to express in prokaryotic organisms in a useful manner. Eukaryotic enzyme-dockerin fusion products could be expressed in some other organisms, like yeast, and displayed extracellularly on the bacterial scaffold, giving a whole new toolkit to researchers and companies alike.</p>
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| − | <h3>Our proof of concept</h3>
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| − | <p>To demonstrate the immediate practicality and usefulness of our project, we decided to tackle a health issue that affects an important number of people all over the world.</p>
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| − | <p>Lactose intolerance is the inability to degrade the sugar lactose normally, due to an insufficient secretion of the enzyme responsible for this process, known as lactase. This inability to break down lactose often results in symptoms of the gastrointestinal tract, such as feeling of discomfort, stomach pain, bloating and diarrhoea, which prevents the people suffering from this condition to consume dairy in which this sugar is found abundantly.</p>
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| − | <p>By introducing a combination of the scaffold-displaying strain of Lactococcus lactis, which is naturally found in dairy products such as yogurt and cheese, and a dockerin-lactase fusion product as a supplement for the gastrointestinal tract of patients suffering from this condition, significant and long-lasting relief could be offered along with the possibility of consuming dairy products regularly. This option is particularly attractive and represents a safe, reliable and longer-lasting alternative for the currently offered solutions for this problem.</p>
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Project Description
“Every step of progress the world has made has been from scaffold to scaffold (...)”
Scaffococcus: Lactococcus scaffold
This year we genetically engineered a strain of the species Latococcus lactis by introducing optimized genes of the bacterium Clostridium thermocellum. Our ultimate goal was to obtain an organism capable of expressing a customizable extracellular platform that could harbour a very wide range of enzymes that, in turn, would be able to carry out a seemingly endless variety of metabolic processes. Due to its high potential for further engineering, we believe the scaffold to be a very important advancement in the biotechnology landscape.
We believe the scaffold to have many serious implications, since it may tackle many different issues such as customizing and facilitating the creation of synthetic metabolic pathways, and the processing of substrates in industrial processes.
The implications are:
For many metabolic processes happening in the cell, the enzymatic substrate must be imported into the cytoplasm for processing. This usually requires energy from the cell, which diminishes the efficiency of the process. In the same way, if the metabolic product of such a reaction would be of interest, the organism would require a way to excrete it, which is, again, energetically consuming. With an external scaffold holding these enzymes in place, the import of substrate and export of product would be bypassed, and therefore the efficiency of a certain enzymatic process could be increased or facilitated.
With the scaffold it is possible to recreate or engineer diverse multi-step processes with high efficiency. Imitating the naturally occurring process of enzyme channeling, emulating multi-enzyme complexes and programming seemingly complex metabolic processes are only some of the many possible advantages of the scaffold.
Many proteins that have potential scientific or industrial interest are difficult or impossible to express in prokaryotic organisms in a useful manner. Eukaryotic enzymes could be expressed in some other organisms, like yeast, and displayed extracellularly on the bacterial scaffold, giving a whole new toolkit to researchers and companies alike.
The Alcohol-to-Carboxylic-Acid Pathway
We believe that one of the strongest points about the scaffold is its ability to harbor proteins in an ordered fashion. This feature alone will have a huge impact on the engineering of processes, since artificial metabolic pathways can be tailored for specific needs, and then displayed on the scaffold for diverse industrial and research purposes. To affirm this presumption, we decided to build a metabolic pathway that could be easily measurable and in which the scaffold’s contribution to its efficiency could be readily measurable.
We prototyped the alcohol-to-carboxylic-acid pathway, which was thought to transform ethanol into acetic acid through a two-step mechanism. We displayed alcohol dehydrogenase and aldehyde dehydrogenase sequentially onto the scaffold which would, in principle, yield the desired compound in an efficient manner. The efficiency of the pathway would be readily measurable by assaying the production of the pathway’s final product, acetic acid.
