Team:NCTU Formosa/BP design

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<p>
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Global energy and environmental problems have stimulated increasing efforts towards synthesizing biofuel from renewable resources. Compared to the traditional biofuel, ethanol, higher alcohols offer advantages as gasoline substitutes because of their higher energy density and lower hygroscopicity<font size="12">﹝1﹞</font>. The biggest benefits butanol offers as an alternative to ethanol in motor fuel derive directly from its chemical and physical properties. The vapor pressure of butanol is well below that of ethanol at room temperature. That means a gasoline-butanol blend would not suffer from the volatility concerns that plague gasoline-ethanol blends, thus reducing evaporative emissions from vehicles using it. Importantly, butanol is much less soluble in water than ethanol. That means it is much less prone to the kind of separation problems that have kept ethanol out of petroleum product pipelines. It also suggests it should be easier to separate from water after fermentation than ethanol, requiring less energy. Overall, butanol raises some fascinating possibilities for our alternate fuels strategy<font size="12">﹝2﹞</font>.</p>
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Global energy and environmental problems have stimulated increasing efforts towards synthesizing biofuel from renewable resources. Compared to the traditional biofuel, ethanol, higher alcohols offer advantages as gasoline substitutes because of their higher energy density and lower hygroscopicity. <sub>(1)</sub> The biggest benefits butanol offers as an alternative to ethanol in motor fuel derive directly from its chemical and physical properties. The vapor pressure of butanol is well below that of ethanol at room temperature. That means a gasoline-butanol blend would not suffer from the volatility concerns that plague gasoline-ethanol blends, thus reducing evaporative emissions from vehicles using it. Importantly, butanol is much less soluble in water than ethanol. That means it is much less prone to the kind of separation problems that have kept ethanol out of petroleum product pipelines. It also suggests it should be easier to separate from water after fermentation than ethanol, requiring less energy. Overall, butanol raises some fascinating possibilities for our alternate fuels strategy. <sub>(2)</sub> </p>
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<hr> <h3>
Reference﹝1﹞:Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.
Reference﹝1﹞:Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.
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Reference﹝2﹞: Geoffrey Styles (2006) Higher Alcohols <a href=”http://energyoutlook.blogspot.com/2006/08/higher-alcohols.html”>http://energyoutlook.blogspot.com/2006/08/higher-alcohols.html</a></font>
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Reference﹝2﹞: Geoffrey Styles (2006) Higher Alcohols <a href="http://energyoutlook.blogspot.com/2006/08/higher-alcohols.html">http://energyoutlook.blogspot.com/2006/08/higher-alcohols.html</a></h3>
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<br>
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<h2>Design<h2>
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<h2> Design
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</h2>
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<p>
<p>
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In traditional genetic engineering method(Figure 1), we use strong promoter to initiate our genes, but this way E.coli will overexpress the proteins we need in synthetic pathway. However, overexpression of target proteins will let E.coli waste its limited growth resources, or the activity and performance of the enzymes may be too low. In this situation, it unbalances the synthetic pathway, and the production of isobutanol will not be optimum. This is also a problem in the production of isobutanol which is poisonous to E.coli. </p>  
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In traditional genetic engineering method(Figure 1), we use strong promoter to initiate our genes, however there are several disadvantages in this method. Firstly, E.coli will overexpress the target proteins which we need in synthetic pathway. However, this over-expression will cause E.coli wastes its limited growth resources. Then on the other hand, the activity and performance of the enzymes could be too low to produce enough product in the end. Therefore, it will unbalance the synthetic pathway, and the production of isobutanol will not be optimum. In addition, this is also a major problem in the production of isobutanol which is poisonous to E.coli. </p>  
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<br>
<div><img src = "http://partsregistry.org/wiki/images/5/59/Butanol-1.png" width="700"></div>
<div><img src = "http://partsregistry.org/wiki/images/5/59/Butanol-1.png" width="700"></div>
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<br><b> Figure 1.</b><br>
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<br><b> Figure 1.</b>Traditional genetic engineering method.
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Traditional genetic engineering method.
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<p>
<p>
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To solve the problem, we need to adjust the expression of the genes, and make sure every intermediate can be catalyzed by the very next enzyme. The intermediates of isobutanol can be catalyzed step by step till they become the target products we want. (the reaction pathway is shown in the picture of Overall pathway) In the new method we design, we control the pathway by stopping the mechanism when it reaches the step to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate, then under specific thermal control, the mechanism would continue to express. The advantage of our new method is that the precursors are much less toxic for E.coli than our target product (isobutanol) is. Applying this new method to our project, we first accumulate lots of the non-toxic intermediate as the precursor, 2-Ketoisovalerate, to a certain amount, and then convert the entire non-toxic precursor into the product, isobutanol, all at once.</p>
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To solve the problem, we need to adjust the expression of the genes, and make sure every intermediates can be catalyzed by the very next enzyme. The intermediates of isobutanol can be catalyzed step by step till they become the target products we want. (the reaction pathway is shown in the picture of <b>Overall pathway</b>) We design a new method with which we can control the pathway by stopping the mechanism when it reaches particular step where it starts to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate, then under specific thermal control, the mechanism would continue to express. The advantage of our new method is that the precursors are much less toxic for E.coli than our target product (isobutanol) is. We then apply this new method to our project. We first accumulate lots of the non-toxic intermediate as the precursor, 2-Ketoisovalerate, to a certain amount, and then convert the entire non-toxic precursor into the product, isobutanol, all at once.</p>
<p>
<p>
We clone the genes which will be translated into enzymes such as AlsS, IlvC, IlvD ,KivD, and assemble the genes into two circuits as following. The enzymes are crucial for producing butanol.(Reference: Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.)
We clone the genes which will be translated into enzymes such as AlsS, IlvC, IlvD ,KivD, and assemble the genes into two circuits as following. The enzymes are crucial for producing butanol.(Reference: Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.)
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Following Figure 2 is about the strains where the alss, ilvC, ilvD and kivd genes are cloned from, with their point mutation sites: </p>
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Following Figure 2 is about the strains where the alss, ilvC, ilvD and kivd genes are cloned from, along with their point mutation sites: </p>
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<div><img src = "http://partsregistry.org/wiki/images/7/7c/Butanol-2.png" width="500"></div>
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<div><img src = "http://partsregistry.org/wiki/images/7/7c/Butanol-2.png" width="600"></div>
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<br><b> Figure 2.</b We cloned four gene, ilvC, ilvD, AlsS, kivd. The figure above are the names, strains, length and point mutation of four genes.
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<br><b> Figure 2.</b> We cloned four gene, ilvC, ilvD, AlsS, kivd. The figure above are the names, strains, length and point mutation of four genes.
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<p>
<p>
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In order to achieve the goal of our experimental design, we apply fermentation of carbohydrate to gain our product. First, we choose glucose as the resource to get through biosynthetic pathway, and we can harvest isobutanol which is the derivative of butanol. Glucose can be catalyzed into isobutanol through afterward enzymes- Alss, Ilvc, Ilvd,and Kivd step by step. We can also stop any step to accumulate the intermediates we want (see picture Overall Reaction). </p>
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In order to achieve the goal of our experimental design, we apply carbohydrate fermentation to gain our product. First, we choose glucose as the resource to get through biosynthetic pathway, and we can harvest isobutanol which is the derivative of butanol. Glucose can be catalyzed into isobutanol through enzymes- Alss, Ilvc, Ilvd,and Kivd step by step. We can also stop any step to accumulate the intermediates we want (see picture <b>Overall Reaction</b>). And we combine our low-temperature release device with butanol pathway because isobutanol will easily vaporize in high temperature, and the specific enzyme involved has better activity at 30℃. </p>
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<div><img src = "http://partsregistry.org/wiki/images/d/d5/RNA_data_2-2.jpg" width="700"></div>
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<div><img src = "https://static.igem.org/mediawiki/2011/2/28/Kivd2.jpg" width="500"></div>
<br><b> Overall Reaction: </b> We choose glucose as the resource to get through biosynthetic pathway. Glucose can be catalyzed into isobutanol through afterward enzymes- Alss, Ilvc, Ilvd,and Kivd. We can also stop any step to accumulate the intermediate we want.
<br><b> Overall Reaction: </b> We choose glucose as the resource to get through biosynthetic pathway. Glucose can be catalyzed into isobutanol through afterward enzymes- Alss, Ilvc, Ilvd,and Kivd. We can also stop any step to accumulate the intermediate we want.
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<b>Circuit A.</b>                                                                               
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<b>Circuit A.</b>
 
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<div><img src = "http://partsregistry.org/wiki/images/5/58/Butanol-3.png" width="700"></div>
<div><img src = "http://partsregistry.org/wiki/images/5/58/Butanol-3.png" width="700"></div>
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<br><b Figure 3. Circuit A( <a href=“http://partsregistry.org/Part: BBa_K539691”>BBa_K539691 </a>, pSB1c3, 6200bp):</b> The expression of alss ilvC and ilvD <br><br>
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<br><b> Figure 3. Circuit A( <a href="http://partsregistry.org/Part:BBa_K539691">BBa_K539691 </a>, pSB1c3, 6200bp):</b> The expression of alss ilvC and ilvD <br><br>
<p>
<p>
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n circuit A (<a href=“http://partsregistry.org/Part: BBa_K539691”>BBa_K539691 </a>),Plac (<a href=“http://partsregistry.org/Part: BBa_R0010”>BBa_R0010</a>) is lacI regulated promoter to initiate the circuit to produce Alss, IlvC and IlvD. Gene alss, ilvC and ilvD will be translated into enzymes and catalyze pyruvate into 2-Ketoisovalerate. And the 37℃induced RBS we use is <a href=“http://partsregistry.org/Part: BBa_K115002”>BBa_K115002 </a>. The terminator is BBa J61048. The host strain is DH5α. This circuit inhibits part <a href=“http://partsregistry.org/Part:BBa_K539742”>BBa_K539742 </a> (Figure 4) by producing TetR which is regulated by 37℃ RBS at 37℃ or highr.
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In circuit A (<a href="http://partsregistry.org/Part:BBa_K539691">BBa_K539691 </a>),Plac (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) is lacI regulated promoter to initiate the circuit to produce Alss, IlvC and IlvD. Gene alss, ilvC and ilvD will be translated into enzymes and catalyze pyruvate into 2-Ketoisovalerate. And the 37℃induced RBS we use is <a href="http://partsregistry.org/Part:BBa_K115002">BBa_K115002 </a>. The terminator is <a href="http://partsregistry.org/Part:BBa_J61048">BBa_J61048</a>. The host strain is DH5α. This circuit inhibits part <a href="http://partsregistry.org/Part:BBa_K539742">BBa_K539742 </a> (Figure 4) by producing TetR which is regulated by 37℃ RBS at 37℃ or higher.
</p>
</p>
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<b>Circuit B.</b>
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<b>Circuit B.</b><hr>
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<div><img src = " http://partsregistry.org/wiki/images/8/8b/Butanol-4.png" width="350"></div>
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<br><b>Figure 4. Circuit B (<a href="http://partsregistry.org/Part:BBa_K539742">BBa_K539742 </a>, pSB1c3, 1864bp):</b> The expression of kivd. <br><br>
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<div><img src = " http://partsregistry.org/wiki/images/8/8b/Butanol-4.png" width="450"></div>
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<br><b> Circuit B (<a href=“http://partsregistry.org/Part:BBa_K539742”>BBa_K539742 </a>, pSB1c3, 1864bp):</b> The expression of kivd. <br><br>
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<p>
<p>
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In circuit B (<a href=“http://partsregistry.org/Part:BBa_K539742”>BBa_K539742 </a>), Ptet <a href=“http://partsregistry.org/Part:BBa_R0040”>BBa_R0040 </a>is TetR repressible promoter, and the RBS we use is <a href=“http://partsregistry.org/Part:BBa_B0034”>BBa_B0034 </a>. Kivd (<a href=“http://partsregistry.org/Part:BBa_K539714”>BBa_K539714 </a>) encodes alpha-ketoisovalerate decarboxylase which is responsible for the decarboxylation into aldehydes of α-keto acids derived from amino acid transamination. The terminator is <a href=“http://partsregistry.org/Part:BBa_B0015”>BBa_B0015</a>.
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In circuit B (<a href="http://partsregistry.org/Part:BBa_K539742">BBa_K539742 </a>), Ptet <a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040 </a>is TetR repressible promoter, and the RBS we use is <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034 </a>. Kivd (<a href="http://partsregistry.org/Part:BBa_K539714">BBa_K539714 </a>) encodes 2-keto-acid decarboxylases which convert 2-Ketoisovalerate into aldehyde and then to isobutanol by alcohol dehydrogenases. Alcohol dehydrogenases are produced by E.coli itself naturally. The terminator is <a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>.
</p>
</p>
<p>
<p>
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By viewing our two circuits (Figure 3 and Figure 4) we could observe one circumstance that when the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet (Figure 5). In this way, part <a href=“http://partsregistry.org/Part:BBa_K539742”>BBa_K539742</a> will not be expressed, and if we keep the temperature over 37℃, we can accumulate the intermediate, 2-Ketoisovalerate, which is non-toxic for E.Coli. In the other hand, when we lower the temperature under 37℃, the37℃ induced RBS (<a href=“http://partsregistry.org/Part:BBa_K115002”>BBa_K115002</a>) will not express so that part <a href=“http://partsregistry.org/Part:BBa_K539742”>BBa_K539742 </a> will not be inhibited by TetR (Figure 6). At the same time, KivD will be produced and start to catalyze 2-Ketoisovalerate into isobutyraldehyde and to isobutanol by another enzyme produced by E.coli naturally (the enzyme is not shown in Overall Reaction). The reason why we use low-temperature released device is because isobutanol is easy to evaporate and the enzyme which catalyze sobutyraldehyde to isobutanol has better activity in lower temperature, 30℃.
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By viewing our two circuits (Figure 3 and Figure 4) we could observe one circumstance that when the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet (Figure 5). In this way, part <a href="http://partsregistry.org/Part:BBa_K539742">BBa_K539742</a> will not be expressed, and if we keep the temperature over 37℃, we can accumulate the intermediate, 2-Ketoisovalerate, which is non-toxic for E.Coli. In the other hand, when we lower the temperature under 37℃, the 37℃ induced RBS (<a href="http://partsregistry.org/Part:BBa_K115002">BBa_K115002</a>) will not express so that part <a href="http://partsregistry.org/Part:BBa_K539742">BBa_K539742 </a> will not be inhibited by TetR (Figure 6). At the same time, KivD will be produced and start to catalyze 2-Ketoisovalerate into isobutyraldehyde and to isobutanol by alcohol dehydrogenases which is produced by E.coli naturally (the enzyme is not shown in <b>Overall Reaction</b>). The reason why we use low-temperature released device is because isobutanol is easy to evaporate and the enzyme which catalyze isobutyraldehyde to isobutanol has better activity in lower temperature, 30℃.
</p>
</p>
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<div><img src = " http://partsregistry.org/wiki/images/c/ca/Butanol-5.png" width="700"></div>
<div><img src = " http://partsregistry.org/wiki/images/c/ca/Butanol-5.png" width="700"></div>
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<br><b> Figure 5. </b> When the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet. In this way, part <a href=“http://partsregistry.org/Part:BBa_K539742”>BBa_K539742 </a> will not be expressed.
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<br><b> Figure 5. </b> When the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet. In this way, part <a href="http://partsregistry.org/Part:BBa_K539742">BBa_K539742 </a> will not be expressed.
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<div><img src = " http://partsregistry.org/wiki/images/b/ba/Butanol-6.png" width="700"></div>
<div><img src = " http://partsregistry.org/wiki/images/b/ba/Butanol-6.png" width="700"></div>
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<br><b> Figure 6.</b> When we lower the temperature under 37℃, the37℃ induced RBS (<a href=“http://partsregistry.org/Part:BBa_K115002”>BBa_K115002</a>) will not express so that part <a href=“http://partsregistry.org/Part:BBa_K539742”>BBa_K539742</a> will not be inhibited by TetR. At the same time, KivD will be produced and start to catalyze 2-Ketoisovalerate into isobutanol.
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<br><b> Figure 6.</b> When we lower the temperature under 37℃, the37℃ induced RBS (<a href="http://partsregistry.org/Part:BBa_K115002">BBa_K115002</a>) will not express so that part <a href="http://partsregistry.org/Part:BBa_K539742">BBa_K539742</a> will not be inhibited by TetR. At the same time, KivD will be produced and start to catalyze 2-Ketoisovalerate into isobutanol.
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Latest revision as of 03:37, 6 October 2011



Butanol pathway

Motivation

Global energy and environmental problems have stimulated increasing efforts towards synthesizing biofuel from renewable resources. Compared to the traditional biofuel, ethanol, higher alcohols offer advantages as gasoline substitutes because of their higher energy density and lower hygroscopicity. (1) The biggest benefits butanol offers as an alternative to ethanol in motor fuel derive directly from its chemical and physical properties. The vapor pressure of butanol is well below that of ethanol at room temperature. That means a gasoline-butanol blend would not suffer from the volatility concerns that plague gasoline-ethanol blends, thus reducing evaporative emissions from vehicles using it. Importantly, butanol is much less soluble in water than ethanol. That means it is much less prone to the kind of separation problems that have kept ethanol out of petroleum product pipelines. It also suggests it should be easier to separate from water after fermentation than ethanol, requiring less energy. Overall, butanol raises some fascinating possibilities for our alternate fuels strategy. (2)


Reference﹝1﹞:Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89. Reference﹝2﹞: Geoffrey Styles (2006) Higher Alcohols http://energyoutlook.blogspot.com/2006/08/higher-alcohols.html


Design

In traditional genetic engineering method(Figure 1), we use strong promoter to initiate our genes, however there are several disadvantages in this method. Firstly, E.coli will overexpress the target proteins which we need in synthetic pathway. However, this over-expression will cause E.coli wastes its limited growth resources. Then on the other hand, the activity and performance of the enzymes could be too low to produce enough product in the end. Therefore, it will unbalance the synthetic pathway, and the production of isobutanol will not be optimum. In addition, this is also a major problem in the production of isobutanol which is poisonous to E.coli.



Figure 1.Traditional genetic engineering method.

To solve the problem, we need to adjust the expression of the genes, and make sure every intermediates can be catalyzed by the very next enzyme. The intermediates of isobutanol can be catalyzed step by step till they become the target products we want. (the reaction pathway is shown in the picture of Overall pathway) We design a new method with which we can control the pathway by stopping the mechanism when it reaches particular step where it starts to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate, then under specific thermal control, the mechanism would continue to express. The advantage of our new method is that the precursors are much less toxic for E.coli than our target product (isobutanol) is. We then apply this new method to our project. We first accumulate lots of the non-toxic intermediate as the precursor, 2-Ketoisovalerate, to a certain amount, and then convert the entire non-toxic precursor into the product, isobutanol, all at once.

We clone the genes which will be translated into enzymes such as AlsS, IlvC, IlvD ,KivD, and assemble the genes into two circuits as following. The enzymes are crucial for producing butanol.(Reference: Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.) Following Figure 2 is about the strains where the alss, ilvC, ilvD and kivd genes are cloned from, along with their point mutation sites:



Figure 2. We cloned four gene, ilvC, ilvD, AlsS, kivd. The figure above are the names, strains, length and point mutation of four genes.

In order to achieve the goal of our experimental design, we apply carbohydrate fermentation to gain our product. First, we choose glucose as the resource to get through biosynthetic pathway, and we can harvest isobutanol which is the derivative of butanol. Glucose can be catalyzed into isobutanol through enzymes- Alss, Ilvc, Ilvd,and Kivd step by step. We can also stop any step to accumulate the intermediates we want (see picture Overall Reaction). And we combine our low-temperature release device with butanol pathway because isobutanol will easily vaporize in high temperature, and the specific enzyme involved has better activity at 30℃.



Overall Reaction: We choose glucose as the resource to get through biosynthetic pathway. Glucose can be catalyzed into isobutanol through afterward enzymes- Alss, Ilvc, Ilvd,and Kivd. We can also stop any step to accumulate the intermediate we want.


Circuit A.

Figure 3. Circuit A( BBa_K539691 , pSB1c3, 6200bp): The expression of alss ilvC and ilvD

In circuit A (BBa_K539691 ),Plac (BBa_R0010) is lacI regulated promoter to initiate the circuit to produce Alss, IlvC and IlvD. Gene alss, ilvC and ilvD will be translated into enzymes and catalyze pyruvate into 2-Ketoisovalerate. And the 37℃induced RBS we use is BBa_K115002 . The terminator is BBa_J61048. The host strain is DH5α. This circuit inhibits part BBa_K539742 (Figure 4) by producing TetR which is regulated by 37℃ RBS at 37℃ or higher.


Circuit B.

Figure 4. Circuit B (BBa_K539742 , pSB1c3, 1864bp): The expression of kivd.

In circuit B (BBa_K539742 ), Ptet BBa_R0040 is TetR repressible promoter, and the RBS we use is BBa_B0034 . Kivd (BBa_K539714 ) encodes 2-keto-acid decarboxylases which convert 2-Ketoisovalerate into aldehyde and then to isobutanol by alcohol dehydrogenases. Alcohol dehydrogenases are produced by E.coli itself naturally. The terminator is BBa_B0015.

By viewing our two circuits (Figure 3 and Figure 4) we could observe one circumstance that when the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet (Figure 5). In this way, part BBa_K539742 will not be expressed, and if we keep the temperature over 37℃, we can accumulate the intermediate, 2-Ketoisovalerate, which is non-toxic for E.Coli. In the other hand, when we lower the temperature under 37℃, the 37℃ induced RBS (BBa_K115002) will not express so that part BBa_K539742 will not be inhibited by TetR (Figure 6). At the same time, KivD will be produced and start to catalyze 2-Ketoisovalerate into isobutyraldehyde and to isobutanol by alcohol dehydrogenases which is produced by E.coli naturally (the enzyme is not shown in Overall Reaction). The reason why we use low-temperature released device is because isobutanol is easy to evaporate and the enzyme which catalyze isobutyraldehyde to isobutanol has better activity in lower temperature, 30℃.



Figure 5. When the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet. In this way, part BBa_K539742 will not be expressed.



Figure 6. When we lower the temperature under 37℃, the37℃ induced RBS (BBa_K115002) will not express so that part BBa_K539742 will not be inhibited by TetR. At the same time, KivD will be produced and start to catalyze 2-Ketoisovalerate into isobutanol.