Team:Tokyo Tech/Modeling/Urea-cooler/urea-cooler

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<ul>
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<li><a href="#intro">1. Introduction</a></li>
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                <li><a href="#abstract">1. Abstract</a></li>
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<li><a href="#results">2. Results</a></li>
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<li><a href="#intro">2. Introduction</a></li>
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<li><a href="#future">3. Future work</a></li>
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                <li><a href="#flux">2.1 What is Elementary Flux Modes?</a></li>
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                <li><a href="#analyzing">2.2 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</a></li>
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                <li><a href="#modes">2.3 Finding Modes to Increase the Urea production by <i>E. coli</i></a></li>
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                <li><a href="#results">3. Results</a></li>
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                <li><a href="#function">3.1 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</a></li>
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                <li><a href="#increase">3.2 Finding Modes to Increase the Urea production by <i>E. coli</i></a></li>
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<li><a href="#future">4. Future work</a></li>
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<h1> Flux analysis for providing more urea </h1>
<h1> Flux analysis for providing more urea </h1>
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<h2 id=abstract>1. Abstract </h2>
<p>
<p>
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<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="750px" />
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This section is about a metabolic engineering study we did about the urea cycle. On the first part we show how we used &ldquo;elementary flux modes&rdquo; (Schuster <i>et al</i>., 2000) to analyze the function of the compounds involved in the urea cycle. Mainly we deduced which compounds act as sources of carbon and sources of nitrogen for the production of urea. On the second part of this study we show how we determined elementary flux modes of the urea cycle to find ways to increase the yield of urea. We focused on a strategy which involves increasing the concentration of four components of the cycle and which we concluded would yield more urea. To confirm our results future experiments will be done.
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<div class="graph_title">
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</p>
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<br />Fig.5 The reactions related with the urea cycle
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</div>
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<div class="graph_title">
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<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="750px" /><br />
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Fig. 5 The reactions related with the urea cycle
 +
</div>
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene">rocF</span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.<br />
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene">rocF</span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.<br />
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<h2 id="intro">1. Introduction</h2>
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<h2 id="intro">2. Introduction</h2>
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<h3 id="flux">2.1 What is Elementary Flux Modes?</h3>
<p>
<p>
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In metabolic engineering, mathematical modeling is the effective way to increase the products.
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In Metabolic Engineering, mathematical modeling is an effective way to increase the products of a reaction. In particular, Flux Analysis, which is based on the hypothesis that the system is in a steady state, is effective to find how to increase these products. The concept of elementary flux mode provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. As a method of metabolic flux analysis, it is based on the hypothesis that the concentration of the reactants and products involved in the cycle does not change.
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Flux analysis, based on the hypothesis that the system is in steady state,  
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is the effective way to find how to increase the products. In this work,
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we firstly focused on the concept of 'elementary flux modes' (<i>Schuster et al, 2000</i>),
+
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which provides metabolic routes both stoichiometrically and thermodynamically feasible. <br />
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One elementary mode shows that the carbon atom of urea derives from HCO<sub>3</sub><sup>-</sup> which abounds
+
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in bacterial cytoplasm. Furthermore, in spite of L-glutamine consumption to transfer
+
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the side-chain ammonium group for production of carbamoyl phosphate which transfers
+
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the ammonium group to the urea cycle, ammonium ion can restore L-glutamine from
+
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L-glutamate which is a byproduct of the carbamoyl phosphate production.  
+
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These findings suggest that there was little difference in the condition of
+
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containing NH<sub>3</sub> or L-glutamine in the culture to obtain more urea. This finding is
+
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proven in previous report's experiment. (<i>Mendel et al, 1996</i>) We also confirmed that
+
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the urea cycle in <span class="name">E.coli</span> is well designed in a stoichiometrically point of view.<br />
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To provide more urea, there are two strategies. First one is to increase
+
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the amount of carbamoyl phosphate which is the reactant of the rate-limiting step of the urea cycle. The second one is to increase
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the amount of four components of the urea cycle: L-ornithine, L-citrulline, N-(L-arginino)succinate, L-arginine.  We thus identified the elementary flux modes which produce these compounds from L-glutamate or compounds in TCA cycle: 2-oxoglutarate, oxaloacetate, L-malate, fumarate. In all modes, ornithine was intermediate or final product to produce the four components of the urea cycle. Furthermore, all modes include the reaction which converts L-glutamate to L-ornithine to produce L-ornithine. We also confirmed that E. coli have no feasible route for production of the four compounds other than those indicated in Fig.5.
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Therefore, ornithine production needs the reaction which requires ATP, NADPH, Acetyl-CoA, and L-glutamine, is the necessary step in this strategy.  We will confirm the effect of L-glutamate supplement in future wet experiments for not only urea production but also the intermediates like L-ornithine. Positions of L-arginine and L-glutamate in the reaction network show that supplement of these compounds has similer effect on urea production. <br />
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From another point of view that L-aspartate is consumed in protein
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biosynthesis, this compounds should be supplied from medium or produced
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by <i>E. coli</i> itself not only for increasing the amount of urea but for
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maintenance of the cycle.
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<br /><br />
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</p>
</p>
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<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="Fig7" width="400px" />
 
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<span style="font-style:italic, bold;"> <br /><big><b>What is elementary flux mode</b></big></span>
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<h3 id="analyzing">2.2 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</h3>
<p>
<p>
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"The concept of elementary flux mode provides a mathematical tool to define
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By determining the elementary flux modes of a cycle we can have a more clear view of the function of each of the compounds involved in the cycle being analyzed. Based on the elementary flux modes of the urea cycle, in this study we could deduce that HCO<sub>3</sub><sup>-</sup> acts as the source of carbon for urea production and that both L-glutamine and NH<sub>3</sub> act as nitrogen sources for the formation of urea.
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and comprehensively describe all metabolic routes that are both stoichiometrically
+
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and thermodynamically feasible for a group of enzymes".  
+
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(<i>Schuster et al, 2000</i>) We can determine the modes which are able to work at steady state
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by this analysis. <br />
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As application of elementary flux modes, we can expect what substrates are needed
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to produce to the substances of interest. Furthermore, we can find expect which
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enzymes to overexpress or knockout so as to maximize the products we want.
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</p>
</p>
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<center>
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<h2 id="results">2. Results</h2>
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<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="193px" />
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<span style="font-size:15;">&rarr;</span>
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<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="193px" />
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<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="Fig7" width="400px" />
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</center>
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<h3 id="modes">2.3 Finding Modes to Increase the Urea production by <i>E. coli</i></h3>
<p>
<p>
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Overall reactions related with the urea cycle
+
In this study we determined elementary flux modes to maximize urea production by <i>E. coli</i>. We found that there are two main strategies to increase urea production: one is to increase the amount of carbamoyl phosphate (which formation is known to be the rate-limiting step of the urea cycle). The other one is to increase the concentration of four components of the urea cycle: L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine. We deduced the latter strategy by determining the elementary modes of the urea cycle, and therefore in this study we will focus on the description of this strategy.
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We considered the enzymatic reactions shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a> to determine the elementary  
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</p>
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flux modes. The scheme of the reaction is shown in Fig.5.<br />
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<div align="center">
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="500px" /><br />
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<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="600px" /></div>
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Fig. 8 Two ways to increase urea production
 +
</div>
 +
<h2 id="results">3. Results</h4>
 +
<p>
 +
In our study, we considered the enzymatic reactions shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a> to determine the elementary flux modes related to urea production by <i>E. coli</i>. The scheme the overall reaction system is shown in Fig. 5 below.
 +
</p>
 +
<div align="center">
 +
<img src="https://static.igem.org/mediawiki/2011/e/e7/TokyoTech_Urea-fig5.png" alt="Fig5" width="600px" /></div>
<div class="graph_title">
<div class="graph_title">
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<br />Fig.5 The reactions related with the urea cycle
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<br />Fig. 5 The reactions related with the urea cycle
-
</div>
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</div>
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*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene">rocF</span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.<br />
+
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing <span class="gene">rocF</span> gene. For complete names of the enzymes see <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table3">Table 3</a>.
 +
<p>
 +
By determining the elementary flux modes to produce urea inside <i>E. coli</i>, we found two important results:
</p>
</p>
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<ol>
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<span style="font-style:italic,borld">
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<li><p>
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<br /><big><b>One of the urea producing cycles without supplying the intermediates</b></big>
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We confirmed both L-glutamine and NH<sub>3</sub> act as nitrogen providers in the urea cycle, as well as deducing that HCO<sub>3</sub><sup>-</sup> acts as the source of carbon for urea production. These modes did not make use of organic intermediates. Even though L-glutamine is consumed in order to to transfer the side-chain ammonium group needed for the production of carbamoyl phosphate (which in turn transfers the ammonium group to the urea cycle), free ammonium ion can restore L-glutamine from L-glutamate (which is a byproduct of the reaction that yields carbamoyl phosphate as a product).
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</span>
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</p></li>
 +
<li><p>
 +
We concluded that increasing the concentration of L-ornithine will increase the concentration of three related compounds (L-citrulline, N-(L-arginino)succinate, and L-arginine) and this will ultimately lead to an increase in the production of urea. We also noted that since the L-aspartate amino acid, which is needed in the urea cycle we considered(Fig. 5), is normally consumed in protein biosynthesis, so it should be supplied in the culture medium or synthetized by ,<span class="name">E. coli</span> in order to be able to increase the amount of urea and to maintain the cycles that produce it.
 +
</p></li>
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</ol>
<p>
<p>
-
At first, we attempted to get the elementary flux modes in the condition whose
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Below is a detailed description of these three results.
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input is L-glutamine like previous reports. (<i>Mendel et al, 1996</i>) We determined the
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elementary flux modes by calculating matrix like. The initial tableau is shown below.
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</p>
</p>
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<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="800px" />
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                <h3 id="function">3.1 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes</h3>
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<br /><br />We calculated and got the final tableau.<br />
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<p>
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<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="800px" />
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The first step was to determine the flux modes which need of L-glutamine as an input (Mendel <i>et al</i>., 1996</i>). We did this by calculations based on a matrix as the tableau shown below.
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<a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/method">The way to calculate is here.</a>.
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</p>
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<center>
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<img src="https://static.igem.org/mediawiki/2011/1/14/T%280%29.png" alt=T(0) width="800px" /><br />
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<img src="https://static.igem.org/mediawiki/2011/2/28/T9.png" alt=T9 width="800px" />
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</center>
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<a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/method" align="right">Details about the calculations can be found here</a>
<p>
<p>
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We got eight modes shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem1">Fig.6</a>. Each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table4">Table 4</a>.  
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We found eight modes that can produce urea without using organic intermediates. These are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem1">Fig. 6</a>. Each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table4">Table 4</a>. In particular, we focused on one the mode displayed in Fig. 7.
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We focused on one of the urea producing modes in these eight modes as shown in Fig.7.
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</p>
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</p>
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<div align="center">
<div align="center">
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<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="fig7" width="750px" /></div>
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<img src="https://static.igem.org/mediawiki/2011/8/83/Urea_modeling.png" alt="fig7" width="750px" />
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</div>
<div class="graph_title">
<div class="graph_title">
<div style="font-size:larger">
<div style="font-size:larger">
<b>2NH<sub>3</sub> + HCO<sub>3</sub><sup>-</sup> + 3ATP + H<sub>2</sub>O + NADPH + NAD<sup>+</sup>  
<b>2NH<sub>3</sub> + HCO<sub>3</sub><sup>-</sup> + 3ATP + H<sub>2</sub>O + NADPH + NAD<sup>+</sup>  
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Urea + 2ADP + AMP + 2Pi + PPi + NADP<sup>+</sup> + NADH</b>
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&rarr; Urea + 2ADP + AMP + 2Pi + PPi + NADP<sup>+</sup> + NADH</b>
</div>
</div>
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Fig.7 One of the urea producing cycles leaded by the concept of elementary flux modes
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Fig. 7 One of the urea producing cycles leaded by the concept of elementary flux modes<br />
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*The numbers indicate the relative flux carried by the enzymes.<br /><br />
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*The numbers indicate the relative flux carried by the enzymes.
</div>
</div>
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<p>
<p>
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If we compared Fig.5 and Fig.7, we can see that in the mode displayed in Fig.7
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As shown in Fig. 7, we deduced that the carbon atom of urea is provided from HCO<sub>3</sub><sup>-</sup> , which is a byproduct of respiration and therefore is already an abundant compound in the bacterial cytoplasm. On the other hand, we also confirmed that carbamoyl phosphate is a nitrogen source for urea production.We also found that the function of L-glutamine in the urea cycle is to provide nitrogen for urea production via carbamoyl phosphate, because ammonium ion can restore L-glutamine from L-glutamate (which is a
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the reaction which converts L-glutamate to L-ornithine is not needed for urea production. <br />
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byproduct of the reaction that yields carbamoyl phosphate as a product). This conclusion was confirmed experimentally by Mendel <i>et al.</i> (1996).  Also, since only providing a nitrogen source is enough to increase urea production by <i>E. coli</i>, we can also conclude that the aritificial urea cycle in <i>E. coli</i> is stoichiometrically well designed. By comparing Fig. 5 and Fig. 7 we can also observe that, in Fig. 7, the reaction which converts L-glutamate to L-ornithine is not needed for urea production.  
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As shown in Fig.7, the carbon atom of urea derives from HCO<sub>3</sub><sup>-</sup> which abounds
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in bacterial cytoplasm.  
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</p>
</p>
 +
 +
<h3 id="increase">3.2 Finding Modes to Increase the Urea production by <i>E. coli</i></h3>
<p>
<p>
-
Considering about nitrogen sources of urea, one of the nitrogen is derived
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There are two ways to obtain more products from a cycle of reactions: increasing the speed the reactions and increasing the concentration of the reactants. This becomes obvious if we think of the cycle as a track which is travelled by cars (the reactants), and the products as the total sum of the number of laps made by every car. If we double the speed of the cars the number of laps will also double (Fig. 8, lower left). Similarly, if we double the number of cars, the number of laps will double as well (Fig. 8, lower right). We applied this analogy to the urea cycle, where the metabolites in the cycle are represented by the cars and the total number of laps represents the total urea yield (as shown in the figure below).<br />
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from carbamoyl phosphate. Carbamoyl phosphate transfers the ammonium group to  
+
Increasing the velocity of the cars corresponds to increasing the amount of carbamoyl phosphate in the urea cycle, because the reaction which converts L-glutamine to carbamoyl phosphate is the rate-limiting reaction of the cycle. On the other hand, increasing the number of the cars correspond to increasing the concentration of the compounds of the urea cycle. We focused on increasing the concentration the compounds of the urea cycle to find ways to increase the urea yield.<br />
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the urea cycle from L-glutamine. Therefore, it seems that L-glutamine is the  
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<div align="center">
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effective nitrogen source. However, free ammonium ion can restore L-glutamine from
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<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="700px" /><br />
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L-glutamate which is a byproduct of the carbamoyl phosphate production.
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Fig. 8 Two ways to increase urea production
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It means that L-glutamine and NH<sub>3</sub> have the same role to be the nitrogen source
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</div>
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for urea. These finding suggest that there was little difference in the  
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condition of containing NH<sub>3</sub> or L-glutamine in the culture to obtain more urea.  
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This finding is proven in previous report's experiment. (<i>Mendel et al, 1996</i>)
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We also confirmed that the urea cycle in <span class="name">E. coli</span> is well designed in a stoichiometrically
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point of view.
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</p>
</p>
<p>
<p>
-
<span style="font-style:bold;"><br /><big><b>Increasing the four components of the urea cycle to provide more urea</b></big><br /></span>
+
L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine are four important compounds of the urea cycle. As can be seen in Fig. 7, these compounds form a sub-cycle that directly yields urea. Therefore, by increasing the yield of this cycle we can increase the production of urea in <i>E. coli</i>.
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To obtain more urea, we introduced the easy allegory. There are 3 cars in the cycle. We count the number of the cars driving in front of us during some period of time. There are two ways to increase in the number we count. One way is increasing the velocity of the cars.(Fig.8 The corner of the left side) The other way is increasing the number of the cars driving in the cycle.(Fig.8 The corner of the right side) The cars in this allegory are compounds of the urea cycle. The number we count are the product, urea. Increasing the velocity of the cars means to increase the amount of carbamoyl phosphate because the reaction which converts L-glutamine to carbamoyl phosphate is the rate-limiting. On the other hand, increasing the number of the cars means to increase the amount of compounds of the urea cycle. We chose the second way to determine how to increase the urea.(Fig.8 The corner of the left side)<br />
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<img src="https://static.igem.org/mediawiki/2011/d/d6/Cars.png" alt=T(0) width="800px" /><br />
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Fig.8 Two ways to increase urea production <br /><br />
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We determined the elementary modes which produce the four components of the urea cycle:L-ornithine, L-citrulline, N-(L-arginino)succinate, L-arginine. All elementary flux modes which produce these components from L-glutamine or components
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in TCA cycle produce L-ornithine as intermediate or final product.  
+
-
These modes are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem2">Fig.9</a>. Each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table5">Table 5</a>.
+
-
One of the L-ornithine producing modes is shown in Fig.10.
+
</p>
</p>
-
+
<p>
-
<img src="https://static.igem.org/mediawiki/2011/8/89/Urea-fig11.png" alt="fig11" width="600px" />
+
We determined the elementary modes which produce these four important compounds. All elementary flux modes which produce these compounds from L-glutamine or from compounds in TCA cycle produce L-ornithine as intermediate or final product (these modes are shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/figures#Elem2">Fig. 9</a> and each reaction formula is shown in <a href="https://2011.igem.org/Team:Tokyo_Tech/Modeling/Urea-cooler/tables#table5">Table 5</a>), it can be concluded that increasing the concentration of L-ornithine will increase the production of urea. One of the L-ornithine producing modes is shown in Fig. 10.
 +
</p>
 +
 
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2011/8/89/Urea-fig11.png" alt="fig11" width="600px" />
 +
</center>
<div class="graph_title">
<div class="graph_title">
<div style="font-size:larger">
<div style="font-size:larger">
-
<small><b>2-oxoglutarate + NH<sub>3</sub> + acetyl-CoA + ATP + 3NADPH + 3H+  
+
<small><b>2-oxoglutarate + NH<sub>3</sub> + acetyl-CoA + ATP + 3NADPH + 3H<sup>+</sup>
-
L-ornithine + CoASH + acetate + ADP + Pi + H<sub>2</sub>O + 3NADP<sup>+</sup></b></small>
+
&rarr; L-ornithine + CoASH + acetate + ADP + Pi + H<sub>2</sub>O + 3NADP<sup>+</sup></b></small>
</div>
</div>
-
Fig.10 One of the L-ornithine producing pathways from intermediates of TCA cycle<br />
+
Fig. 10 One of the L-ornithine producing pathways from intermediates of TCA cycle<br />
*The numbers indicate the relative flux carried by the enzymes.<br /><br />
*The numbers indicate the relative flux carried by the enzymes.<br /><br />
</div>
</div>
-
<p>
 
-
As shown in Fig.9, we can provide L-ornithine by using a reaction which converts
 
-
L-glutamate to L-ornithine.  Furthermore, all modes include this reaction to produce L-ornithine. We also confirmed that <i>E. coli</i> has no feasible route for production of the four components other than those indicated in Fig.5. Therefore, the reaction which convert L-glutamate to L-ornithine is a key reaction to increase the reaction rates in the urea cycle. This reaction requires ATP, NADPH, Acetyl-CoA, and L-glutamate. We will confirm that the effect of L-glutamate supplement in future wet experiments for not only urea production but also the intermediates like L-ornithine.
 
-
</p>
 
-
<img src="https://static.igem.org/mediawiki/2011/a/a6/Urea_modeling_overview.png" alt="fig.11a" width="600px" />
 
-
Fig.10
 
-
 
-
 
<p>
<p>
-
<big><b>The effect of protein biosynthesis to urea production</b><br /></big>
+
The reactions we determined increase the above mentioned four compounds of the urea cycle are shown in Fig. 9. All modes include the reaction that yields L-ornithine by converting L-glutamate to L-ornithine. <br />
-
From another point of view that L-aspartate is consumed in protein
+
We also confirmed that <span class="name">E. coli</span> has no feasible routes for production of these four components other than those indicated in Fig. 5. Therefore, we can conclude that the reaction which converts L-glutamate to L-ornithine is a key reaction to increase the reaction rates in the urea cycle and thereby to increase urea production. It should be noted that one of the reactions of the cycle shown in Fig. 5 (the one in the lowest part of the image) requires ATP, NADPH, Acetyl-CoA, and L-glutamate. With the exception of L-glutamate, all of these compounds are already abundant in the cell. Therefore, in future wet experiments, we will focus on studying the effects of supplying L-glutamate to <i>E. coli</i>. We will confirm that by supplying L-glutamate the concentration of intermediates like L-ornithine can be increased and therefore urea production can be increased.<br />
-
biosynthesis, this amino acid should be supplied from medium or produced
+
</p>
-
by <i>E. coli</i> itself not only for increasing the amount of urea but for
+
-
maintenance of the cycle.  
+
<p>
<p>
-
<br />In conclusion, we confirmed that
+
Furthermore, to supply L-glutamine, L-glutamate and L-arginine is effective way to increase the amount of ornithine.(Fig. 11)
-
the urea cycle in <span class="name">E.coli</span> is well designed in a stoichiometrically point of view.<br />
+
-
Furthermore, to supply L-glutamine, L-glutamate, L-arginine and L-aspartate is effective way to increase the amount of urea.
+
</p>
</p>
-
+
<center>
-
<h2 id="future">3. Future work</h2>
+
<img src="https://static.igem.org/mediawiki/2011/a/a6/Urea_modeling_overview.png" alt="fig.11a" width="600px" /><br />
-
<p>According to our results, we found out that supply of L-glutamine, L-glutamate, L-arginine and L-aspartate is supposed to increase the amount of urea produced. For future work, to activate the reactions which supply these amino acids is considered to be the effective way to obtain more urea.<br />
+
Fig. 11 Ornithine is made from L-glutamine, L-glutamate and L-arginine<br />
 +
</center>
 +
<p>
 +
We also noted that since L-aspartate is consumed in protein biosynthesis, this amino acid should be supplied from in the medium or produced by <i>E. coli</i> itself not only for increasing the amount of urea production, but also for maintaining the cycle.
 +
</p>
 +
<p>
 +
In conclusion, increasing the concentration of L-glutamine, L-glutamate, L-arginine and L-aspartate is an effective way to increase the amount of urea produced.
 +
</p>
-
 
-
</p> <b><big>Reference</big></b><br />
 
-
[1] Stefan Schuster, et al. A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic network, Nat Biotechnol(2000) 18:326-32<br />
 
-
[2] Mendel Tuchman, et al. Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains, Apple Environ Microbiol(1997) 63: 38-8<br />
 
-
</p>
+
<h2 id="future">4. Future Work</h2>
 +
<p>
 +
As a future work, we will experimentally confirm our results to show that activating the reactions which supply these amino acids is an effective way to increase the production of urea by <i>E. coli</i>.
 +
</p>
 +
 
 +
<h2>Reference</h2>
 +
[1] Stefan Schuster, <i>et al</i>. A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic network, Nat Biotechnol(2000) 18:326-32<br />
 +
[2] Mendel Tuchman, <i>et al</i>. Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains, Apple Environ Microbiol(1997) 63: 38-8<br />
 +
<br />
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Latest revision as of 13:45, 28 October 2011

Tokyo Tech 2011

Flux analysis for providing more urea

1. Abstract

This section is about a metabolic engineering study we did about the urea cycle. On the first part we show how we used “elementary flux modes” (Schuster et al., 2000) to analyze the function of the compounds involved in the urea cycle. Mainly we deduced which compounds act as sources of carbon and sources of nitrogen for the production of urea. On the second part of this study we show how we determined elementary flux modes of the urea cycle to find ways to increase the yield of urea. We focused on a strategy which involves increasing the concentration of four components of the cycle and which we concluded would yield more urea. To confirm our results future experiments will be done.

Fig5
Fig. 5 The reactions related with the urea cycle
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing rocF gene. For complete names of the enzymes see Table 3.

2. Introduction

2.1 What is Elementary Flux Modes?

In Metabolic Engineering, mathematical modeling is an effective way to increase the products of a reaction. In particular, Flux Analysis, which is based on the hypothesis that the system is in a steady state, is effective to find how to increase these products. The concept of elementary flux mode provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. As a method of metabolic flux analysis, it is based on the hypothesis that the concentration of the reactants and products involved in the cycle does not change.

2.2 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes

By determining the elementary flux modes of a cycle we can have a more clear view of the function of each of the compounds involved in the cycle being analyzed. Based on the elementary flux modes of the urea cycle, in this study we could deduce that HCO3- acts as the source of carbon for urea production and that both L-glutamine and NH3 act as nitrogen sources for the formation of urea.

T(0) T9 Fig7

2.3 Finding Modes to Increase the Urea production by E. coli

In this study we determined elementary flux modes to maximize urea production by E. coli. We found that there are two main strategies to increase urea production: one is to increase the amount of carbamoyl phosphate (which formation is known to be the rate-limiting step of the urea cycle). The other one is to increase the concentration of four components of the urea cycle: L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine. We deduced the latter strategy by determining the elementary modes of the urea cycle, and therefore in this study we will focus on the description of this strategy.

T(0)
Fig. 8 Two ways to increase urea production

3. Results

In our study, we considered the enzymatic reactions shown in Table 3 to determine the elementary flux modes related to urea production by E. coli. The scheme the overall reaction system is shown in Fig. 5 below.

Fig5

Fig. 5 The reactions related with the urea cycle
*The orange letters are the abbreviated names of the enzymes involved. The red letters are the enzyme expressed by introducing rocF gene. For complete names of the enzymes see Table 3.

By determining the elementary flux modes to produce urea inside E. coli, we found two important results:

  1. We confirmed both L-glutamine and NH3 act as nitrogen providers in the urea cycle, as well as deducing that HCO3- acts as the source of carbon for urea production. These modes did not make use of organic intermediates. Even though L-glutamine is consumed in order to to transfer the side-chain ammonium group needed for the production of carbamoyl phosphate (which in turn transfers the ammonium group to the urea cycle), free ammonium ion can restore L-glutamine from L-glutamate (which is a byproduct of the reaction that yields carbamoyl phosphate as a product).

  2. We concluded that increasing the concentration of L-ornithine will increase the concentration of three related compounds (L-citrulline, N-(L-arginino)succinate, and L-arginine) and this will ultimately lead to an increase in the production of urea. We also noted that since the L-aspartate amino acid, which is needed in the urea cycle we considered(Fig. 5), is normally consumed in protein biosynthesis, so it should be supplied in the culture medium or synthetized by ,E. coli in order to be able to increase the amount of urea and to maintain the cycles that produce it.

Below is a detailed description of these three results.

3.1 Analyzing the function of the compounds involved in the Urea Cycle by determining the elementary flux modes

The first step was to determine the flux modes which need of L-glutamine as an input (Mendel et al., 1996). We did this by calculations based on a matrix as the tableau shown below.

T(0)
T9
Details about the calculations can be found here

We found eight modes that can produce urea without using organic intermediates. These are shown in Fig. 6. Each reaction formula is shown in Table 4. In particular, we focused on one the mode displayed in Fig. 7.

fig7
2NH3 + HCO3- + 3ATP + H2O + NADPH + NAD+ → Urea + 2ADP + AMP + 2Pi + PPi + NADP+ + NADH
Fig. 7 One of the urea producing cycles leaded by the concept of elementary flux modes
*The numbers indicate the relative flux carried by the enzymes.

As shown in Fig. 7, we deduced that the carbon atom of urea is provided from HCO3- , which is a byproduct of respiration and therefore is already an abundant compound in the bacterial cytoplasm. On the other hand, we also confirmed that carbamoyl phosphate is a nitrogen source for urea production.We also found that the function of L-glutamine in the urea cycle is to provide nitrogen for urea production via carbamoyl phosphate, because ammonium ion can restore L-glutamine from L-glutamate (which is a byproduct of the reaction that yields carbamoyl phosphate as a product). This conclusion was confirmed experimentally by Mendel et al. (1996). Also, since only providing a nitrogen source is enough to increase urea production by E. coli, we can also conclude that the aritificial urea cycle in E. coli is stoichiometrically well designed. By comparing Fig. 5 and Fig. 7 we can also observe that, in Fig. 7, the reaction which converts L-glutamate to L-ornithine is not needed for urea production.

3.2 Finding Modes to Increase the Urea production by E. coli

There are two ways to obtain more products from a cycle of reactions: increasing the speed the reactions and increasing the concentration of the reactants. This becomes obvious if we think of the cycle as a track which is travelled by cars (the reactants), and the products as the total sum of the number of laps made by every car. If we double the speed of the cars the number of laps will also double (Fig. 8, lower left). Similarly, if we double the number of cars, the number of laps will double as well (Fig. 8, lower right). We applied this analogy to the urea cycle, where the metabolites in the cycle are represented by the cars and the total number of laps represents the total urea yield (as shown in the figure below).
Increasing the velocity of the cars corresponds to increasing the amount of carbamoyl phosphate in the urea cycle, because the reaction which converts L-glutamine to carbamoyl phosphate is the rate-limiting reaction of the cycle. On the other hand, increasing the number of the cars correspond to increasing the concentration of the compounds of the urea cycle. We focused on increasing the concentration the compounds of the urea cycle to find ways to increase the urea yield.

T(0)
Fig. 8 Two ways to increase urea production

L-ornithine, L-citrulline, N-(L-arginino)succinate and L-arginine are four important compounds of the urea cycle. As can be seen in Fig. 7, these compounds form a sub-cycle that directly yields urea. Therefore, by increasing the yield of this cycle we can increase the production of urea in E. coli.

We determined the elementary modes which produce these four important compounds. All elementary flux modes which produce these compounds from L-glutamine or from compounds in TCA cycle produce L-ornithine as intermediate or final product (these modes are shown in Fig. 9 and each reaction formula is shown in Table 5), it can be concluded that increasing the concentration of L-ornithine will increase the production of urea. One of the L-ornithine producing modes is shown in Fig. 10.

fig11
2-oxoglutarate + NH3 + acetyl-CoA + ATP + 3NADPH + 3H+ → L-ornithine + CoASH + acetate + ADP + Pi + H2O + 3NADP+
Fig. 10 One of the L-ornithine producing pathways from intermediates of TCA cycle
*The numbers indicate the relative flux carried by the enzymes.

The reactions we determined increase the above mentioned four compounds of the urea cycle are shown in Fig. 9. All modes include the reaction that yields L-ornithine by converting L-glutamate to L-ornithine.
We also confirmed that E. coli has no feasible routes for production of these four components other than those indicated in Fig. 5. Therefore, we can conclude that the reaction which converts L-glutamate to L-ornithine is a key reaction to increase the reaction rates in the urea cycle and thereby to increase urea production. It should be noted that one of the reactions of the cycle shown in Fig. 5 (the one in the lowest part of the image) requires ATP, NADPH, Acetyl-CoA, and L-glutamate. With the exception of L-glutamate, all of these compounds are already abundant in the cell. Therefore, in future wet experiments, we will focus on studying the effects of supplying L-glutamate to E. coli. We will confirm that by supplying L-glutamate the concentration of intermediates like L-ornithine can be increased and therefore urea production can be increased.

Furthermore, to supply L-glutamine, L-glutamate and L-arginine is effective way to increase the amount of ornithine.(Fig. 11)

fig.11a
Fig. 11 Ornithine is made from L-glutamine, L-glutamate and L-arginine

We also noted that since L-aspartate is consumed in protein biosynthesis, this amino acid should be supplied from in the medium or produced by E. coli itself not only for increasing the amount of urea production, but also for maintaining the cycle.

In conclusion, increasing the concentration of L-glutamine, L-glutamate, L-arginine and L-aspartate is an effective way to increase the amount of urea produced.

4. Future Work

As a future work, we will experimentally confirm our results to show that activating the reactions which supply these amino acids is an effective way to increase the production of urea by E. coli.

Reference

[1] Stefan Schuster, et al. A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic network, Nat Biotechnol(2000) 18:326-32
[2] Mendel Tuchman, et al. Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains, Apple Environ Microbiol(1997) 63: 38-8