Evan pepper (Talk | contribs) |
|||
Line 756: | Line 756: | ||
<br> | <br> | ||
<h2 style="text-align: left; font-weight: 500;">Overview</h2> | <h2 style="text-align: left; font-weight: 500;">Overview</h2> | ||
− | <p>To predict acetaminophen biosynthesis, we analyzed the abundance of the acetaminophen's precursor, chorismate. Chorismate is a precursor in our organism for the three aromatic amino acids and folate[1]; also producing alkaloids, salicylic acid, and vitamin K in other organisms<sup>[2]</sup>. Our inserted gene <i>4ABH</i> takes intermediates from tryptophan and folate pathways and converts it to 4-aminophenol, processing it to acetaminophen with the help of <i>nhoA</i><sup>[3]</sup>. We used published data on these intermediates to quantifiy the precursors available for converting to acetaminophen. To understand how much precursor our enzyme <i>4ABH</i> would process, we compared enzyme K<sub>m</sub>s against competiting enzymes using ratios and creating a simulation of chorismate metabolism in Python<sup>[4]</sup>. Insufficient data on enzyme rates, quantities, and K<sub>m</sub> binding affinity values necessitated using limited data from other bacterial species and comparing sequence identities to find data for the closest sequence to <i>Arthrospira platensis</i>. With these data, we made several estimates for the amount of acetaminophen precursor available and how much would go down each pathway.</p> | + | <p>To predict acetaminophen biosynthesis, we analyzed the abundance of the acetaminophen's precursor, chorismate. Chorismate is a precursor in our organism for the three aromatic amino acids and folate<sup>[1]</sup>; also producing alkaloids, salicylic acid, and vitamin K in other organisms<sup>[2]</sup>. Our inserted gene <i>4ABH</i> takes intermediates from tryptophan and folate pathways and converts it to 4-aminophenol, processing it to acetaminophen with the help of <i>nhoA</i><sup>[3]</sup>. We used published data on these intermediates to quantifiy the precursors available for converting to acetaminophen. To understand how much precursor our enzyme <i>4ABH</i> would process, we compared enzyme K<sub>m</sub>s against competiting enzymes using ratios and creating a simulation of chorismate metabolism in Python<sup>[4]</sup>. Insufficient data on enzyme rates, quantities, and K<sub>m</sub> binding affinity values necessitated using limited data from other bacterial species and comparing sequence identities to find data for the closest sequence to <i>Arthrospira platensis</i>. With these data, we made several estimates for the amount of acetaminophen precursor available and how much would go down each pathway.</p> |
<br> | <br> | ||
Line 763: | Line 763: | ||
<h2 style="text-align: left; font-weight: 500;">Enzyme Competition</h2> | <h2 style="text-align: left; font-weight: 500;">Enzyme Competition</h2> | ||
− | <p>Precursor concentrations would be the first limit to how much acetaminophen could be produced. The second would be how effective our enzyme <i>4ABH</i> was at converting precursors from the tryptophan and folate pathways into 4-aminophenol. To answer the competition question, we can either assume all three competing enzymes will have similar precursor affinity and produce each product in equal quantities or use ratios of each competing enzyme's K<sub>m</sub> for the limiting intermediates. This enzyme kinetics based method is difficult because there is so little data on rates, quantities, and affinities for our particular enzyme sequences. For that reason, all of the following calulations will be computed using a 33% assumed precursor to product conversion rate. </p> | + | <p>Precursor concentrations would be the first limit to how much acetaminophen could be produced. The second would be how effective our enzyme <i>4ABH</i> was at converting precursors from the tryptophan and folate pathways into 4-aminophenol. To answer the competition question, we can either assume all three competing enzymes will have similar precursor affinity and produce each product in equal quantities or use ratios of each competing enzyme's K<sub>m</sub> for the limiting intermediates. This enzyme kinetics based method is difficult because there is so little data on rates, quantities, and affinities for our particular enzyme sequences. For that reason, all of the following calulations will be computed using a 33% assumed precursor to product conversion rate.</p> |
− | <p>Since few cyanobacterial proteins have been isolated and tested for kinetic data, many of the K<sub>m</sub>s compared are from other species. <i>4ABH</i> has a K<sub>m</sub> of 20.4 µM for p-amino benzoate(PABA), while the folate processing enzyme folp1 has a K<sub>m</sub> of 0.37 µM <sup>[5,6]</sup>. Assuming the rate and quantity are the same, the folate enzyme will be fully saturated at a much lesser concentration of PABA than <i>4ABH</i>. You can assume that the logrithmic K<sub>m</sub> ratio of 20.4:0.37 (or 55:1) will represent how many moles of PABA goes to folate versus to acetaminophen. For the tryptophan intermediate anthranilate, no K<sub>m</sub> is given but <i>4ABH</i> is assayed as having a rate of 24% of PABA, equal to 34 µM for anthranilate, this compares to TrpD's K<sub>m</sub> for chorismate of 40nM, meaning we'd have 850:1 tryptophan to acetaminophen. Since these ratios are based on poorly matching enzymes and make acetaminophen production undetectably small, we will keep this enzyme competition in mind while using 33% conversion for simplicity. | + | |
+ | <p>Since few cyanobacterial proteins have been isolated and tested for kinetic data, many of the K<sub>m</sub>s compared are from other species. <i>4ABH</i> has a K<sub>m</sub> of 20.4µM for p-amino benzoate(PABA), while the folate processing enzyme folp1 has a K<sub>m</sub> of 0.37µM<sup>[5,6]</sup>. Assuming the rate and quantity are the same, the folate enzyme will be fully saturated at a much lesser concentration of PABA than <i>4ABH</i>. You can assume that the logrithmic K<sub>m</sub> ratio of 20.4:0.37 (or 55:1) will represent how many moles of PABA goes to folate versus to acetaminophen. For the tryptophan intermediate anthranilate, no K<sub>m</sub> is given but <i>4ABH</i> is assayed as having a rate of 24% of PABA, equal to 34µM for anthranilate, this compares to <i>TrpD's</i> K<sub>m</sub> for chorismate of 40nM, meaning we'd have 850:1 tryptophan to acetaminophen. Since these ratios are based on poorly matching enzymes and make acetaminophen production undetectably small, we will keep this enzyme competition in mind while using 33% conversion for simplicity.</p> | ||
+ | |||
<h2 style="text-align: left; font-weight: 400;">Assumptions</h2> | <h2 style="text-align: left; font-weight: 400;">Assumptions</h2> | ||
<ul style="font-family: 'objektiv-mk1'; font-size: inherit; text-align: left;"> | <ul style="font-family: 'objektiv-mk1'; font-size: inherit; text-align: left;"> | ||
<li>We assume that our genes were designed, inserted, and translated successfully and in reasonable quantities.</li> | <li>We assume that our genes were designed, inserted, and translated successfully and in reasonable quantities.</li> | ||
+ | <br> | ||
<li>K<sub>m</sub> values for <i>Arthrospira platensis</i> can be approximated by using other organisms' enzyme rates. Sequences were BLAST compared in each case, resulting in a range of similarity between 52% and 63% positive alignment, suggesting that enzyme orthologs might be significantly different.</li> | <li>K<sub>m</sub> values for <i>Arthrospira platensis</i> can be approximated by using other organisms' enzyme rates. Sequences were BLAST compared in each case, resulting in a range of similarity between 52% and 63% positive alignment, suggesting that enzyme orthologs might be significantly different.</li> | ||
+ | <br> | ||
<li>We assume that enzyme rate and quantity is the same for each <i>4ABH, folp1, and TrpD</i>, else we could not compare K<sub>m</sub> ratios and calculate approximate the K<sub>m</sub> of <i>4ABH</i> for anthranilate.</li> | <li>We assume that enzyme rate and quantity is the same for each <i>4ABH, folp1, and TrpD</i>, else we could not compare K<sub>m</sub> ratios and calculate approximate the K<sub>m</sub> of <i>4ABH</i> for anthranilate.</li> | ||
</ul> | </ul> | ||
Line 774: | Line 778: | ||
<h2 style="text-align: left; font-weight: 500;">Amino Acid Method</h2> | <h2 style="text-align: left; font-weight: 500;">Amino Acid Method</h2> | ||
− | <p>Once we found out that acetaminophen was produced from the same precursors as the tryptophan and folate, we found published amino acid composition data for <i>Arthrospira platensis</i> and back converted those amino acids to moles of acetaminophen precursors anthranilate and PABA. These molecules are the direct substrate for our enzyme <i>4ABH</i>, converting it to 4-aminophenol before <i> | + | <p>Once we found out that acetaminophen was produced from the same precursors as the tryptophan and folate, we found published amino acid composition data for <i>Arthrospira platensis</i> and back converted those amino acids to moles of acetaminophen precursors anthranilate and PABA. These molecules are the direct substrate for our enzyme <i>4ABH</i>, converting it to 4-aminophenol before <i>nhoA</i> converts it to acetaminophen. We show several different calculations below using different sources of data.</p> |
<h2 style="text-align: left; font-weight: 400;">Assumptions</h2> | <h2 style="text-align: left; font-weight: 400;">Assumptions</h2> | ||
<ul style="font-family: 'objektiv-mk1'; font-size: inherit; text-align: left;"> | <ul style="font-family: 'objektiv-mk1'; font-size: inherit; text-align: left;"> | ||
<li><i>Synechococcus</i> and <i>Arthrospira platensis</i> have similar amino acid ratios. Since there was no available amino acid data for our transformed <i>Synechococcus</i>, we must assume our species has a similar ratio to the more well described <i>Arthrospira platensis</i>.</li> | <li><i>Synechococcus</i> and <i>Arthrospira platensis</i> have similar amino acid ratios. Since there was no available amino acid data for our transformed <i>Synechococcus</i>, we must assume our species has a similar ratio to the more well described <i>Arthrospira platensis</i>.</li> | ||
+ | <br> | ||
<li>The amount of anthranilate and PABA precursors are equal to that of their products tryptophan and folate.</li> | <li>The amount of anthranilate and PABA precursors are equal to that of their products tryptophan and folate.</li> | ||
<li>Of the available precursors, 33% will go down our pathway. This is based off the idea that the enzymes may have similar affinities for the precursor.</li> | <li>Of the available precursors, 33% will go down our pathway. This is based off the idea that the enzymes may have similar affinities for the precursor.</li> | ||
− | <li>Folate is present in such small amounts, at 0. | + | <br> |
+ | <li>Folate is present in such small amounts, at 0.21nM of PABA, that its effect on acetaminophen production is neglegible<sup>[7]</sup>.</li> | ||
</ul> | </ul> | ||
− | |||
<br> | <br> | ||
<br> | <br> | ||
$$\frac{0.442\ µmol\ Trp}{1g\ biomass}\approx \frac{0.455\ µmol\ anth}{1g\ biomass}\times\frac{1\ mol\ acet}{3\ moles\ anth}\time\frac{151g\ acet.}{1 mol acet.}=\frac{2.3mg\ acet.}{1g\ biomass}$$ | $$\frac{0.442\ µmol\ Trp}{1g\ biomass}\approx \frac{0.455\ µmol\ anth}{1g\ biomass}\times\frac{1\ mol\ acet}{3\ moles\ anth}\time\frac{151g\ acet.}{1 mol acet.}=\frac{2.3mg\ acet.}{1g\ biomass}$$ | ||
− | <figcaption>This estimate for acetaminophen(acet.) production from tryptophan(Trp) in <i>Arthrospira platensis</i> gives us an approximation for acetaminophen's precursor anthranilate(anth.). The starting Trp is the average of two literature values for tryptophan concentration <sup>[7,8]</sup> and assuming 33% of the anthranilate precursor goes to our inserted enzyme, <i>4ABH</i>.</figcaption> | + | <figcaption>This estimate for acetaminophen (acet.) production from tryptophan (Trp) in <i>Arthrospira platensis</i> gives us an approximation for acetaminophen's precursor anthranilate(anth.). The starting Trp is the average of two literature values for tryptophan concentration<sup>[7,8]</sup> and assuming 33% of the anthranilate precursor goes to our inserted enzyme, <i>4ABH</i>.</figcaption> |
<br> | <br> | ||
<h2 style="text-align: left; font-weight: 500;">Sequence Analysis Method</h2> | <h2 style="text-align: left; font-weight: 500;">Sequence Analysis Method</h2> | ||
− | <p>To validate our organism's quantity of tryptophan precursor, we used a custom Python program[9] to convert DNA sequences to amino acids and calculate molar and mass percentages of tryptophan. We ran both the genome and all 55 listed ribosomal protein sequences through our program, which resulted in 0.9% and 0.6% tryptophan by moles. Knowing that about 60% of <i>Arthrospira platensis</i> is protein by mass, we can predict acetaminophen production.</p> | + | <p>To validate our organism's quantity of tryptophan precursor, we used a custom Python program<sup>[9]</sup> to convert DNA sequences to amino acids and calculate molar and mass percentages of tryptophan. We ran both the genome and all 55 listed ribosomal protein sequences through our program, which resulted in 0.9% and 0.6% tryptophan by moles. Knowing that about 60% of <i>Arthrospira platensis</i> is protein by mass, we can predict acetaminophen production.</p> |
− | + | <br> | |
+ | <br> | ||
$$\frac{0.056\ g\ Trp}{1\ biomass}\rightarrow\frac{0.27\ mmol\ Trp}{1\ g\ biomass}\times\frac{1\ mol\ acet}{3\ mol\ Trp}\times\frac{151.163g\ acet}{1\ mol\ acet.}=\frac{13.7mg\ acet.}{1g\ biomass}$$ | $$\frac{0.056\ g\ Trp}{1\ biomass}\rightarrow\frac{0.27\ mmol\ Trp}{1\ g\ biomass}\times\frac{1\ mol\ acet}{3\ mol\ Trp}\times\frac{151.163g\ acet}{1\ mol\ acet.}=\frac{13.7mg\ acet.}{1g\ biomass}$$ | ||
Line 801: | Line 807: | ||
$$\frac{0.036 g\ Trp}{1 g\ biomass}\rightarrow \frac{0.0.175\ mmol\ Trp}{1\ g\ biomass} * \frac{1\ mol\ acet}{3\ mol\ chor} *\frac{151.163\ g}{1 mol\ acet} = \frac{8.8\ mg\ acet}{1 g\ biomass}$$ | $$\frac{0.036 g\ Trp}{1 g\ biomass}\rightarrow \frac{0.0.175\ mmol\ Trp}{1\ g\ biomass} * \frac{1\ mol\ acet}{3\ mol\ chor} *\frac{151.163\ g}{1 mol\ acet} = \frac{8.8\ mg\ acet}{1 g\ biomass}$$ | ||
<figcaption>This equation uses translated ribosomal amino acid composition to approximate total cellular amino acid composition since ribosomal proteins are highly expressed in cells, composing 9-22% of all proteins by mass<sup>[10]</sup>.</figcaption> | <figcaption>This equation uses translated ribosomal amino acid composition to approximate total cellular amino acid composition since ribosomal proteins are highly expressed in cells, composing 9-22% of all proteins by mass<sup>[10]</sup>.</figcaption> | ||
+ | |||
+ | <br> | ||
+ | |||
<h2 style="text-align: left; font-weight: 400;">Assumptions</h2> | <h2 style="text-align: left; font-weight: 400;">Assumptions</h2> | ||
<ul style="font-family: 'objektiv-mk1'; font-size: inherit; text-align: left;"> | <ul style="font-family: 'objektiv-mk1'; font-size: inherit; text-align: left;"> | ||
<li>Codon composition from the genome and ribosomal proteins alone apprimates the amino acid composition of the cyanobacteria.</li> | <li>Codon composition from the genome and ribosomal proteins alone apprimates the amino acid composition of the cyanobacteria.</li> | ||
+ | <br> | ||
<li>The amount of anthranilate and PABA precursors are equal to that of their products tryptophan and folate.</li> | <li>The amount of anthranilate and PABA precursors are equal to that of their products tryptophan and folate.</li> | ||
+ | <br> | ||
<li>Of the available precursors, 33% will go down our pathway. This is based off the idea that the enzymes may have similar affinities for the precursor.</li> | <li>Of the available precursors, 33% will go down our pathway. This is based off the idea that the enzymes may have similar affinities for the precursor.</li> | ||
− | <li>Folate is present in such small amounts, at 0.21 nanomoles of PABA, that its effect on acetaminophen production is neglegible [7]. | + | <br> |
+ | <li>Folate is present in such small amounts, at 0.21 nanomoles of PABA, that its effect on acetaminophen production is neglegible<sup>[7]</sup>.</li> | ||
</ul> | </ul> | ||
Line 812: | Line 824: | ||
<p>These numbers show that there will probably be enough precursor to produce a useful, detectable quantity of acetaminophen. Based on literature and sequence estimates of aromatic amino acids, we can assume there would be at least that many moles of chorismate from which our added pathway pushes towards acetaminophen. The three calculations above can be averaged to finally predict 8.26mg ± 2.77mg acetaminophen per gram of <i>Arthrospira platensis</i> biomass.</p> | <p>These numbers show that there will probably be enough precursor to produce a useful, detectable quantity of acetaminophen. Based on literature and sequence estimates of aromatic amino acids, we can assume there would be at least that many moles of chorismate from which our added pathway pushes towards acetaminophen. The three calculations above can be averaged to finally predict 8.26mg ± 2.77mg acetaminophen per gram of <i>Arthrospira platensis</i> biomass.</p> | ||
− | <p>We used different tryptophan estimates to reach several different predictions for acetaminophen, averaging 8.26mg ± 2.77mg acetaminophen per gram of <i>Arthrospira platensis</i> biomass or 8.26 µg per mL. This would be significantly above the limit of detection for our HPLC, at 50ng per ml, and serve as a starting point for optimizing production. This means that one 325mg dose of acetaminophen could be obtained in ~39g of biomass, meaning a 12 by 3 feet round pool could produce enough acetaminophen for more than 200 people every 10 days. While 39 grams isn't an ideal amount of medicine to consume, it does show that <i>Arthrospira platensis</i> has significant potential as a molecular factory for acetaminophen.</p> | + | <p>We used different tryptophan estimates to reach several different predictions for acetaminophen, averaging 8.26mg± 2.77mg acetaminophen per gram of <i>Arthrospira platensis</i> biomass or 8.26µg per mL. This would be significantly above the limit of detection for our HPLC, at 50ng per ml, and serve as a starting point for optimizing production. This means that one 325mg dose of acetaminophen could be obtained in ~39g of biomass, meaning a 12 by 3 feet round pool could produce enough acetaminophen for more than 200 people every 10 days. While 39 grams isn't an ideal amount of medicine to consume, it does show that <i>Arthrospira platensis</i> has significant potential as a molecular factory for acetaminophen.</p> |
<div class="container"> | <div class="container"> | ||
Line 827: | Line 839: | ||
</div> | </div> | ||
− | <p>The quantity of | + | <p>The quantity of DMB B<sub>12</sub> produced depends on a successful production and integration of the active B<sub>12</sub> lower ligand, 5,6-dimethyl-benzimidazole (5,6-DMB). For phytoplankton in the wild, cobalt is often the limiting factor for growth and production of B<sub>12</sub><sup>[11,12]</sup>, while B<sub>12</sub> production is limited by growth need in optimal media<sup>[12]</sup>. With <i>ssuE</i> and <i>bluB</i> genes inserted and regulated using a strong PrtC promoter, the activating lower ligand 5,6-DMB will be created in abundance<sup>[13]</sup>. <i>Synechococcus</i> and <i>Arthrospira platensis</i> both have <i>CobS</i>, <i>bluB</i>, and <i>pGam</i> genes that code for proteins which bind 5,6-DMB to the cobalt<sup>[14,15]</sup>. If these proteins work as well as in their origin organism, then assays report that 5,6-DMB has at least 100 times higher affinity for cobalt than the B<sub>12</sub> analog ligand, adenine<sup>[16,17]</sup>, meaning the DMB B<sub>12</sub> to B<sub>12</sub> analog ration would be 100:1. Published HPLC results show that that <i>Arthrospira platensis</i> produces between 1.5-2.5µg B<sub>12</sub> analogs per gram dry weight<sup>[18]</sup>.</p> |
− | $$\frac{2.5µg\ B_{12}\ analog}{1g\ drymass} * \frac{100\ DMB\ bindings}{101\ DMB+adenine\ binding} = \frac{2.47µg | + | <br> |
− | <figcaption>This equation shows an example of the calculation which models conversion from B<sub>12</sub> analogs to active DMB | + | <br> |
+ | |||
+ | $$\frac{2.5µg\ B_{12}\ analog}{1g\ drymass} * \frac{100\ DMB\ bindings}{101\ DMB+adenine\ binding} = \frac{2.47µg\ DMB B_{12}}{1g\ biomass}$$ | ||
+ | <figcaption>This equation shows an example of the calculation which models conversion from B<sub>12</sub> analogs to active DMB B<sub>12</sub>. This equation assumes the cobalt binding affinity ratio of CobS and pGam for DMB and adenine in <i>Arthrospira platensis</i> are the same as their orthologs assayed on their origin organism <i>Propionibacterium freudenreichii</i><sup>[14,15]</sup>.</figcaption> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <p>An additional paper assayed <i> Synechococcus elongatus sp WH 7803</i> at 10<sup>-18</sup> moles per cell<sup>[16]</sup>, and at a reported density 10<sup>9</sup> cells per liter (about a gram)<sup>[16,17]</sup>, <i>Synechococcus</i> would produce 1.3µg B<sub>12</sub> per liter dry mass. Another older paper used microbiological assays along with TLC and HPLC, finding maximums of 2.4, 1.47, and 1.27µg B<sub>12</sub> per gram dry mass. Averaging the 6 data points and multiplying by a 100:1 conversion ratio results in a predicted production of 1.74 ± 0.23µg DMB B<sub>12</sub> per gram of <i>Arthrospira platensis</i> drymass, meaning the USDA’s recommended daily value of 6µg could be obtained in one 3.5 gram serving.</p> | ||
− | |||
<h2 style="text-align: left; font-weight: 400;">Assumptions</h2> | <h2 style="text-align: left; font-weight: 400;">Assumptions</h2> | ||
<ul style="font-family: 'objektiv-mk1'; font-size: inherit; text-align: left;"> | <ul style="font-family: 'objektiv-mk1'; font-size: inherit; text-align: left;"> | ||
− | <li>Gene inserts will be expressed, converting riboflavin-5′-phosphate to 5,6-DMB in excess [13].</li> | + | <li>Gene inserts will be expressed, converting riboflavin-5′-phosphate to 5,6-DMB in excess<sup>[13]</sup>.</li> |
− | <li>The bluB/CobS protein complex in <i>Arthrospira platensis</i> will attach 5,6-DMB to cobalt at rates similar to those assayed in <i>Propionibacterium freudenreichii</i><sup>[14,15]</sup>.</li> | + | <br> |
− | <li>Cobalt will be provided in excess of 0. | + | <li>The <i>bluB/CobS</i> protein complex in <i>Arthrospira platensis</i> will attach 5,6-DMB to cobalt at rates similar to those assayed in <i>Propionibacterium freudenreichii</i><sup>[14,15]</sup>.</li> |
− | <li><i>Synechococcus 7942, 7803</i> and <i>Arthrospira platensis</i> will have similar rates of B< | + | <br> |
+ | <li>Cobalt will be provided in excess of 0.3mM according the BG-11 recipe, ensuring maximum precursor availability<sup>[16,17]</sup></li> | ||
+ | <br> | ||
+ | <li><i>Synechococcus 7942, 7803</i> and <i>Arthrospira platensis</i> will have similar rates of B<sub>12</sub> production. | ||
</ul> | </ul> | ||
− | <p>Future B<sub>12</sub> projects might use chemo-trophic bacteria such as <i>Methanosarcina barkeri</i> which produces more than 1000 times more B<sub>12</sub> and could be converted to active form using a similar bioengineering proccess[21]. One exceptional use of cyanobacterial B<sub>12</sub> is growing cyanobacteria in the water used to grow rice, increasing the carbon fixation, nitrogen fixation, and bioenriching the rice with B<sub>12</sub> [18].</p> | + | |
+ | <br> | ||
+ | |||
+ | <p>Future B<sub>12</sub> projects might use chemo-trophic bacteria such as <i>Methanosarcina barkeri</i> which produces more than 1000 times more B<sub>12</sub> and could be converted to active form using a similar bioengineering proccess<sup>[21]</sup>. One exceptional use of cyanobacterial B<sub>12</sub> is growing cyanobacteria in the water used to grow rice, increasing the carbon fixation, nitrogen fixation, and bioenriching the rice with B<sub>12</sub><sup>[18]</sup>.</p> | ||
<br> | <br> | ||
<br> | <br> | ||
Line 864: | Line 888: | ||
<!-- <div class="row no-gutters justify-content-md-center"> --> | <!-- <div class="row no-gutters justify-content-md-center"> --> | ||
<div class="row"> | <div class="row"> | ||
− | <div class="col-lg-12" style="margin-left: | + | <div class="col-lg-12" style="margin-left: 0px; margin-right: 0px;"> |
<div id="graph"></div> | <div id="graph"></div> | ||
</div> | </div> |
Revision as of 16:17, 1 November 2017
![](https://static.igem.org/mediawiki/2017/f/fd/UCSCmodeling.png)
MODELING
Predict and optimize yield
Background
The purpose of modeling is to carefully examine the pathways of each intended biosynthetic products, look for ways to optimize production, understand limiting factors, and to support and innovate for the team in wet lab. To accomplish these goals, we read dozens of different academic papers, sorted through metabolic pathways, and used several different methods to model acetaminophen, B12, and biomass production. Each of these modeling methods has different assumptions which allow these data points to be averaged; providing reasonable quantitative estimates of our biosynthetic products.
ACETAMINOPHEN
![](https://upload.wikimedia.org/wikipedia/commons/thumb/2/29/Paracetamol-skeletal.svg/1200px-Paracetamol-skeletal.svg.png)
Overview
To predict acetaminophen biosynthesis, we analyzed the abundance of the acetaminophen's precursor, chorismate. Chorismate is a precursor in our organism for the three aromatic amino acids and folate[1]; also producing alkaloids, salicylic acid, and vitamin K in other organisms[2]. Our inserted gene 4ABH takes intermediates from tryptophan and folate pathways and converts it to 4-aminophenol, processing it to acetaminophen with the help of nhoA[3]. We used published data on these intermediates to quantifiy the precursors available for converting to acetaminophen. To understand how much precursor our enzyme 4ABH would process, we compared enzyme Kms against competiting enzymes using ratios and creating a simulation of chorismate metabolism in Python[4]. Insufficient data on enzyme rates, quantities, and Km binding affinity values necessitated using limited data from other bacterial species and comparing sequence identities to find data for the closest sequence to Arthrospira platensis. With these data, we made several estimates for the amount of acetaminophen precursor available and how much would go down each pathway.
![](https://static.igem.org/mediawiki/2017/7/7a/Chorismate-metabolism.png)
Enzyme Competition
Precursor concentrations would be the first limit to how much acetaminophen could be produced. The second would be how effective our enzyme 4ABH was at converting precursors from the tryptophan and folate pathways into 4-aminophenol. To answer the competition question, we can either assume all three competing enzymes will have similar precursor affinity and produce each product in equal quantities or use ratios of each competing enzyme's Km for the limiting intermediates. This enzyme kinetics based method is difficult because there is so little data on rates, quantities, and affinities for our particular enzyme sequences. For that reason, all of the following calulations will be computed using a 33% assumed precursor to product conversion rate.
Since few cyanobacterial proteins have been isolated and tested for kinetic data, many of the Kms compared are from other species. 4ABH has a Km of 20.4µM for p-amino benzoate(PABA), while the folate processing enzyme folp1 has a Km of 0.37µM[5,6]. Assuming the rate and quantity are the same, the folate enzyme will be fully saturated at a much lesser concentration of PABA than 4ABH. You can assume that the logrithmic Km ratio of 20.4:0.37 (or 55:1) will represent how many moles of PABA goes to folate versus to acetaminophen. For the tryptophan intermediate anthranilate, no Km is given but 4ABH is assayed as having a rate of 24% of PABA, equal to 34µM for anthranilate, this compares to TrpD's Km for chorismate of 40nM, meaning we'd have 850:1 tryptophan to acetaminophen. Since these ratios are based on poorly matching enzymes and make acetaminophen production undetectably small, we will keep this enzyme competition in mind while using 33% conversion for simplicity.
Assumptions
- We assume that our genes were designed, inserted, and translated successfully and in reasonable quantities.
- Km values for Arthrospira platensis can be approximated by using other organisms' enzyme rates. Sequences were BLAST compared in each case, resulting in a range of similarity between 52% and 63% positive alignment, suggesting that enzyme orthologs might be significantly different.
- We assume that enzyme rate and quantity is the same for each 4ABH, folp1, and TrpD, else we could not compare Km ratios and calculate approximate the Km of 4ABH for anthranilate.
Amino Acid Method
Once we found out that acetaminophen was produced from the same precursors as the tryptophan and folate, we found published amino acid composition data for Arthrospira platensis and back converted those amino acids to moles of acetaminophen precursors anthranilate and PABA. These molecules are the direct substrate for our enzyme 4ABH, converting it to 4-aminophenol before nhoA converts it to acetaminophen. We show several different calculations below using different sources of data.
Assumptions
- Synechococcus and Arthrospira platensis have similar amino acid ratios. Since there was no available amino acid data for our transformed Synechococcus, we must assume our species has a similar ratio to the more well described Arthrospira platensis.
- The amount of anthranilate and PABA precursors are equal to that of their products tryptophan and folate.
- Of the available precursors, 33% will go down our pathway. This is based off the idea that the enzymes may have similar affinities for the precursor.
- Folate is present in such small amounts, at 0.21nM of PABA, that its effect on acetaminophen production is neglegible[7].
$$\frac{0.442\ µmol\ Trp}{1g\ biomass}\approx \frac{0.455\ µmol\ anth}{1g\ biomass}\times\frac{1\ mol\ acet}{3\ moles\ anth}\time\frac{151g\ acet.}{1 mol acet.}=\frac{2.3mg\ acet.}{1g\ biomass}$$
Sequence Analysis Method
To validate our organism's quantity of tryptophan precursor, we used a custom Python program[9] to convert DNA sequences to amino acids and calculate molar and mass percentages of tryptophan. We ran both the genome and all 55 listed ribosomal protein sequences through our program, which resulted in 0.9% and 0.6% tryptophan by moles. Knowing that about 60% of Arthrospira platensis is protein by mass, we can predict acetaminophen production.
$$\frac{0.056\ g\ Trp}{1\ biomass}\rightarrow\frac{0.27\ mmol\ Trp}{1\ g\ biomass}\times\frac{1\ mol\ acet}{3\ mol\ Trp}\times\frac{151.163g\ acet}{1\ mol\ acet.}=\frac{13.7mg\ acet.}{1g\ biomass}$$
$$\frac{0.036 g\ Trp}{1 g\ biomass}\rightarrow \frac{0.0.175\ mmol\ Trp}{1\ g\ biomass} * \frac{1\ mol\ acet}{3\ mol\ chor} *\frac{151.163\ g}{1 mol\ acet} = \frac{8.8\ mg\ acet}{1 g\ biomass}$$
Assumptions
- Codon composition from the genome and ribosomal proteins alone apprimates the amino acid composition of the cyanobacteria.
- The amount of anthranilate and PABA precursors are equal to that of their products tryptophan and folate.
- Of the available precursors, 33% will go down our pathway. This is based off the idea that the enzymes may have similar affinities for the precursor.
- Folate is present in such small amounts, at 0.21 nanomoles of PABA, that its effect on acetaminophen production is neglegible[7].
These numbers show that there will probably be enough precursor to produce a useful, detectable quantity of acetaminophen. Based on literature and sequence estimates of aromatic amino acids, we can assume there would be at least that many moles of chorismate from which our added pathway pushes towards acetaminophen. The three calculations above can be averaged to finally predict 8.26mg ± 2.77mg acetaminophen per gram of Arthrospira platensis biomass.
We used different tryptophan estimates to reach several different predictions for acetaminophen, averaging 8.26mg± 2.77mg acetaminophen per gram of Arthrospira platensis biomass or 8.26µg per mL. This would be significantly above the limit of detection for our HPLC, at 50ng per ml, and serve as a starting point for optimizing production. This means that one 325mg dose of acetaminophen could be obtained in ~39g of biomass, meaning a 12 by 3 feet round pool could produce enough acetaminophen for more than 200 people every 10 days. While 39 grams isn't an ideal amount of medicine to consume, it does show that Arthrospira platensis has significant potential as a molecular factory for acetaminophen.
VITAMIN B12
![](https://static.igem.org/mediawiki/2017/6/62/B12structure.png)
The quantity of DMB B12 produced depends on a successful production and integration of the active B12 lower ligand, 5,6-dimethyl-benzimidazole (5,6-DMB). For phytoplankton in the wild, cobalt is often the limiting factor for growth and production of B12[11,12], while B12 production is limited by growth need in optimal media[12]. With ssuE and bluB genes inserted and regulated using a strong PrtC promoter, the activating lower ligand 5,6-DMB will be created in abundance[13]. Synechococcus and Arthrospira platensis both have CobS, bluB, and pGam genes that code for proteins which bind 5,6-DMB to the cobalt[14,15]. If these proteins work as well as in their origin organism, then assays report that 5,6-DMB has at least 100 times higher affinity for cobalt than the B12 analog ligand, adenine[16,17], meaning the DMB B12 to B12 analog ration would be 100:1. Published HPLC results show that that Arthrospira platensis produces between 1.5-2.5µg B12 analogs per gram dry weight[18].
$$\frac{2.5µg\ B_{12}\ analog}{1g\ drymass} * \frac{100\ DMB\ bindings}{101\ DMB+adenine\ binding} = \frac{2.47µg\ DMB B_{12}}{1g\ biomass}$$
An additional paper assayed Synechococcus elongatus sp WH 7803 at 10-18 moles per cell[16], and at a reported density 109 cells per liter (about a gram)[16,17], Synechococcus would produce 1.3µg B12 per liter dry mass. Another older paper used microbiological assays along with TLC and HPLC, finding maximums of 2.4, 1.47, and 1.27µg B12 per gram dry mass. Averaging the 6 data points and multiplying by a 100:1 conversion ratio results in a predicted production of 1.74 ± 0.23µg DMB B12 per gram of Arthrospira platensis drymass, meaning the USDA’s recommended daily value of 6µg could be obtained in one 3.5 gram serving.
Assumptions
- Gene inserts will be expressed, converting riboflavin-5′-phosphate to 5,6-DMB in excess[13].
- The bluB/CobS protein complex in Arthrospira platensis will attach 5,6-DMB to cobalt at rates similar to those assayed in Propionibacterium freudenreichii[14,15].
- Cobalt will be provided in excess of 0.3mM according the BG-11 recipe, ensuring maximum precursor availability[16,17]
- Synechococcus 7942, 7803 and Arthrospira platensis will have similar rates of B12 production.
Future B12 projects might use chemo-trophic bacteria such as Methanosarcina barkeri which produces more than 1000 times more B12 and could be converted to active form using a similar bioengineering proccess[21]. One exceptional use of cyanobacterial B12 is growing cyanobacteria in the water used to grow rice, increasing the carbon fixation, nitrogen fixation, and bioenriching the rice with B12[18].
BIOMASS
![](https://static.igem.org/mediawiki/2017/5/5c/Spirulina_powder.png)
To understand the production capacity of our organisms, we aggregated growth data from published papers[22,23,24] and our lab’s daily optical density growth data. Using carrying-capacity-limited logistic growth curves to fit our data to an equation, we modelled dried biomass with respect to time. We also added additional dependent variables of temperature and light intensity to our equation so that we can better understand how minimalist growth conditions might affect the maximum culture density and the maximum growth rate.
Light Intensity: μE m-2 s-1
Temperature: ℃