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 support 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 production of acetaminophen and B₁₂. Each of these modeling methods has different assumptions which allow these data-points to be averaged; providing reasonable quantitative estimates of our biosynthetic products.

Overview

To predict acetaminophen biosynthesis, we analyzed the abundance of the acetaminophen's precursor, chorismate. Chorismate is a precursor primarily for the three aromatic amino acids, as well as salicylic acid, folate, vitamins, and many alkaloids ^[1]. We used published data on these products and gene transcription of amino acids for multiple species of cyanobacteria to assume the chorismate pool available to be processed into acetaminophen. Following that, we compared enzyme binding affinity K_m's by creating a simulation of chorismate metabolism in Python to approximate enzyme rates. Insufficient data on cyanobacterial K_m values necessitated using data from other bacterial species and comparing sequence identities to find the best match with our enzymes. We assumed the highest matching sequence K_m's would be analagous to our enzyme's rate. With this information, we made a quantitative metabolic model which estimates how much of our precursor goes down each pathway.

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 tryptophan and folate are equal to the moles of their precursors, anthranilate and PABA.


• Of the available precursors, 30% will go down our pathway. This is based off of K_m ratios of 4ABH and its competitor TrpD.

$$\frac{0.449\ mmol\ FWY}{1g\ biomass}\approx \frac{0.449\ mmol\ chor.}{1g\ biomass}\rightarrow\frac{1\ mol\ acet.}{3\ mol\ chor.}=\frac{0.15\ mmoles\ acet}{1\ g\ biomass}\times\frac{151.163g\ acet.}{1\ mol\ acet.}=\frac{22.62mg\ acet.}{1g\ biomass}$$

To validate our organism's quantity of chorismate precursor, we used a custom Python program to convert DNA sequences to amino acids and calculate molar and mass percentages of chorismate derived aromatic amino acids. We ran both the genome and ribosomal protein sequences through our program, which resulted in 9.3% and 5.14% by mass aromatic amino acids. Using our higher sequence value of 9.3% and the assumption that our enzymes would take a third of the acetaminophen precursor, we estimate an acetaminophen concentration would be around 18mg per gram dried biomass.

$$\frac{0.093\ g\ FYW}{1\ g\ protein}\times\frac{0.6g protein}{1\ biomass}=\frac{0.056\ g\ FYW}{1g\ biomass}\rightarrow\frac{0.37\ mmol\ chor}{1\ g\ biomass}\times\frac{1\ mol\ acet}{3\ mol\ chor}\times\frac{151.163g\ acet}{1\ mol\ acet.}=\frac{18.61mg\ acet.}{1g\ biomass}$$
$$\frac{0.051 g\ FYW}{1 g\ protein} * \frac{0.6 g\ protein}{1g\ biomass} = \frac{0.031g\ FWY}{1 g\ biomass} \rightarrow \frac{0.0.2973\ mmol\ chor}{1\ g\ biomass} * \frac{1\ mol\ acet}{3\ mol\ chor} *\frac{151.163\ g}{1 mol\ acet} = \frac{14.9\ mg\ acet}{1 g\ biomass}$$

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 18.61mg ± 1.63mg acetaminophen per gram of Synechococcus biomass.

We used different chorismate concentration estimates to reach several different estimates for acetaminophen, averaging 18.61 ± 1.63mg acetaminophen per gram of Synechococcus biomass. This would be a sufficient quantity to detect through HPLC and serve as a starting point for optimizing production. This means that one 325mg dose of acetaminophen could be obtained in ~17g of biomass, meaning a 12 by 3 feet round pool could produce enough acetaminophen for more than 500 people every 10 days.

The quantity of active form of 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^[5,6], while B₁₂ production is limited by growth need in optimal media^[6]. 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[7]. Synechococcus and Arthrospira platensis both have CobS, bluB, and pGam genes that code for proteins which bind 5,6-DMB to the cobalt [8,9]. 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 [10,11], 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^[12].

$$\frac{0.051 g\ FYW}{1 g\ protein} * \frac{0.6 g\ protein}{1g\ biomass} = \frac{0.031g\ FWY}{1 g\ biomass} \rightarrow \frac{0.0.2973\ mmol\ chor}{1\ g\ biomass} * \frac{1\ mol\ acet}{3\ mol\ chor} *\frac{151.163\ g}{1 mol\ acet} = \frac{14.9\ mg\ acet}{1 g\ biomass}$$ $$\frac{2.5µg\ B₁₂\ analog}{1 g\ drymass} * \frac{100\ DMB bindings}{101\ total binding} = \frac{2.47µg\ active DMB-B₁₂}{1g\ biomass}$$

An additional paper assayed Synechococcus elongatus sp WH 7803 at 10^-18 moles per cell^[13], and at a reported density 10⁹ cells per liter (about a gram) ^[13,14], Synechococcus would produce 1.3 mµg B₁₂ per liter dry mass. Another older paper used microbiological assays along with TLC and HPLC, finding maximums of 2.4, 1.47, and 1.27 µg 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 active DMB-B₁₂ per gram of Arthrospira platensis drymass, meaning the USDA’s recommended daily value of 6 µg 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 [7].


• The bluB/CobS protein complex in Arthrospira platensis will attach 5,6-DMB to cobalt at rates similar to those assayed in Propionibacterium freudenreichii^[8,9].


• Cobalt will be provided in excess of 0.3 mM according the BG-11 recipe, ensuring maximum precursor availability [5,16]


• 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[15]. 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 [12].

BIOMASS

To understand the production capacity of our organism, we aggregated growth data from published papers and all of our lab’s growth data. Using carrying-capacity-limited logistic growth curves to fit our data to an equation, we modelled dried biomass and cell count with respect to time. We have also used growth optimization papers ^[9,10] to add additional dependent variables of temperature, light intensity, and starter culture density to our equation.

Timescale: < days >
Light Intensity: < μE m^-2 s^-1 >
Temperature: < ℃ >
Starting Density: < g biomass/ L >

P R O J E C T

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HOMEPAGE

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ACETAMINOPHEN

VITAMIN B₁₂

RESULTS

TARGET ORGANISM

PARTS

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[2] Food Composition Databases Show Foods -- Seaweed, spirulina, dried. (n.d.). Retrieved October 27, 2017, from https://ndb.nal.usda.gov/ndb/foods/show/3306?fgcd=&manu=&lfacet=&format=Full&count=&max=50&offset=&sort=default\&order=asc\&qlookup=11667&ds=&qt=&qp=&qa=&qn=&q=&ing=

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[4] Dennis, P. P., & Bremer, H. (1974). Macromolecular Composition During Steady-State Growth of Escherichia coli B/r. Journal of Bacteriology, 119(1), 270–281.

[5] Panzeca, C., Beck, A. J., Leblanc, K., Taylor, G. T., Hutchins, D. A., & Sañudo-Wilhelmy, S. A. (2008). Potential cobalt limitation of vitamin B12 synthesis in the North Atlantic Ocean. Global Biogeochemical Cycles, 22(2), GB2029. https://doi.org/10.1029/2007GB003124

[6] Helliwell, K. E., Lawrence, A. D., Holzer, A., Kudahl, U. J., Sasso, S., Kräutler, B., … Smith, A. G. (2016). Cyanobacteria and Eukaryotic Algae Use Different Chemical Variants of Vitamin B12. Current Biology, 26(8), 999–1008. https://doi.org/10.1016/j.cub.2016.02.041

[7] Huang, H.-H., Camsund, D., Lindblad, P., & Heidorn, T. (2010). Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Research, 38(8), 2577–2593. https://doi.org/10.1093/nar/gkq164

[8] Deptula, P., Kylli, P., Chamlagain, B., Holm, L., Kostiainen, R., Piironen, V., … Varmanen, P. (2015). BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B12 by Propionibacterium freudenreichii. Microbial Cell Factories, 14. https://doi.org/10.1186/s12934-015-0363-9

[9] KEGG PATHWAY: Porphyrin and chlorophyll metabolism - Synechococcus elongatus PCC7942. (n.d.). Retrieved November 1, 2017, from http://www.genome.jp/kegg-bin/show_pathway?syf00860

[10] Anderson, P. J., Lango, J., Carkeet, C., Britten, A., Kräutler, B., Hammock, B. D., & Roth, J. R. (2008). One pathway can incorporate either adenine or dimethylbenzimidazole as an alpha-axial ligand of B12 cofactors in Salmonella enterica. Journal of Bacteriology, 190(4), 1160–1171. https://doi.org/10.1128/JB.01386-07

[11] Stupperich, E., & Nexø, E. (1991). Effect of the cobalt-N coordination on the cobamide recognition by the human vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin. European Journal of Biochemistry, 199(2), 299–303. https://doi.org/10.1111/j.1432-1033.1991.tb16124.x

[12] Bonnet, S., Webb, E. A., Panzeca, C., Karl, D. M., Capone, D. G., & Wilhelmy, S. A. S. (2010). Vitamin B12 excretion by cultures of the marine cyanobacteria Crocosphaera and Synechococcus. Limnology and Oceanography, 55(5), 1959–1964. https://doi.org/10.4319/lo.2010.55.5.1959

[13] Wang, K., Wommack, K. E., & Chen, F. (2011). Abundance and Distribution of Synechococcus spp. and Cyanophages in the Chesapeake Bay▿. Applied and Environmental Microbiology, 77(21), 7459–7468. https://doi.org/10.1128/AEM.00267-11

[14] Watanabe, F., Katsura, H., Takenaka, S., Fujita, T., Abe, K., Tamura, Y., … Nakano, Y. (1999). Pseudovitamin B12 Is the Predominant Cobamide of an Algal Health Food, Spirulina Tablets. Journal of Agricultural and Food Chemistry, 47(11), 4736–4741. https://doi.org/10.1021/jf990541b

[15] Mazumder, T. K., Nishio, N., Fukuzaki, S., & Nagai, S. (1987). Production of extracellular vitamin B-12 compounds from methanol by Methanosarcina barkeri. Applied Microbiology and Biotechnology, 26(6), 511–516. https://doi.org/10.1007/BF00253023

[16] Algae, U. C. C. of. (n.d.). BG-11 Trace Metals Solution Recipe. Retrieved November 1, 2017, from https://utex.org/products/bg-11-trace-metals-solution-recipe

[8] Deptula, P., Kylli, P., Chamlagain, B., Holm, L., Kostiainen, R., Piironen, V., … Varmanen, P. (2015). BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B12 by Propionibacterium freudenreichii. Microbial Cell Factories, 14. https://doi.org/10.1186/s12934-015-0363-9

[9] Kuan, D., Duff, S., Posarac, D., & Bi, X. (2015). Growth optimization of Synechococcus elongatus PCC7942 in lab flasks and a 2-D photobioreactor. The Canadian Journal of Chemical Engineering, 93(4), 640–647. https://doi.org/10.1002/cjce.22154

[10] Yan, R., Zhu, D., Zhang, Z., Zeng, Q., & Chu, J. (2012). Carbon metabolism and energy conversion of Synechococcus sp. PCC 7942 under mixotrophic conditions: comparison with photoautotrophic condition. Journal of Applied Phycology, 24(4), 657–668. https://doi.org/10.1007/s10811-011-9683-2