Kelly Hillen, Luke O’Malley, Matteo Ricci, Amanda Stewart, TC ShiBIOE120: Chemical Engineering Group Project

We investigated the production of taxol, which is used as an anti-cancer drug. Taxol is an uncommon molecule, and is conventionally obtained from the bark of the yew tree. Obtaining taxol with the tree as the sole source is not ideal because the yew tree is a fairly rare tree, only located in a small region of the Pacific Northwest of the United States. In addition, the trees are not large in size, the layer of bark covering them is not thick, and extracting the taxol from the bark kills the tree. On top of that, the yield of taxol per yew tree is low, so obtaining large amounts of taxol is a difficult process, which is a problem because taxol has been shown to be effective in fighting many different forms of cancer. We explored alternative ways to produce taxol, and the method that we chose involves bioengineering a synthetic pathway that would culminate in the synthesis of taxolthrough a more cost-effective process. We will ultimately determine the reduction in cost from theconventional path to the bioengineered one. The major reactions in the pathway are as follows:

1) Glycolysis: D-glucose (DegR = 24) → D-glyceraldehyde-3-P (DegR = 12)C6H12O6 → 2C3H7O6P

2) Pentose phosphate: D-glucose (DegR = 24) → D-glyceraldehyde-3-P (DegR = 12)C6H12O6 → C3H7O6P + 3CO2 + 6NADH

3) MEP: D-glyceraldehyde-3-P (DegR = 12) → geranylgeranyl diphosphate (DegR = 112)7C3H7O6P + 14NADH → C20H36O7P2 + CO2

4) Taxol synthesis: geranylgeranyl diphosphate (DegR = 112) → taxol (DegR = 208)47C20H36O7P2 → 20C47H51NO14 + 552NADH

Maximum Yield Possible (D-glucose → taxol):● 47C6H12O6 → 6C47H51NO14

Carbon maximum yield = 6/47 = 0.128 mol taxol/mol D-glucose● DegR = 24 → DegR = 208

Degree of reduction maximum yield = 24/208 = 0.115 mol taxol/mol D-glucose

0.115 < 0.128 so the maximum yield possible for the pathway is 0.115 mol taxol/mol D-glucose.Reaction 1 requires an input of 2 ATP and Reaction 3 requires an input of 14 ATP, so we will engineer the pathway to transfer electrons to oxygen via electron transport phosphorylation in orderto supply the energy necessary for ATP synthesis.

NADH → NAD+ + 2e-

½ O2 + 2e- → H2O ΔGo’= -36 kcal/molADP + Pi → ATP ΔGo’ = 7.3 kcal/mol

Assuming standard cellular conditions:

2 ATP × 7.3 kcalmol ATP

× mol NADH36 kcal = 0.406 NADH Reaction 1 requires 0.406 NADH.

14 ATP × 7.3 kcalmol ATP

× mol NADH36kcal = 2.84 NADH Reaction 3 requires (14 + 2.84) = 16.84 NADH.

The pathway can be maximized for the synthesis of taxol by consuming all intermediates with minimal loss of carbon to CO2 and having no net production of NADH. The following system of equations were used to maximize yield, with x, y, and z as the multiplying factors of Reactions 1, 2,and 3, respectively, and 1 set as the multiplying factor of Reaction 4:

NADH balance: 0.406x + 16.84z = 6z + 552D-glyceraldehyde balance: 2x + y = 7zgeranylgeranyl balance: z = 47

After solving the system we found x = 139.8, y = 49.4, z = 47, which yielded the followingequations for the pathway:

1) Glycolysis: D-glucose → D-glyceraldehyde-3-P56.8 NADH + 139.8C6H12O6 → 279.6C3H7O6P

2) Pentose phosphate: D-glucose → D-glyceraldehyde-3-P 49.4C6H12O6 → 49.4C3H7O6P + 148.2CO2 + 296.4NADH

3) MEP: D-glyceraldehyde-3-P → geranylgeranyl diphosphate329C3H7O6P + 791.5NADH → 47C20H36O7P2 + 47CO2

4) Taxol synthesis: geranylgeranyl diphosphate → taxol47C20H36O7P2 → 20C47H51NO14 + 552NADH

Overall Pathway: 189.2C6H12O6 → 20C47H51NO14 + 195.2CO2

Theoretical yield = 20/189.2 = 0.106 mol taxol/mol D-glucoseThe theoretical yield we obtained is slightly less than what was found in the maximum yield calculations, which is due to the fact that a small amount of additional NADH was necessary in order to obtain enough energy to supply ATP to Reactions 1 and 3 through electron transportphosphorylation.

Note: This pathway is only feasible at steady-state because Reaction 1 requires an input of NADH,but the overall pathway has a net NADH consumption of zero.

Assumptions for Cost Analysis:● 2 grams of taxol are necessary to treat the average cancer patient.● 255 grams of taxol can be obtained from 1 hectare of yew trees.● The costs associated with taxol production from yew trees are as follows (in thousands of yen per

hectare): 7,648 (seedling production), 315 (preparation), 4,688 (extraction). ● The costs associated with taxol biosynthesis from glucose are $225 per 25 kg glucose and

an extraction cost assumed to be equivalent to that of taxol production from yew trees.

Yew tree production:

2 g taxolpatient

× hectare yew trees255 g taxol

× (7648+315+4688) yenhectare

× $ 0.0083yen

= $ 0.82patient

Biosynthesis from glucose:2 g taxolpatient

× mol taxol853.906 g

× 189.2 mol glucose20 mol taxol

× 180.156 gmol glucose

× 1kg1000 g

× $22525 kg glucose

=$ 0.036∈raw materialspatient

2 g taxolpatient

× 4688 yen225 g taxol

× $ 0.0083yen

=$ 0.346 for extractionpatient

$ 0.036∈raw materialspatient +$ 0.346 for extraction

patient=¿ $ 0.38

patient

Reduction in cost = $ 0.82−$ 0.38

$ 0.82 ×100% ¿54%

Conclusion: The cost of obtaining taxol through the conventional yew tree method is $0.82 per patient, while the biosynthesis of taxol from glucose through the engineered pathway yields a cost of $0.38 per patient. This 54% reduction in cost represents a significant savings to the manufacturers of the drug, which has the potential to drastically reduce costs to patients in need of treatment. It is likely that even if the engineered pathway did not produce 100% yield, the cost is still overestimated because we assumed that extraction costs for the same yield of taxol would be equal for both methods. However, a much higher yield of taxol can be obtained per quantity of materials discarded during the extraction process for the engineered pathway, so extraction costs per patient would likely be lower for the engineered pathway. In addition, the engineered pathway provides scientists with more control over the reactions taking place within the organisms, enabling them to reduce the production of unwanted compounds that would contaminate the product, so less purification may be necessary.

Sources

1. Pujar, Anuradha. "MetaCyc Taxol Biosynthesis." MetaCyc Taxol Biosynthesis. SRIInternational, 18 Sept. 2007. Web. Apr. 2015.2. Tissier, Christopher, and Ron Caspi. "MetaCyc Superpathway of Geranylgeranyl Diphosphate Biosynthesis II (via MEP)." MetaCyc Superpathway of Geranylgeranyl Diphosphate Biosynthesis II (via MEP). The Arabidopsis Information Resource, 26 Feb. 2006. Web.3. Ingraham, John L. "MetaCyc Pentose Phosphate Pathway." MetaCyc Pentose Phosphate Pathway. UC Davis, 14 July 2006. Web.4. "Taxol® (NSC 125973)." Success Story: Taxol. National Cancer Institute, n.d. Web.Itokawa, Hideji, and Kuo-Hsiung Lee. Taxus the Genus Taxus. London: Taylor & Francis, 2003. 173-75. Print.5. Caspi, Ron. "MetaCyc TCA Cycle I (prokaryotic)." MetaCyc TCA Cycle I (prokaryotic). SRI International, 19 Dec. 2011. Web.