The model of resveratrol biosynthesis
Resveratrol is an antioxidant found predominantly in the peel of the grapes, and is a part of the plant’s antibacterial and antifungal defense. Resveratrol is one of the components of red wine which are beneficial to human health.
Research has shown that it improves cardiovascular health and affects, among other things, various factors of cellular ageing. Our vision for Wonder Wine is that it should encapsulate a multitude of beneficial attributes, primarily health-related.
Recently, advancement has been achieved in creating engineered yeast which contained two foreign enzymes essential to resveratrol biosynthesis[1]. These enzymes, originally found in grapes, are 4CL (P-coumarate-CoA ligase) and RS (Resveratrol Synthase). 4CL turns p-coumarate into p-coumaroyl-CoA, which is then used by RS to produce resveratrol. Since p-coumarate is not found naturally in yeast, the scientists RS had to add it to the medium in order for resveratrol synthesis to occur. That's where we come in. We would like to demonstrate, with the help of a model, how the introduction of a third enzyme from grapes – TAL (Tyrosine-Ammonia-Lyase) can obviate the need to supply p-coumarate externally. TAL can convert L-tyrosine, found naturally in yeast, into p-coumarate, thereby supplying the missing building block for resveratrol biosynthesis, with no need for specialized media.
Our model is meant to predict the overall success (or otherwise) of the yeast resveratrol pathway with the addition of TAL (Fig. 1). It was created by using Copasi[2] – a software for the simulation of biochemical networks, which implements MCA – metabolic control analysis. MCA is a comprehensive mathematical framework invented to deal with the response of metabolic pathways, at steady state, to small perturbations in the activity of enzymes and the concentrations of the various metabolites. It can calculate the steady-state flux through the pathway, the control coefficients of the participating enzymes and the steady-state concentration of each metabolite.
The model includes four enzymes (in this order):
PAH - Phenylalanine hydroxylase(E.C 1.14.16.1)
The reaction:
L-phenylalanine + BH4 + O2 → L-tyrosine + 4-hydroxy-BH4
Rate equation:
TAL-Tyrosine-Ammonia-Lyase (EC 4.3.1.23)
The reaction:
L-tyrosine → p-coumaric acid+NH3+H
Rate equation:
4CL - P-coumarate-CoA ligase (EC 6.2.1.12)
The reaction:
ATP+p-coumarat+CoA → AMP+diphosphate+p-coumaroyl-CoA
Rate equation:
RS – Resveratrol Synthase (EC 2.3.1.95)
The reaction:
3malonyl-CoA → 4CoA+resveratrol+4co2
Rate equation:
Fig. 1: the yeast resveratrol pathway
Only PAH (phenylalanine hydroxylase) is a yeast enzyme. The other three are from Vitis vinifera - the wine grape. The reactions of 4CL and RS were treated as irreversible under normal cellular conditions. The 4CL (p-coumarate-CoA ligase) reaction involves ATP hydrolysis. The RS (trihydroxy-stilbene synthase) reaction generates much entropy (4 molecules turn into 9) and releases CO2 [3], which can readily diffuse and dissipate (Fig. 2). In order to allow the model to represent, as much as possible, the real situation inside the yeast cell, we have supplied it with the known intracellular concentrations of the relevant metabolites and the maximal reaction rate (Vmax) of each enzyme (Vmax values were taken from the Brenda website [4]). Unknown rate and Michaelis constants were given default values. According to our model, if the intracellular concentration of the four enzymes is similar (~1 micromolar), the very low turnover number of RS, which is 2-3 orders of magnitude smaller than that of the other enzymes, does not allow the pathway to reach steady state and results in uncontrolled accumulation of p-coumaroyl CoA, which might become toxic (Fig. 3). Only when the turnover number of RS was artificially (and unrealistically) increased thousandfold, was the model able to reach steady state with a small flux of 1.15e-5 mmol/sec (Fig. 4). At this steady state, 4CL holds most of the control over the resveratrol flux (i.e. it has the larger flux control coefficient), PAH has only a small control coefficient, and the control coefficient of RS is 0 (Table 1 and Fig. 5) . Under such conditions, the resveratrol flux becomes sensitive to the concentrations of ammonia (NH3) and malonyl CoA (Figs. 6-7). Therefore, the model predicts that the main obstacle to the introduction of these three new enzymes into the yeast is the need to tailor the individual activities of each enzyme (especially RS) to fit the constraints of the entire pathway. This task, however, is currently beyond the scope of the IGEM project
Fig. 2: the reactions of resveratrol biosynthesis
Fig. 3: uncontrolled accumulation of p-coumaroyl-CoA at low RS activity
Fig. 4: the calculated flux of the resveratrol pathway
Fig. 5: the influence of a 10x increase in 4CL’s activity on the resveratrol flux
Table 1: the flux control coefficients of the enzymes of the resveratrol pathway
Fig. 6: the influence of NH3 intracellular concentration on the resveratrol flux
Fig. 7: the influence of malonyl-CoA intracellular concentration on the resveratrol flux
References:
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Sun, P., Liang, J.L., Kang, L.Z., Huang, X.Y., Huang, J.J., Ye, Z.W., Guo, L.Q., and Lin, J.F., (2015). Increased resveratrol production in wines using engineered wine strains Saccharomyces cerevisiae EC1118 and relaxed antibiotic or auxotrophic selection. Biotechnol. Prog. 31 (3): 650-655.
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Hoops S., Sahle S., Gauges R., Lee C., Pahle J., Simus N., Singhal M., Xu L., Mendes P. and Kummer U. (2006). COPASI: a COmplex PAthway SImulator. Bioinformatics 22, 3067-74.
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Kreuzaler, F. and Hahlbrock, K., (1975). Enzymic synthesis of an aromatic ring from acetate units. Partial purification and some properties of flavanone synthase from cell-suspension cultures of Petroselinum hortense. European journal of biochemistry. 56 (1): 205-213.
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Schomburg, I., Chang, A., Hofmann, O., Ebeling, C., Ehrentreich, F., and Schomburg, D., (2002). BRENDA: a resource for enzyme data and metabolic information. Trends Biochem Sci. 27 (1): 54-56.
