Team:Edinburgh/Engineering
Engineering: Cell free biosynthesis of glutathione with ATP-regenerating system - poultry waste upcycle
Introduction:
Glutathione (GSH) is a tripeptide consisting of glycine, L-glutamic acid and L- cysteine. The versatility of GSH to the human body contributes to its vast application in the pharmaceutical, food and cosmetics industries, leading to the anticipated market value of US$ 198.2 million by 2027 [1].
In regarding to one of the recalcitrant waste The SuperGrinder is focusing, the poultry waste. The mixture of amino acids from chicken feathers were reported as rich in glycine, glutamic acid and cysteine, which were the precursors of GSH [2]. Therefore the degraded keratin can potentially apply in the glutathione synthesis platform to upcycle this resource.
Figure 1.Scheme showing glutathione production via (i) GshA and GshB, (ii) GshF.
With the simple composition of GSH, the biosynthesis is simply done by a single bifunctional enzyme, gshF (bifunctional glutathione synthesis enzyme) (Figure 1). However, the bottleneck of the process is the ATP supply which is also the main factor causing the costly production. For this reason, this part of the SuperGrinder looks into the establishment of a glutathione bioproduction platform, furnished with ATP-regeneration in the cell-free system (Figure 2).
Figure 2.Multiple enzyme ATP regeneration with glutathione production introduced in this study (GCS = g-L-glutamyl-L-cysteine, GSH = glutathione).
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1. Design:
Figure 3.Graphical overview of computational analysis pipeline of this study. (A) The pathway analysis pipeline of both MDF and ECM analysis. (B) GSH-ATP simulation and optimization pipeline.
The cell-free system was designed as depicted in figure 2. However, before deciding to go forward with these enzymes, the pathway was analysed by Equilibrator [3] to observe the feasibility and potential bottleneck of the whole cascade, along with the free energy analysis under physiological conditions. The result indicates that the overall pathway is feasible but acetate kinase (AckA) is the limiting step (Figure 4).
Figure 4.The pathway analysis results from Equilibrator. R000000 and R000001 are gshF reaction, R000002 is the pyruvate oxidase (pox5) and R000003 is the acetate kinase (ackA)
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2. Built - Test
The GSH-ATP system is then simulated via the kinetic modelling disregarding the physiological condition and using the parameters found from literature search - in vitro condition (Table 1). The simulation applied the Ordinary Differential Equations (ODEs) to create an exploratory model by speculating the system progression and the experimental result [4] (Figure 5).
Table 1.Parameters, mechanism and source of organisms used for GSH-ATP modelling
Figure 5.The GSH-ATP system simulated for 300 minutes, 50 timesteps. (A) The overall plot showing all species involved, including intermediates (glu = L- glutamic acid, cys = L-cysteine, gly = glycine, gcs = g-L-glutamyl-L-cysteine, gsh = reduced glutathione, atp = ATP, adp = ADP, pyr = pyruvate, Ppi = orthophosphate, acp = acetyl phosphate, co2 = CO2, h2o2 = H2O2, ace = acetate). (B-C) are subplots of (A) where species involved in GSH production (B) and species in ATP-regeneration (C) were displayed.
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3. Learn
The exploratory model allows an investigation of each species corresponding to the initial concentration. Observing the model after simulating several initial values and varying limiting agents, we found that pyruvate supply is the limiting substrate of the overall pathway. Additionally, altering parameters like Vmax of each enzyme is also affecting the system differently. The Vmax of enzymes in the pathway is vastly different in magnitude which implies that some enzymes are more active than others. Therefore, the question of balanced enzyme concentration was brought up. Since enzymes are one of the main costs in cell-free production, finding the minimum enzyme needed for the maximum production will make the pathway substantially efficient.
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4. Design
The next design iteration is based on the prediction of enzyme ratio needed for the maximum titer of GSH. The fabricated model and SciPy (45) library were used to further predict the character of the system under various enzyme levels. For this experiment, the aim is to do concave maximisation, which is simply to search for the optimum Vmax, that can be assumed relevant to the in vitro enzyme level, for the maximum glutathione production. However, using the SciPy library, only the minimise function was available. So, the model was transposed into a quasiconvex function by returning the highest negative glutathione flux and report enzyme level at the point.
Figure 6.Global optimisation heatmap arranges along the x-axis by the descending summation of Vmax. The ODE was set to compute at time 30 minutes where the system reached equilibrium.
The GshF low-level requirement was recommended from the global optimisation (Figure 6). On average, GSH was produced at 10.993 ± 0.005 mM and the minimum total Vmax discovered were 11.62, 725.69 and 1621.17 mM/min of GshF, Pox5 and AckA, respectively. Various Vmax of AckA and Pox5 indicate a low sensitivity to the pathway to some extent. The ratio between VmaxPox5/ VmaxAckA is 1.43 ± 0.9.
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5. Built
The GSH-ATP system is then simulated via the kinetic modelling again with the initial condition similar to that being used in practice and applying the amount of enzyme as the Vmax suggested.
Figure 7.The GSH-ATP system simulated for 60 minutes, 50 timesteps.
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6. Test-Learn
The predicted condition was demonstrated experimentally using the exact initial concentration of substrate and incubation time as found in the in silico model. However, due to the time constraints of this project, enzymes applied in the experiment are from the clarified cell lysates but not the purified enzyme as predicted.
The conclusion of whether the system regenerates ATP cannot be clear cut because reaction 8, another positive control, is only giving 0.43% yield (calculated from theoretical product titre predicted from the model). Therefore, GSH production in sample 6, which contain lower ATP, might be hindered by the detection limit. Interestingly, sample 7 with higher pyruvate is showing a slightly higher GSH. Even though the GSH amount in reactions 6 and 7 is not significantly different, this unidentical result could possibly indicate ATP regeneration, which is limited by pyruvate. The low level of GSH obtained is possibly caused by the endogenous enzyme. Therefore, the experiment should be perform again with either an excessive amount of pyruvate or a more purified enzyme.
Figure 8.GSH titer from GSH production-coupled ATP regeneration system. Refer to the table for the composition in each setting. Reactions 1, 2, 3, and 5 are Negative control are reactions 1, 2, 3 and 5, while reactions 4 and 8 are positive controls. Reaction 6 demonstrate the system with moderate pyruvate while reaction 7 was supplied with a high concentration of pyruvate [available, available in high level, not available/negative control]. The error bar shows the standard deviation of duplicated GSH detection noted that reactions 1-3 are single replicate.
References:
[1] Acumen Research and Consulting. Glutathione Market Value Anticipated To Reach US$ 198.2 Million By 2027: Acumen Research And Consulting. 2021; [online]; IntradoGlobeNewswire. [Updated 2021 Mar 11, Cited 2021 Aug 17]. Available from: https://www.globenewswire.com/news-release/2021/03/11/2191543/0/en/Glutathione-Market-Value-Anticipated-To-Reach-US-198-2-Million-By-2027-Acumen-Research-And-Consulting.html
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