Limonene Synthase
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Model construction progress :
Enzyme | Pathway | Keq | Kcat | VmaxF | KmGPP | KmSub2 | [GPP] | [Sub2] | VmaxR | KmLim | KmPP | [Limonene] | [PP] |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
02_LimSynth | Limonene Biosynthesis | 1 | 5 | 3 | 4 | 1 |
Legend:
Have not started ·· | 1 -2 data found ·· | 3-4 data found ·· | sufficient data found/estimated ·· | data distribution generated ·· | data sampled |
to do | DONE! |
Contents
What we know
Issues
Strategies
Reaction catalysed
Metabolite Background Information
Long metabolite names are abbreviated in the model for clarity and standard identification purposes.
Metabolite | Abbreviation | Chemical Formula | Molar mass (g/mol) | ChEBI | ChEMBL | PubChem |
---|---|---|---|---|---|---|
geranyl diphosphate | GPP | C10H20O7P2 | 314.209 | 17211 | 41432 | 445995 |
(-)-4S-limonene | Limonene | C10H16 | 136.24 | 15384 | 449062 | 22311 or 439250 |
diphosphate | PP | O7P2 | 173.94 | 644102 |
Equation Rate
Parameter | Description | Units |
---|---|---|
VLimSynth | Reaction rate for Limonene Synthase | |
Vmaxforward | Maximum reaction rate towards the production of limonene | ref |
KmGPP | Michaelis-Menten constant for GPP | mM |
KmLimonene | Michaelis-Menten constant for Limonene | mM |
KmPP | Michaelis-Menten constant for PP | mM |
Keq | Equilibrium constant | |
[GPP] | GPP concentration | mM |
[Limonene] | Limonene concentration | mM |
[PP] | PP concentration | mM |
Strategies for estimating the kinetic parameter values
Calculating the Equilibrium Constant
Standard Gibbs Free energy
Standard Gibbs Free energy for Limonene Synthase from MetaCyc (EC 4.2.3.16) is -28.049988 kcal/mol [1].
SI derived unit for Gibbs free energy is Joules per mol (J mol-1). 1 kJ·mol−1 is equal to 0.239 kcal·mol−1.
Therefore, the Gibbs free energy for Limonene synthase in kJ mol-1 is:
- Failed to parse (Cannot store math image on filesystem.): \cfrac {1}{0.239 kcal.mol^-1} * -28.049988 kcal.mol^-1
- Failed to parse (Cannot store math image on filesystem.): = -117.36396 kJmol^-1
The Equilibrium Constant
The equilibrium constant can be calculated using the Van't Hoff Isotherm equation:
Failed to parse (Cannot store math image on filesystem.): = exp \left ( \cfrac {-(- 117.36396 \text { kJmol}^{-1})}{ (8.31 \text{ JK}^{-1} \text { mol}^{-1} * 289 K} \right )
Failed to parse (Cannot store math image on filesystem.): = exp \left ( \cfrac { + 117.36396 \text { kJmol}^{-1} }{ 2401.59 \text{ JK}^{-1}\text { mol}^{-1} }\right)
Failed to parse (Cannot store math image on filesystem.): = exp \left ( \cfrac{ 117.364 * 10^3 \text { Jmol}^{-1}}{2401.59 \text{ JK}^{-1}\text { mol}^{-1}} \right)
Failed to parse (Cannot store math image on filesystem.): =exp \left ( 48.8693 \right )
Failed to parse (Cannot store math image on filesystem.): = 1.6736 * 10^{21}
where;
Keq | Equilibrium constant |
-ΔG° | Gibbs free energy change. For Limonene Synthase it is -117.364 kJmol-1 |
R | Gas constant with a value of 8.31 JK-1mol-1 |
T | Temperature which is always expressed in Kelvin |
Kinetic Parameter Values
Values for the kinetic parameter required to simulate this model can be obtained from published and unpublished literature.
A more detailed descriptions of the values listed above can be found here , where I've linked and highlighted where these data came from.
Km Values
Km (mM) | Unit | Substrate / Product | Directionality | Organism | References |
---|---|---|---|---|---|
0.00125 | mM | GPP | forward | Ricciocarpos natans | Ref |
0.0018 | mM | GPP | forward | Mentha piperita | Ref |
0.00625 | mM | GPP | forward | Cannabis sativa L. | Ref |
0.00496 | mM | GPP | forward | Cannabis sativa L. | Ref |
0.0031 | mM | GPP | forward | Citrus limon | ref |
0.016 | mM | GPP | forward | Escherichia coli | Ref |
0.0068 | mM | GPP | forward | Cannabis sativa L. | [2] |
0.0067 | mM | GPP | forward | Cannabis sativa L. | Ref |
Vmax values
Vmax | Unit | Directionality | Organism | References |
---|---|---|---|---|
0.08 | µmol/min/mg | forward | Cannabis sativa L. | References |
0.13 | µmol/min/mg | forward | Cannabis sativa L. | References |
0.4748 | µmol/min/mg | forward | Citrus limon | References |
Vmax | Unit | Directionality | Organism | References |
Vmax | Unit | Directionality | Organism | References |
Vmax | Unit | Directionality | Organism | References |
Kcat values
Kcat | Unit | Organism | Reference |
---|---|---|---|
0.3 | s-1 | Mentha piperita & Mentha spicata | [3] |
0.08 | s-1 | Cannabis sativa L. | Reference |
0.14 | s-1 | Cannabis sativa L. | Reference |
0.02 | s-1 | E. coli | Reference |
0.082 | s-1 | Cannabis sativa L. | [2] |
Extracting Information from Limonene Production Rates
The production rates would reflect on the flux for this enzyme, and this would provide provide the insights on the Vmax of this enzyme.
Amount produced (mg/L) | Time (H) | Organism | Description | Reaction Flux (µM/s) |
---|---|---|---|---|
5 | 24 | Escherichia coli | Possible reason for the low limonene production might due to the insufficient supply of IPP and DMAPP [4]. | 0.0255 |
335 | 48 | Escherichia coli | Engineered E.coli in which heterologous MVA pathway was installed [5]. | 0.8537 |
35.8 | 48 | Escherichia coli | E.coli was engineered to express GPPS, LS, DXS, and IDI [6] . | 0.0912 |
4.87 | 48 | Escherichia coli | This was the initial titer. The study established a limonene biosynthesis pathway in E.coli using four different polycistronic operons based on 3 vectors with varied expression strength [6]. | 0.0124 |
17.4 | 48 | Escherichia coli | Using a plasmid with DXS and IDI over expressed [6]. | 0.0445 |
430 | 72 | Escherichia coli | [5] | 0.7306 |
Simulations
these are mock simulation results
References
- ↑ Latendresse M. (2013). "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."
- ↑ 2.0 2.1 Günnewich et. al. 2006. "Functional expression and characterization of trichome-specific (-)-limonene synthase and (+)-α-pinene synthase from Cannabis sativa", Natural Product Communications, 0(0):1-10
- ↑ Alonso et. al. 1992. "Purification of 4S-Limonene Synthase, a Monoterpene Cyclase from the Glandular Trichomes of Peppermint (Mentha x piperita) and Spearmint (Mentha spicata)", The Journal of Biological Chemistry, 267(11):7582-7587
- ↑ Carter, Ora A. et. al.2013. "Monoterpene biosynthesis pathway construction in Escherichia coli",Phytochemistry, 64:425–433, 2003.
- ↑ 5.0 5.1 Alonso-Gutierez et. al. 2013. "Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production", Metabolic Engineering, 19:33-41 Cite error: Invalid
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tag; name "AlonsoGutierez2013" defined multiple times with different content - ↑ 6.0 6.1 6.2 Du et. al. 2014. "Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E.coli", Bioprocessing and Bioprocessing, 1:10