Limonene Synthase

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Reaction catalysed

$geranyl diphosphate \rightleftharpoons (−)-(4S)-limonene + diphosphate$

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

Failed to parse (Cannot store math image on filesystem.): V_\mathrm{LimSynth} = Vmax_\mathrm{forward} * \cfrac {\cfrac{[GPP]}{Km_\mathrm{GPP}} * \left ( 1 - \cfrac {[Limonene]*[PP]}{[GPP]*K_\mathrm{eq}} \right )}{\left (1 + \cfrac {[GPP]}{Km_\mathrm{GPP}} + \cfrac {[Limonene]}{Km_\mathrm{Limonene}} \right) * \left (1 + \cfrac {[PP]}{Km_\mathrm{PP}} \right)}

Parameter	Description	Reference
VLimSynth	Reaction rate for Limonene Synthase	ref
Vmaxforward	Maximum reaction rate towards the production of limonene	ref
KmGPP	Michaelis-Menten constant for GPP	ref
KmLimonene	Michaelis-Menten constant for Limonene	ref
KmPP	Michaelis-Menten constant for PP	ref
Keq	Equilibrium constant	ref
[GPP]	GPP concentration	ref
[Limonene]	Limonene concentration	ref
[PP]	PP concentration	ref

Strategies for estimating the kinetic parameter values

Calculating the Equilibrium Constant

The equlibrium constant, K_eq, can be calculated from the Gibbs free energy of a reaction, ΔG_r, using the Van't Hoff isotherm equation:

$-ΔG^° = -RT ln K$

and by dividing both sides of the equation with RT, and later take the exponents of both sides, the K_eq can be calculated by this equation:

$K_\mathrm{eq} = exp \left ( \cfrac {-ΔG^{°'}}{RT} \right )$

where;

Keq	Equilibrium constant
-ΔG°	Gibbs free energy change
R	Gas constant with a value of 8.31 JK-1mol-1
T	Temperature which is always expressed in kelvin

Standard Gibbs Free energy

Standard Gibbs Free energy for Limonene Synthase from MetaCyc (rxn: 4.2.3.16) is -28.049988 kcal/mol .

Extracting Information from Limonene Production Rates

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 .	0.0255
335	48	Escherichia coli	Engineered E.coli in which heterologous MVA pathway was installed .	0.8537
35.8	48	Escherichia coli	E.coli was engineered to express GPPS, LS, DXS, and IDI .	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 .	0.0124
17.4	48	Escherichia coli	Using a plasmid with DXS and IDI over expressed .	0.0445
430	72	Escherichia coli		0.7306

Published Kinetic Parameter Values

Km (mM)	Vmax	Kcat (s-1)	Kcat/Km	Organism	Description
0.00125	-	-	-	Ricciocarpos natans	GPP -> Limonene
0.0018	-	-	-	Mentha piperita	GPP -> Limonene
0.00625	0.08 µmol/min/mg	0.08	1.5	Cannabis sativa L.	GPP -> Limonene
0.00496	0.13 µmol/min/mg	0.14	2.9	Cannabis sativa L.	GPP -> Limonene
0.0031	28.49 µmol/h/mg	-	-	Citrus limon	GPP -> Limonene
0.016	-	0.02	-	Escherichia coli	GPP -> Limonene
0.0068	-	0.082	-	Cannabis sativa L.	GPP -> Limonene
0.0067	-	0.081	-	Cannabis sativa L.	GPP -> Limonene