Difference between revisions of "Limonene Synthase"

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You can go back to main page of the kinetic model [http://www.systemsbiology.ls.manchester.ac.uk/wiki/index.php/Kinetic_Model_of_Monoterpenoid_Biosynthesis_Wiki '''here'''].
 
You can go back to main page of the kinetic model [http://www.systemsbiology.ls.manchester.ac.uk/wiki/index.php/Kinetic_Model_of_Monoterpenoid_Biosynthesis_Wiki '''here'''].
  
−
In this model, LIMS is modelled in Escherichia coli and this model is replicating the bacterial system in vivo. As such, in vivo-like conditions such as pH of 7.5 and temperature of 30°C in E. coli is set as the ideal conditions when assigning weights to its parameter. Limonene synthase catalyses the formation of limonene and pyrophosphate from one molecule of geranyl diphosphate (GPP). The following equations show LIMS’s reaction stoichiometry and its corresponding reaction rate using the Michaelis-Menten rate law:
+
Limonene synthase (LIMS) is not native to ''E. coli'' and a heterologous LIMS gene from ''Mentha spicata'' was engineered into the cell <ref name="AlonsoGutierrez2013">[https://www.sciencedirect.com/science/article/pii/S109671761300058X Alonso-Gutierrez, J., et al.](2013). "Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production." Metabolic Engineering 19: 33-41.</ref>.
−
 
 
  
 
:<math>
 
:<math>
Line 11: Line 10:
  
  
 +
== Reaction rate ==
  
−
== Equation Rate ==
+
The reaction rate for LIMS is modelled using the reversible Michaelis-Menten equation <ref name="Sauro2011">Sauro, H. M. (2011). Appendix B: List of Common Rate Laws. Enzyme Kinetics for Systems Biology. United States of America, Future Skills Software: 279-290.</ref>, and is shown below:
−
 
 
−
The reversible Michaelis-Menten equation to model the dynamic changes of LimSynth is:
 
  
 
:<math>
 
:<math>
  
−
V_\mathrm{LimSynth} =  Vmax_\mathrm{forward} * \cfrac {\cfrac{[GPP]}{Km_\mathrm{GPP}} * \left ( 1 - \cfrac {[Limonene]*[PP]}{[GPP]*K_\mathrm{eq}} \right )}{1 + \cfrac {[GPP]}{Km_\mathrm{GPP}} + \cfrac {[Limonene]}{Km_\mathrm{Limonene}}  + \cfrac {[PP]}{Km_\mathrm{PP}}  + \cfrac {[Limonene]*[PP]}{Km_\mathrm{Limonene}*Km_\mathrm{PP}}}
+
V_\mathrm{LimSynth} =  Kcat_\mathrm{LIMS}*[LIMS] * \cfrac {\cfrac{[GPP]}{Km_\mathrm{GPP}} * \left ( 1 - \cfrac {[Limonene]*[PP]}{[GPP]*K_\mathrm{eq}} \right )}{1 + \cfrac {[GPP]}{Km_\mathrm{GPP}} + \cfrac {[Limonene]}{Km_\mathrm{Limonene}}  + \cfrac {[PP]}{Km_\mathrm{PP}}  + \cfrac {[Limonene]*[PP]}{Km_\mathrm{Limonene}*Km_\mathrm{PP}}}
  
 
</math>
 
</math>
Line 28: Line 26:
 
! scope="col" style="width: 50px; background: #ADD8E6;" | Units
 
! scope="col" style="width: 50px; background: #ADD8E6;" | Units
 
|-
 
|-
−
| V<sub>LimSynth</sub> || Reaction rate for Limonene Synthase ||  
+
| V<sub>LimSynth</sub> || Reaction rate for Limonene Synthase || μM/min
 
|-
 
|-
−
| V<sub>max<sub>forward</sub></sub> || Maximum reaction rate towards the production of limonene || ref
+
| K<sub>cat<sub>LIMS</sub></sub> || Turnover number for limonene synthase || min<sup>-1</sup>
 
|-
 
|-
−
| Km<sub>GPP</sub> || Michaelis-Menten constant for GPP || mM
+
| Km<sub>GPP</sub> || Michaelis-Menten constant for GPP || μM
 
|-
 
|-
−
| Km<sub>Limonene</sub> || Michaelis-Menten constant for Limonene || mM
+
| Km<sub>Limonene</sub> || Michaelis-Menten constant for Limonene ||μM
 
|-
 
|-
−
| Km<sub>PP</sub> || Michaelis-Menten constant for PP || mM
+
| Km<sub>PP</sub> || Michaelis-Menten constant for PP || μM
 
|-
 
|-
−
| K<sub>eq</sub> || Equilibrium constant ||  
+
| K<sub>eq</sub> || Equilibrium constant || dimensionless
 
|-
 
|-
−
| [GPP] || GPP concentration || mM
+
| [GPP] || GPP concentration || μM
 
|-
 
|-
−
| [Limonene] || Limonene concentration || mM
+
| [Limonene] || Limonene concentration || μM
 
|-
 
|-
−
| [PP]|| PP concentration || mM
+
| [PP]|| PP concentration || μM
 
|}
 
|}
  
−
== Metabolite Background Information ==
 
−
 
−
Long metabolite names are abbreviated in the model for clarity and standard identification purposes.
 
−
 
−
{| class="wikitable" style="text-align:center"
 
−
|-
 
−
! Metabolite
 
−
! Abbreviation
 
−
! Chemical Formula
 
−
! Molar mass (g/mol)
 
−
! ChEBI
 
−
! ChEMBL
 
−
! PubChem
 
−
|-
 
−
| geranyl diphosphate
 
−
| GPP
 
−
| C<sub>10</sub>H<sub>20</sub>O<sub>7</sub>P<sub>2</sub>
 
−
| 314.209
 
−
| 17211
 
−
| 41432
 
−
| 445995
 
−
|-
 
−
| (-)-4S-limonene
 
−
| Limonene
 
−
| C<sub>10</sub>H<sub>16</sub>
 
−
| 136.24
 
−
| 15384
 
−
| 449062
 
−
| 22311 or 439250
 
−
|-
 
−
| diphosphate
 
−
| PP
 
−
| O<sub>7</sub>P<sub>2</sub>
 
−
| 173.94
 
−
|
 
−
|
 
−
| 644102
 
−
|-
 
−
| limonene synthase
 
−
| LimSynth
 
−
|
 
−
| 70.03 kDa <ref name ="Gunnewich2008"></ref>, 72.4 kDa <ref name="Turner1999"> [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC59327/ Turner,G. et. al.]1999. "Limonene synthase, the enzyme responsible for monoterpene biosynthesis in peppermint, is localized to leucoplasts of oil gland secretory cells", Plant Physiology 120(3): 879-886</ref> ; 60kDa <ref name="Maruyama2002">[http://www.ncbi.nlm.nih.gov/pubmed/12033511 Maruyama, T. et. al. ]2002. "Molecular cloning, functional expression and characterization of ''d''-Limonene synthase from ''Agastache rugosa''" Biol. Pharm. Bull. 25(5): 661-665</ref>; 56 kDa <ref name="Alonso1992"></ref>
 
−
|
 
−
|
 
−
|
 
−
|-
 
−
|}
 
  
 
== Parameterisation ==
 
== Parameterisation ==
−
 
+
The parameterisation of LIMS for this model was performed according to our DIPPER protocol <ref name="Tsig2018>[https://www.nature.com/articles/s41596-018-0056-z Tsigkinopoulou, A., et al.]  (2018). "Defining informative priors for ensemble modeling in systems biology." Nature protocols 13: 2643-2663.</ref>. Here, we list the thermodynamic and kinetic paramater values obtained from the literature, weights assigned and  the log-normal distribution parameter values calculated.
−
=== Calculating the Equilibrium Constant ===
+
===Thermodynamic parameter values===
−
 
 
−
Unlike the kinetic parameter values, thermodynamic parameter values such as for equilibrium constant (Keq) are not easily found in literature reports. However, Keq can be calculated from Gibbs Free Energy (ΔG°) using the following equation:
 
−
 
 
−
:<math>
 
−
 
 
−
K_\mathrm{eq} = exp \left ( \cfrac {-ΔG^{°'}}{RT} \right )
 
−
 
 
−
</math>
 
−
 
 
−
where;
 
−
 
 
−
{|
 
−
|-
 
−
| style="width: 30pt;" | '''K<sub>eq</sub>'''
 
−
| style="width: 400pt;" | Equilibrium constant
 
−
 
 
−
|-
 
−
| '''-ΔG<sup>°</sup>''' || Gibbs free energy change (kcal/mol)
 
−
|-
 
−
| '''R''' || Gas constant (0.0019859 kcal/K/mol)
 
−
|-
 
−
| '''T''' || Absolute temperature (298 K)
 
−
|}
 
−
 
 
−
 
 
 
Gibbs free energy values for LIMS are obtained from MetaCyc [http://biocyc.org/META/new-image?object=4.2.3.16-RXN (EC 4.2.3.16)] is -28.049988 kcal/mol <ref name="Latendresse2013"> Latendresse M. (2013). [http://www.biocyc.org/PGDBConceptsGuide.shtml#gibbs. "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."] </ref> and Equilibrator [http://equilibrator.weizmann.ac.il/]. Table 2 summarizes the Δ<sub>r</sub>G° values found for LIMS and the calculated Keq. These Keq values are given an arbitrary equal weight of 1 as the Δ<sub>r</sub>G° values obtained were calculated from a group contribution method and do not have any measurement conditions that would allow us to assess the Keq values according to the weighting scheme set out in (insert link here).
 
Gibbs free energy values for LIMS are obtained from MetaCyc [http://biocyc.org/META/new-image?object=4.2.3.16-RXN (EC 4.2.3.16)] is -28.049988 kcal/mol <ref name="Latendresse2013"> Latendresse M. (2013). [http://www.biocyc.org/PGDBConceptsGuide.shtml#gibbs. "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."] </ref> and Equilibrator [http://equilibrator.weizmann.ac.il/]. Table 2 summarizes the Δ<sub>r</sub>G° values found for LIMS and the calculated Keq. These Keq values are given an arbitrary equal weight of 1 as the Δ<sub>r</sub>G° values obtained were calculated from a group contribution method and do not have any measurement conditions that would allow us to assess the Keq values according to the weighting scheme set out in (insert link here).
  
Line 145: Line 71:
 
|-
 
|-
 
|}
 
|}
−
 
−
 
  
 
=== Kinetic Parameter Values ===
 
=== Kinetic Parameter Values ===
−
 
−
Values for kinetic parameters such as for KmGPP and Kcatforward can be obtained from extensive literature searches. Our findings from literature searches of these parameters are summarised in Table 1. In addition to the parameter values, the measurement conditions such as the pH, temperature and gene source used for the kinetic evaluations are also taken into consideration. Then, in order to assess the plausibility of the parameter values collected, weights are assigned according to different weighting categories as described in Table 2.  Final weight for each parameter value is calculated by taking each resulting weight from each category and multiplying it. For example, Lücker et. al. isolated limonene synthase cDNA from lemon (Citrus limon) and functionally expressed them in Escherichia coli. Enzyme assays were conducted at pH 7.0 and 30°C, and the resulting measured KmGPP is at 0.7 µM. This parameter value (KmGPP) is assigned a total weight of 32 (1x1x4x2x4=32) after assessing that:- a) this value was measured in vitro (weight=1), b) limonene synthase isolated was from lemon which is unrelated to E. coli (weight=1), c) limonene synthase measured was from the same EC (EC 4.2.3.16, weight=4), d) measurement pH was within ± 0.5 from the ideal pH of 7.5 (weight=2), and finally e) measurement temperature was at the ideal 30°C (weight=4). The breakdown of weights assigned to each kinetic parameter value found for limonene synthase is shown in Table below.
 
−
 
−
A more detailed descriptions of the values listed above can be found [[ Limonene Synthase: Detailed information on the kinetic values obtained from the literature | '''HERE''' ]], where I've linked and highlighted where these data came from.
 
−
 
  
 
{| class="wikitable"
 
{| class="wikitable"
−
|+ Table 2: Summary of kinetic parameter values retrieved from literature for LIMS
+
|+ Table 2: Km for GPP values for LIMS
−
! style="text-align: center; font-weight: bold;" | Name
+
| align="center" style="background:#f0f0f0;"|'''Parameter'''
−
! style="text-align: center; font-weight: bold;" | Value
+
| align="center" style="background:#f0f0f0;"|'''Value'''
−
! style="text-align: center; font-weight: bold;" | Units
+
| align="center" style="background:#f0f0f0;"|'''Error'''
−
! style="text-align: center; font-weight: bold;" | Remarks-References
+
| align="center" style="background:#f0f0f0;"|'''Weight'''
 +
| align="center" style="background:#f0f0f0;"|'''Error type'''
 +
| align="center" style="background:#f0f0f0;"|'''Description'''
 +
| align="center" style="background:#f0f0f0;"|'''References'''
 +
|-
 +
| Km_gpp_LIMS||47.4||3.8||128||0||LIMS gene from Lavandula angustifolia was expressed in E. coli. The kinetics were measured in vitro at 30°C||<ref name="Landmann2007">[https://www.sciencedirect.com/science/article/pii/S0003986107003013 Landmann, C., et al.] (2007). "Cloning and characterization of three terpene synthases from lavender (''Lavandula angustifolia'')." Archives of Biochemistry and Biophysics 465: 417-429.</ref>
 
|-
 
|-
−
| style="text-align: center;" | KmGPP
+
| Km_gpp_LIMS||130||NaN||32||0||LIMS gene from Citrus sinensis (orange) was expressed in E. coli. The Km was measured in vitro at 20°C.||<ref name="Entova2013">[Entova, S.](2013). Kinetic characterization, crystallization, and photosynthetic expression of (+)-$R-limonene synthase from C. sinensis. Department of Biochemistry. Massachusetts, Brandeis University. Master's: 55.</ref>
−
| style="text-align: center;" | 6.8
 
−
| style="text-align: center;" | µM
 
−
| rowspan="2" | This study has isolated and characterised a (-)-limonene synthase recombinant encoded by Cannabis sativa L. cv. ‘Skunk’ trichome mRNA. pH optimum was determined at pH 6.5 and a temperature optimum at 40°C. (Günnewich,, N., 2007)
 
 
|-
 
|-
−
| style="text-align: center;" | Kcatforward
+
| Km_gpp_LIMS||6.8||NaN||32||0||LIMS gene from Cannabis sativa L. var. 'Skunk' plants was expressed in E. coli. The kinetics were measured at 40°C.||<ref name="Gunnewich2007"> [https://www.researchgate.net/publication/42089356_Functional_expression_and_characterization_of_trichome-specific_--limonene_synthase_and_-a-pinene_synthase_from_Cannabis_sativa Günnewich, N., Page, J.E., Köllner, T.G., Degenhardt, J., & Kutchan, T.M] 2007. "Functional expression and characterization of trichome-specific (-)-limonene synthase and (+)-α-pinene synthase from ''Cannabis sativa'' ". Nat. Prod. Comm. 2(3): 223-232.  </ref>
−
| style="text-align: center;" | 0.082
 
−
| style="text-align: center;" | s-1
 
 
|-
 
|-
−
| style="text-align: center;" | KmGPP
+
| Km_gpp_LIMS||0.7||NaN||128||0||LIMS gene from Citrus limon (lemon) was expressed in E. col. Kinetics were measured in vitro at 30°C.||<ref name="Lucker2002">[https://febs.onlinelibrary.wiley.com/doi/full/10.1046/j.1432-1033.2002.02985.x Lücker, J., et al.] (2002). "Monoterpene biosynthesis in lemon (''Citrus limon'') cDNA isolation and functional analysis of four monoterpene synthases." Eur. J. Biochem. 269: 3160-3171.
−
| style="text-align: center;" | 47.4 ± 3.8
+
</ref>
−
| style="text-align: center;" | µM
 
−
| rowspan="2" | In this study,limonene synthase were cloned from lavender (Lavandula angustifolia) leaves and flowers. Temperature was determined at 30°C and pH at 7.0. (Landmann,C 2007)
 
 
|-
 
|-
−
| style="text-align: center;" | Kcatforward
+
| Km_gpp_LIMS||1.25||NaN||16||0||LIMS isolated from Ricciocarpos natans. Kinetics measured at 32°C and pH7.0. ||<ref name="Adam1996">[https://www.sciencedirect.com/science/article/pii/S0003986196903525 Adam, K.-P., et al.] (1996). "Partial purification and characterization of a monoterpene cyclase, limonene synthase, from the liverwort ''Ricciocarpus natans''." Archives of Biochemistry and Biophysics 332(2): 352-356. </ref>
−
| style="text-align: center;" | 0.012
 
−
| style="text-align: center;" | s-1
 
 
|-
 
|-
−
| style="text-align: center;" | KmGPP
+
| Km_gpp_LIMS||1.8||NaN||32||0||LIMS isolated from Mentha x piperita (peppermint). Kinetics were measured at 30°C.||<ref name="Rajaonarivony1992">[https://www.sciencedirect.com/science/article/pii/0003986192905436 Rajaonarivony, J. I. M., et al.] (1992). "Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha X piperita)." Archives of Biochemistry and Biophysics 296(1): 49-57.</ref>
−
| style="text-align: center;" | 6.7
+
|}
−
| style="text-align: center;" | µM
+
 
−
| rowspan="2" | This study reports the kinetic evaluation the native form of LIMS from Mentha spicata. Reaction mixtures were determined at pH 7.0 and temperature of 30°C. Kinetic constants are averaged data sets of triplicate analyses (n=3). (Williams 1998)
+
{| class="wikitable"
 +
|+ Table 3: Turnover number values for LIMS
 +
| align="center" style="background:#f0f0f0;"|'''Parameter'''
 +
| align="center" style="background:#f0f0f0;"|'''Value'''
 +
| align="center" style="background:#f0f0f0;"|'''Error'''
 +
| align="center" style="background:#f0f0f0;"|'''Weight'''
 +
| align="center" style="background:#f0f0f0;"|'''Error type'''
 +
| align="center" style="background:#f0f0f0;"|'''Description'''
 +
| align="center" style="background:#f0f0f0;"|'''References'''
 
|-
 
|-
−
| Kcatforward
+
| Kcat_LIMS||0.72||NaN||128||0||LIMS gene from Lavandula angustifolia was expressed in E. coli. The kinetics were measured in vitro at 30°C||<ref name="Landmann2007" />
−
| 0.024
 
−
| s-1
 
−
|  
 
 
|-
 
|-
−
| Kcatforward
+
| Kcat_LIMS||7.8||NaN||32||0||LIMS gene from Citrus sinensis (orange) was expressed in E. coli. The enzyme activities were measured in vitro at 20°C and 37°C.||<ref name="Entova2013" />
−
| 0.3
 
−
| s-1
 
−
| In this study, the authors have isolated and purified limonene synthase from both peppermint (Mentha x piperita) and spearmint (Mentha spicata). Enzyme activity was determined at pH 7.0m and temperature 30°C. (Alonso 1992)
 
−
|  
 
 
|-
 
|-
−
| KmGPP
+
| Kcat_LIMS||2.4||NaN||32||0||LIMS gene from Citrus sinensis (orange) was expressed in E. coli. The enzyme activities were measured in vitro at 20°C and 37°C.||<ref name="Entova2013" />
−
| 1.25
 
−
| µM
 
−
| In this study,
 
−
  limonene synthase is characterised and extracted from liverwort (Ricciocarpus natans). The enzyme assay
 
−
  was conducted at pH 6.5 and at temperature of 32°C. (Adam 1996)
 
−
|  
 
 
|-
 
|-
−
| KmGPP
+
| Kcat_LIMS||4.92||NaN||32||0||LIMS gene from Cannabis sativa L. var. 'Skunk' plants was expressed in E. coli. The kinetics were measured at 40°C.||<ref name="Gunnewich2007" />
−
| 0.7
 
−
| µM
 
−
| This study isolated limonene synthase cDNA from lemon (Citrus limon) and functionally expressed them in Escherichia coli. Enzyme assays were
 
−
  conducted at pH 7.0 and 30°C. (Lucker 2002)
 
−
|  
 
 
|-
 
|-
−
| Km<sub>GPP</sub>
 
−
| 1.8
 
−
  ± 0.3
 
−
| µM
 
−
| Limonene synthase from purified preparation of glandular trichome seceratory cell clusters from peppermint (Mentha x piperita). Enzyme assays were carried out in buffer pH 7.0, and incubated at 30°C. Data reported are the averages of three independent assays (n=3).  (Rajaonarivony 1992)
 
−
|
 
 
|}
 
|}
  
−
=== Vmax values ===
+
=== BRENDA data ===
 +
To further enrich the kinetic parameter values for LIMS, parameter values from EC 4 to EC 4.2.3.* that can be obtained from BRENDA is downloaded. These BRENDA data is integrated with the rest of the kinetic parameter values using our ‘BRENDA Add-on’ protocol. In the BRENDA Add-On protocol, we’ve specified six different ‘EC cases’ that are arranged in order of rank. These EC cases are essentially six different datasets of parameter values downloaded from BRENDA that are filtered according to the specific enzyme class and organism of interest. For this case study example, six different ‘EC case’ datasets were downloaded from BRENDA each for Km and Kcat parameters (Tables below).
  
−
{| class="wikitable sortable"
+
{| class="wikitable"
 +
|+ Table 7: Input matrix for Km values from BRENDA for EC 4.2.3.16
 +
|+  align="bottom" style="caption-side: bottom; text-align: left;" |Parameter value and uncertainty is in µM. Each row represents EC case 2 – 6 respectively. EC case 1 is discarded as number of values obtained were <50. .
 +
|+  align="bottom" style="caption-side: bottom; text-align: left;" |*Uncertainty values in this table correspond to the standard deviation calculated from SEM reported in literature
 +
|+  align="bottom" style="caption-side: bottom; text-align: left;" |** Uncertainty type is multiplicative, therefore ‘1’ is defined.
 +
 
 +
! style="font-weight: bold;" | Parameter value
 +
! style="font-weight: bold;" | Uncertainty*
 +
! style="font-weight: bold;" | Weight
 +
! style="font-weight: bold;" | Uncertainty type**
 
|-
 
|-
−
! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | Vmax
+
| 6.80
−
! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | Unit
+
| 14.9566
−
! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 50px;" | Directionality
+
| 11.9024
−
! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 150px;" | Organism
+
| 1
−
! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 300px;" | Organism
 
−
! style="border: 1px solid black; padding: 5px; background: #ffdead; width: 10px;" | References
 
 
|-
 
|-
−
| 0.08
+
| 49
−
| µmol/min/mg
+
| 69.0555
−
| forward
+
| 7.4390
−
| ''Cannabis sativa'' L.
+
| 1
−
|
 
−
| <ref name="Gunnewich2008"> Gunnewich, N. 2008. "Expression and characterization of terpene synthases from ''Cannabis sativa'' L. and ''Salvia sclarea'' L. Doctoral thesis.  </ref>
 
 
|-
 
|-
−
| 0.12 ± 0.01
+
| 121
−
| µmol/min/mg
+
| 36.1126
−
| forward
+
| 2.9756
−
| ''Cannabis sativa''
+
| 1
−
|
 
−
| <ref name="Gunnewich2008"> </ref>
 
 
|-
 
|-
−
| 0.4748
+
| 500
−
| µmol/min/mg
+
| 36.9586
−
| forward
+
| 1.4878
−
| ''Citrus limon''
+
| 1
−
|
 
−
| References
 
−
|-
 
−
| 0.53
 
−
| µM/min
 
−
| forward
 
−
| ''Citrus sinensis''
 
−
| 200µM GPP, 20°C, [E]: 0.07µM, pH7.5
 
−
| <ref name= "Entova2013"></ref>
 
−
|-
 
−
| 0.64
 
−
| µM/min
 
−
| forward
 
−
| ''Citrus sinensis''
 
−
| 200µM GPP, 37°C, [E]: 0.26µM, pH7.5
 
−
| <ref name= "Entova2013"></ref>
 
−
|-
 
−
| 19
 
−
| µmol/h/mg
 
−
| forward
 
−
| Mentha x piperita & Mentha spicata
 
−
| Maximum specific activity, 1-10µg protein per mixture, pH &.0, 30°C
 
−
| References
 
 
|-
 
|-
 +
| 300
 +
| 23.8770
 +
| 0.5951
 +
| 1
 
|}
 
|}
  
−
=== Kcat values ===
+
{| class="wikitable"
−
{| class="wikitable sortable"
+
|+ Input matrix for K<sub>cat</sub> values from BRENDA for EC 4.2.3.16
−
! Parameter || Direction || Substrate || Value || Unit || Method || M_w || Organism || O_w || Expression_vector || E_w || Enzyme || En_w || pH || P_w || Temperature || T_w || Total_weight || Description || Reference
+
|+  align="bottom" style="caption-side: bottom; text-align: left;" |Parameter value and uncertainty is in µM. Each row represents EC case 2 – 6 respectively. EC case 1 is discarded as number of values obtained were <50. .
 +
|+  align="bottom" style="caption-side: bottom; text-align: left;" |*Uncertainty values in this table correspond to the standard deviation calculated from SEM reported in literature
 +
|+  align="bottom" style="caption-side: bottom; text-align: left;" |** Uncertainty type is multiplicative, therefore ‘1’ is defined.
 +
! style="font-weight: bold;" | Parameter value
 +
! style="font-weight: bold;" | Uncertainty*
 +
! style="font-weight: bold;" | Weight
 +
! style="font-weight: bold;" | Uncertainty type**
 
|-
 
|-
−
| Kcat || Forward || GPP || 0.0004 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp, expressed in E.coli and truncated (R59), pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
+
| 2.040
 +
| 38.2439
 +
| 9.5610
 +
| 1
 
|-
 
|-
−
| Kcat || Forward || GPP || 0.0004 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli , truncated R58P59, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
+
| 17.400
 +
| 109.1960
 +
| 5.9756
 +
| 1
 
|-
 
|-
−
| Kcat || Forward || GPP || 0.0004 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli , truncated R58A59, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
+
| 200.698
 +
| 177.0428
 +
| 2.3902
 +
| 1
 
|-
 
|-
−
| Kcat || Forward || GPP || 0.0004 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli , S60, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
+
| 192.000
 +
| 44.6715
 +
| 1.1951
 +
| 1
 
|-
 
|-
−
| Kcat || Forward || GPP || 0.002 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli preprotein, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
+
| 168.000
−
|-
+
| 62.4126
−
| Kcat || Forward || GPP || 0.0024 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli wt, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
+
| 0.4780
−
|-
+
| 1
−
| Kcat || Forward || GPP || 0.0034 || 1/s || in vitro (+2) || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli E57, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
 
−
|-
 
−
| Kcat || Forward || GPP || 0.0036 || 1/s || in vitro  || 2 || Unrelated (+0) || 0 || E.coli (+4) || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli Q54, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
 
−
|-
 
−
| Kcat || Forward || GPP || 0.0037 || 1/s || in vitro  || 2 || Unrelated (+0) || 0 || E.coli  || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli R58, pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
 
−
|-
 
−
| Kcat || Forward || GPP || 0.02 || 1/s || in vitro  || 2 || Unrelated || 0 || E.coli  || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Protein from Mentha sp. expressed in E.coli recombinant preprotein , pH7.0, 1-50 mg [E], 25 µM [GPP], limonene produced 94%, 2% myrcene, pinene  || Williams1998
 
−
|-
 
−
| Kcat || Forward || GPP || 0.04 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli  || 4 || Identical (+4) || 4 || close range (+2) || 2 || 37°C (+4) || 4 || 256 || Citrus sinensis, expressed in E.coli 200µM GPP, 37°C, [E]: 0.26µM, pH7.5 || Entova2013
 
−
|-
 
−
| Kcat || Forward || GPP || 0.082 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli  || 4 || Identical (+4) || 4 || close range (+2) || 2 || 36°C - 40°C (+2) || 2 || 128 || Cannabis sativa L., expressed in E.coli,CsTPS1, pH6.5, 40°C, 1.25 mg [LimSynth] per 500ml assay mixture, 10mM [GPP] || Gunnewich2008
 
−
|-
 
−
| Kcat || Forward || GPP || 0.09 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli  || 4 || Identical (+4) || 4 || close range (+2) || 2 || 36°C - 40°C (+2) || 2 || 128 || Cannabis sativa L., expressed in E.coli,Km of 6.25 ± 0.41 µM, pH 6.5,  40°C, [GPP] 0.5 - 25.0 µM used for kinetic parameter determination, kcat 0.09 /s || Gunnewich2008
 
−
|-
 
−
| Kcat || Forward || GPP || 0.13 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli  || 4 || Identical (+4) || 4 || close range (+2) || 2 || 36°C - 40°C (+2) || 2 || 128 || Cannabis sativa L., expressed in E.coli,Km of 6.25 ± 0.41 µM, pH 6.5,  40°C, [GPP] 0.5 - 25.0 µM used for kinetic parameter determination, kcat 0.09 /s || Gunnewich2008
 
−
|-
 
−
| Kcat || Forward || GPP || 0.13 || 1/s || in vitro  || 2 || Unrelated || 0 || E.coli || 4 || Identical (+4) || 4 || close range (+2) || 2 || different temperature (+0) || 0 || 128 || Citrus sinensis, expressed in E.coli 200µM GPP, 20°C, [E]: 0.07 mM, pH7.5 || Entova2013
 
−
|-
 
−
| Kcat || Forward || GPP || 0.3 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || different temperature (+0) || 0 || 128 || Mentha piperita &amp; Mentha spicata, expressed in E.coli , pH7.0, 30°C, enzyme assay, 1 -10 mg protein per mixture || Alonso1992
 
−
|-
 
−
| Kcat || Forward || GPP || 0.000039 || 1/s || in vitro  || 2 || Unrelated  || 0 || E.coli  || 4 || Identical (+4) || 4 || 7.0 (+4) || 4 || 37°C (+4) || 4 || 512 || Citrus sinensis, expressed in E.coli40mM GPP, 37°C, [E]: 0.3 mM, pH7.0 (check units) || Olsen2011
 
 
|}
 
|}
  
−
=== Extracting Information from Limonene Production Rates ===
+
=== Log-normal distribution parameters ===
  
−
The production rates would reflect on the flux for this enzyme, and this would provide provide the insights on the Vmax of this enzyme.  
+
The Mode, Confidence Interval (CI) factor, mu and sigma for each parameter distribution is calculated using our scripts as detailed in the DIPPER protocol <ref name="Tsig2018>[https://www.nature.com/articles/s41596-018-0056-z Tsigkinopoulou, A., et al.]  (2018). "Defining informative priors for ensemble modeling in systems biology." Nature protocols 13: 2643-2663.</ref>.  
  
−
{| class="wikitable" style="width: 60%; height: 200px;"
+
{| class="wikitable"
 +
| align="center" style="background:#f0f0f0;"|'''Parameter'''
 +
| align="center" style="background:#f0f0f0;"|'''Brenda Add-on'''
 +
| align="center" style="background:#f0f0f0;"|'''Mode'''
 +
| align="center" style="background:#f0f0f0;"|'''CI Factor'''
 +
| align="center" style="background:#f0f0f0;"|'''Mu'''
 +
| align="center" style="background:#f0f0f0;"|'''Sigma'''
 +
|-
 +
| Km_gpp_LIMS||✘||6.8||7.6504||3.3719||1.2062
 
|-
 
|-
−
! style="border: 1px solid black; padding: 5px; background: #ffdead;"| Amount produced (mg/L)
+
| Kcat_LIMS||✘||0.8001||2.6307||0.3424||0.7519
−
! style="border: 1px solid black; padding: 5px; background: #ffdead;"| Time (H)
 
−
! style="border: 1px solid black; padding: 5px; background: #ffdead;"| Organism
 
−
! style="border: 1px solid black; padding: 5px; background: #ffdead;"| Description
 
−
! style="border: 1px solid black; padding: 5px; background: #ffdead;"| Reaction Flux (µM/s)
 
−
|- style="height: 100px;"
 
−
| 5  || 24 || style="width: 150px;" | Escherichia coli || style="width: 1200px;" |  Possible reason for the low limonene production might due to the insufficient supply of IPP and DMAPP <ref name="Carter2013">Carter, Ora A. et. al.2013. [http://www.sciencedirect.com/science/article/pii/S0031942203002048 "Monoterpene biosynthesis pathway construction in ''Escherichia coli''",Phytochemistry, 64:425–433, 2003.]</ref>. || 0.0255
 
−
|- style="height: 100px;"
 
−
| 335 || style="width: 50px;" | 48 || Escherichia coli || Engineered E.coli in which heterologous MVA pathway was installed <ref name= "AlonsoGutierez2013">Alonso-Gutierez et. al. 2013. [http://www.sciencedirect.com/science/article/pii/S109671761300058X "Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production", Metabolic Engineering, 19:33-41]</ref>. || 0.8537
 
−
|- style="height: 100px;"
 
−
| 35.8 || 48 || Escherichia coli || E.coli was engineered to express GPPS, LS, DXS, and IDI <ref name="Du2014"> Du et. al. 2014. [http://www.bioresourcesbioprocessing.com/content/1/1/10 "Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E.coli", Bioprocessing and Bioprocessing, 1:10]</ref> . || 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 <ref name="Du2014"></ref>. || 0.0124
+
| Km_gpp_LIMS||✔||||||||
 
|-
 
|-
−
| 17.4 || 48 || Escherichia coli || Using a plasmid with DXS and IDI over expressed <ref name="Du2014"></ref>. || 0.0445
+
| Kcat_LIMS||✔||||||||
 
|-
 
|-
−
| 430 || 72 || Escherichia coli || <ref name= "AlonsoGutierez2013"> </ref> || 0.7306
 
 
|}
 
|}
  
−
=== Parameter estimation ===
+
=== Log-normal distribution parameter estimation ===
  
 
This section can be found [[ LimSynth: Parameter Estimation | ''' HERE ''']]
 
This section can be found [[ LimSynth: Parameter Estimation | ''' HERE ''']]

Latest revision as of 14:14, 29 November 2018

You can go back to main page of the kinetic model here.

Limonene synthase (LIMS) is not native to E. coli and a heterologous LIMS gene from Mentha spicata was engineered into the cell [1].

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


Reaction rate

The reaction rate for LIMS is modelled using the reversible Michaelis-Menten equation [2], and is shown below:

V_\mathrm{LimSynth} = Kcat_\mathrm{LIMS}*[LIMS] * \cfrac {\cfrac{[GPP]}{Km_\mathrm{GPP}} * \left ( 1 - \cfrac {[Limonene]*[PP]}{[GPP]*K_\mathrm{eq}} \right )}{1 + \cfrac {[GPP]}{Km_\mathrm{GPP}} + \cfrac {[Limonene]}{Km_\mathrm{Limonene}} + \cfrac {[PP]}{Km_\mathrm{PP}} + \cfrac {[Limonene]*[PP]}{Km_\mathrm{Limonene}*Km_\mathrm{PP}}}

where :

Parameter Description Units
VLimSynth Reaction rate for Limonene Synthase μM/min
KcatLIMS Turnover number for limonene synthase min-1
KmGPP Michaelis-Menten constant for GPP μM
KmLimonene Michaelis-Menten constant for Limonene μM
KmPP Michaelis-Menten constant for PP μM
Keq Equilibrium constant dimensionless
[GPP] GPP concentration μM
[Limonene] Limonene concentration μM
[PP] PP concentration μM


Parameterisation

The parameterisation of LIMS for this model was performed according to our DIPPER protocol [3]. Here, we list the thermodynamic and kinetic paramater values obtained from the literature, weights assigned and the log-normal distribution parameter values calculated.

Thermodynamic parameter values

Gibbs free energy values for LIMS are obtained from MetaCyc (EC 4.2.3.16) is -28.049988 kcal/mol [4] and Equilibrator [1]. Table 2 summarizes the ΔrG° values found for LIMS and the calculated Keq. These Keq values are given an arbitrary equal weight of 1 as the ΔrG° values obtained were calculated from a group contribution method and do not have any measurement conditions that would allow us to assess the Keq values according to the weighting scheme set out in (insert link here).

ΔrG°(kcal/mol) Keq Error (±) Source Weight
-28.050 3.843E+20 N/A MetaCyc 1
-42.161 ± 2.844 8.711E+30 5.876E+29 Equilibrator 1

Kinetic Parameter Values

Table 2: Km for GPP values for LIMS
Parameter Value Error Weight Error type Description References
Km_gpp_LIMS 47.4 3.8 128 0 LIMS gene from Lavandula angustifolia was expressed in E. coli. The kinetics were measured in vitro at 30°C [5]
Km_gpp_LIMS 130 NaN 32 0 LIMS gene from Citrus sinensis (orange) was expressed in E. coli. The Km was measured in vitro at 20°C. [6]
Km_gpp_LIMS 6.8 NaN 32 0 LIMS gene from Cannabis sativa L. var. 'Skunk' plants was expressed in E. coli. The kinetics were measured at 40°C. [7]
Km_gpp_LIMS 0.7 NaN 128 0 LIMS gene from Citrus limon (lemon) was expressed in E. col. Kinetics were measured in vitro at 30°C. [8]
Km_gpp_LIMS 1.25 NaN 16 0 LIMS isolated from Ricciocarpos natans. Kinetics measured at 32°C and pH7.0. [9]
Km_gpp_LIMS 1.8 NaN 32 0 LIMS isolated from Mentha x piperita (peppermint). Kinetics were measured at 30°C. [10]
Table 3: Turnover number values for LIMS
Parameter Value Error Weight Error type Description References
Kcat_LIMS 0.72 NaN 128 0 LIMS gene from Lavandula angustifolia was expressed in E. coli. The kinetics were measured in vitro at 30°C [5]
Kcat_LIMS 7.8 NaN 32 0 LIMS gene from Citrus sinensis (orange) was expressed in E. coli. The enzyme activities were measured in vitro at 20°C and 37°C. [6]
Kcat_LIMS 2.4 NaN 32 0 LIMS gene from Citrus sinensis (orange) was expressed in E. coli. The enzyme activities were measured in vitro at 20°C and 37°C. [6]
Kcat_LIMS 4.92 NaN 32 0 LIMS gene from Cannabis sativa L. var. 'Skunk' plants was expressed in E. coli. The kinetics were measured at 40°C. [7]

BRENDA data

To further enrich the kinetic parameter values for LIMS, parameter values from EC 4 to EC 4.2.3.* that can be obtained from BRENDA is downloaded. These BRENDA data is integrated with the rest of the kinetic parameter values using our ‘BRENDA Add-on’ protocol. In the BRENDA Add-On protocol, we’ve specified six different ‘EC cases’ that are arranged in order of rank. These EC cases are essentially six different datasets of parameter values downloaded from BRENDA that are filtered according to the specific enzyme class and organism of interest. For this case study example, six different ‘EC case’ datasets were downloaded from BRENDA each for Km and Kcat parameters (Tables below).

Table 7: Input matrix for Km values from BRENDA for EC 4.2.3.16 Parameter value and uncertainty is in µM. Each row represents EC case 2 – 6 respectively. EC case 1 is discarded as number of values obtained were <50. . *Uncertainty values in this table correspond to the standard deviation calculated from SEM reported in literature ** Uncertainty type is multiplicative, therefore ‘1’ is defined.
Parameter value Uncertainty* Weight Uncertainty type**
6.80 14.9566 11.9024 1
49 69.0555 7.4390 1
121 36.1126 2.9756 1
500 36.9586 1.4878 1
300 23.8770 0.5951 1
Input matrix for Kcat values from BRENDA for EC 4.2.3.16 Parameter value and uncertainty is in µM. Each row represents EC case 2 – 6 respectively. EC case 1 is discarded as number of values obtained were <50. . *Uncertainty values in this table correspond to the standard deviation calculated from SEM reported in literature ** Uncertainty type is multiplicative, therefore ‘1’ is defined.
Parameter value Uncertainty* Weight Uncertainty type**
2.040 38.2439 9.5610 1
17.400 109.1960 5.9756 1
200.698 177.0428 2.3902 1
192.000 44.6715 1.1951 1
168.000 62.4126 0.4780 1

Log-normal distribution parameters

The Mode, Confidence Interval (CI) factor, mu and sigma for each parameter distribution is calculated using our scripts as detailed in the DIPPER protocol [3].

Parameter Brenda Add-on Mode CI Factor Mu Sigma
Km_gpp_LIMS ✘ 6.8 7.6504 3.3719 1.2062
Kcat_LIMS ✘ 0.8001 2.6307 0.3424 0.7519
Km_gpp_LIMS ✔
Kcat_LIMS ✔

Log-normal distribution parameter estimation

This section can be found HERE

Simulations

Simulations performed can be found HERE.

References

  1. ↑ Alonso-Gutierrez, J., et al.(2013). "Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production." Metabolic Engineering 19: 33-41.
  2. ↑ Sauro, H. M. (2011). Appendix B: List of Common Rate Laws. Enzyme Kinetics for Systems Biology. United States of America, Future Skills Software: 279-290.
  3. ↑ 3.0 3.1 Tsigkinopoulou, A., et al. (2018). "Defining informative priors for ensemble modeling in systems biology." Nature protocols 13: 2643-2663.
  4. ↑ Latendresse M. (2013). "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc."
  5. ↑ 5.0 5.1 Landmann, C., et al. (2007). "Cloning and characterization of three terpene synthases from lavender (Lavandula angustifolia)." Archives of Biochemistry and Biophysics 465: 417-429.
  6. ↑ 6.0 6.1 6.2 [Entova, S.](2013). Kinetic characterization, crystallization, and photosynthetic expression of (+)-$R-limonene synthase from C. sinensis. Department of Biochemistry. Massachusetts, Brandeis University. Master's: 55.
  7. ↑ 7.0 7.1 Günnewich, N., Page, J.E., Köllner, T.G., Degenhardt, J., & Kutchan, T.M 2007. "Functional expression and characterization of trichome-specific (-)-limonene synthase and (+)-α-pinene synthase from Cannabis sativa ". Nat. Prod. Comm. 2(3): 223-232.
  8. ↑ Lücker, J., et al. (2002). "Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases." Eur. J. Biochem. 269: 3160-3171.
  9. ↑ Adam, K.-P., et al. (1996). "Partial purification and characterization of a monoterpene cyclase, limonene synthase, from the liverwort Ricciocarpus natans." Archives of Biochemistry and Biophysics 332(2): 352-356.
  10. ↑ Rajaonarivony, J. I. M., et al. (1992). "Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha X piperita)." Archives of Biochemistry and Biophysics 296(1): 49-57.
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