Team:Shenzhen/Result/YAO.Factory

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Contents

1 Introduction
2 Simulation
3 Result
4 References

Introduction

In order to estimate to what extent our YAO system has improved the ability of producing IPP, FPP and GPP in the engineered cell, we construct a model for biochemical reactions of mevalonate pathway in yeast.

At first we plan to model the reactions at equilibrium states. However, as Gibbs free energy for most of the reactions in mevalonate pathway are unavailable, we decide to model them by Michealis-Menton kinetics at the beginning of the reactions when no products have been accumulated.

Simulation

For acetoacetyl-CoA thiolase, the reaction is

The rate expression is defined as

Table 1. Parameters for acetoacetyl-CoA thiolase

Parameter	Defination	Value(mM)	Organism
KmA	binding constant of AcCoA	0.33	Zoogloea ramigera
KiA	Inhibit constant of AcCoA	0.0014	Rattus norvegicus

For 3-hydroxy-3-methylglutaryl coenzyme A synthase, the reaction is

The rate expression is defined as

Table 2. Parameters for 3-hydroxy-3-methylglutaryl coenzyme A synthase

Parameter	Definition	Value(mM)	Organism
KmA(mM)	binding constant for AcCoA	0.014	Saccharomyces cerevisiae
KmB(mM)	binding constant for AcacCoA	0.003	Saccharomyces cerevisiae
KiB(mM)	inhibit constant for AcacCoA	0.008	Saccharomyces cerevisiae
KiC1(mM)	inhibit constant for CoA on AcCoA	0.038	Saccharomyces cerevisiae
KiC2(mM)	Inhibit constant for CoA on AcacCoA	0.06	Saccharomyces cerevisiae

For HMG-CoA Reductase, the reaction is

The rate expression is defined as

Table 3. Parameters for HMG-CoA Reductase

Parameter	Definition	Value(mM)	Organism
KmA	binding constant for HMG-CoA	0.045	Sulfolobus solfataricus
KmB	Binding constant for NADPH	0.023	Sulfolobus solfataricus

For mevalonate kinase, the reference reaction is

The rate expression is defined as

Table 4. Parameters for mevalonate kinase

Parameter	Definition	Value(mM)	Organism
KmA	binding constant for MVA	0.0408	Homo sapiens
KmB	binding constant for ATP	7.4	Saccharomyces cerevisiae
KiADP	inhibit constant for ADP	2.7	Enterococcus faecalis

For phosphomevalonate kinase, the reference reaction is

The rate expression is defined as

Table 5. Parameters for phosphomevalonate kinase

Parameter	Definition	Value(mM)	Organism
KmA	binding constant for MVA-5P	0.034	Homo sapiens
KmB	binding constant for ATP	0.107	Homo sapiens
KiB	inhibit constant for ATP	0.137	Streptococcus pneumoniae
KiADP	Inhibit constant for ADP	0.41	Streptococcus pneumoniae

For mevalonate pyrophosphate decarboxylase, the reference reaction is

The rate expression is defined as

Table 6. Parameters of mevalonate pyrophosphate decarboxylase

Parameter	Definition	Value(mM)	Organism
KmA	binding constant for MVA-5PP	0.123	Saccharomyces cerevisiae
KmB	binding constant for ATP	0.061	Saccharomyces cerevisiae

For isopentenyl diphosphate:dimethylallyl diphosphate isomerase, the reaction is

The rate expression is defined as

Table 7.

Parameter	Definition	Value(mM)	Organism
Km	Binding constant for IPP	0.035	Saccharomyces cerevisiae

For farnesyl diphosphate synthase, the first reaction is:

The rate expression is defined as

The second reaction is

The rate expression is

Table 7. Parameters for the ERG20

Parameter	Definition	Value(mM)	Organism
KmA	binding constant for DMA-PP	0.009	Abies grandis
KmB	binding constant for I-PP	0.0018	Abies grandis
KmC	binding constant for GPP	0.0153	Abies grandis

Table 8. The turnover rate for each enzyme

Enzyme	Substrate	Turnover rate(1/s)	Organism
acetoacetyl-CoA thiolase	Acetyl-CoA	2.1	Zoogloea ramigera
3-hydroxy-3-methylglutaryl coenzyme A synthase	Acetyl-CoA	0.0667	Gallus gallus
hydroxymethylglutaryl-CoA reductase	hydroxymethylglutaryl-CoA	0.023	Rattus norvegicus
mevalonate kinase	(R)-mevalonate	21.9	Rattus norvegicus
phosphomevalonate kinase	phosphomevalonate	10.2	Sus scrofa
mevalonate pyrophosphate decarboxylase	5-diphosphomevalonate	4.9	Saccharomyces cerevisiae
isopentenyl diphosphate:dimethylallyl diphosphate isomerase	isopentenyl diphosphate	0.2	Sulfolobus shibatae
farnesyl diphosphate synthase	isopentenyl diphosphate	0.49	Mycobacterium tuberculosis H37Rv
farnesyl diphosphate synthase	geranyl diphosphate	0.2	Mycobacterium tuberculosis H37Rv

We set the concentration of Actyl-CoA to 1000μM, and consider it as a constant. For simplicity, we assume the concentration of Actyl-CoA is the same in YAO and cytoplasm. The concentration of ATP, ADP and NADPH is set to 1500μM, 1500μM and 100μM respectively and considered as constants. Concentrations of all other metabolite are set to 0 in the beginning.

Result

Our Yao clustered the enzymes in a small space, so that the concentration of enzymes in YAO will be higher than in cytoplasm. We assume that the volume of YAO is 1/10000 of that of cell, and a cell often contains 20 YAOs. Thus the concentrations of enzymes in YAO should be 500 times of that in cytoplasm. However, the transported rate of enzymes with signal peptides should be considered. We let it range from 0.002 to 1, thus ratio of concentrations of enzymes between YAO and cytoplasm range from 0 to 500.

We then plot the concentration of IPP, GPP and FPP as a function of time at different concentration of enzymes.

Figure 1. Production of IPP at different concentrations of enzymes.

Figure 2. Production of GPP at different concentrations of enzymes.

Figure 3. Production of FPP at different concentrations of enzymes.

We can see from the plot that the production of IPP, GPP and FPP in a certain period of time after the beginning of the reactions increase dramatically as the concentrations of enzymes increase.

As the concentrations of products are increasing, need for ATP and NADPH also increase. The concentrations of NADPH and ATP will limit the produce of IPP, GPP and FPP. We then let the constant concentrations of NADPH and ATP range from 0 to maximum to see their affects on production.

Figure 4. Production of IPP at different concentrations of NADPH.

Figure 5. Production of IPP at different concentrations of ATP.

Our results show that concentrations of both ATP and NADPH do not have significant affects on production at the beginning of the reactions.

References

[1] Ro, D.K., et al., Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature, 2006. 440(7086): p. 940-3.

[2] Middleton, B., The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver. Biochem J, 1974. 139(1): p. 109-21.

[3] Tanzawa, K. and A. Endo, Kinetic analysis of the reaction catalyzed by rat-liver 3-hydroxy-3-methylglutaryl-coenzyme-A reductase using two specific inhibitors. Eur J Biochem, 1979. 98(1): p. 195-201.

[4]Middleton, B., The kinetic mechanism of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker's yeast. Biochem J, 1972. 126(1): p. 35-47.

[5] Voynova, N.E., S.E. Rios, and H.M. Miziorko, Staphylococcus aureus mevalonate kinase: isolation and characterization of an enzyme of the isoprenoid biosynthetic pathway. J Bacteriol, 2004. 186(1): p. 61-7.

[6] Scheer, Maurice. BRENDA. 2011. http://www.brenda-enzymes.org/

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