Enzyme constrained models
Enzyme constrained metabolic models include the effect of enzyme kinetics (v = k * e) and a protein capacity limitation (∑e = Etotal) on conventional mass balance (FBA) models.
using COBREXAHere we will construct an enzyme constrained variant of the E. coli "core" model. We will need the model, which we can download if it is not already present.
download_model(
"http://bigg.ucsd.edu/static/models/e_coli_core.json",
"e_coli_core.json",
"7bedec10576cfe935b19218dc881f3fb14f890a1871448fc19a9b4ee15b448d8",
)"e_coli_core.json"Additionally to COBREXA and the model format package, we will need a solver – let's use HiGHS here:
import AbstractFBCModels as A
import JSONFBCModels
import HiGHS
model = load_model("e_coli_core.json")JSONFBCModels.JSONFBCModel(#= 95 reactions, 72 metabolites =#)Enzyme constrained models require parameters that are usually not used by conventional constraint based models. These include reaction specific turnover numbers, molar masses of enzymes, and protein capacity bounds.
Reaction turnover numbers
Enzyme constrained models require reaction turnover numbers, which are often isozyme specfic. Many machine learning tools, or experimental data sets, can be used to estimate these parameters.
Data for reaction turnover numbers
This data is taken from: Heckmann, David, et al. "Machine learning applied to enzyme turnover numbers reveals protein structural correlates and improves metabolic models." Nature communications 9.1 (2018): 1-10.
const ecoli_core_reaction_kcats = Dict(
"ACALD" => 568.11,
"PTAr" => 1171.97,
"ALCD2x" => 75.95,
"PDH" => 529.76,
"MALt2_2" => 234.03,
"CS" => 113.29,
"PGM" => 681.4,
"TKT1" => 311.16,
"ACONTa" => 191.02,
"GLNS" => 89.83,
"ICL" => 17.45,
"FBA" => 373.42,
"FORt2" => 233.93,
"G6PDH2r" => 589.37,
"AKGDH" => 264.48,
"TKT2" => 467.42,
"FRD7" => 90.20,
"SUCOAS" => 18.49,
"ICDHyr" => 39.62,
"AKGt2r" => 234.99,
"GLUSy" => 33.26,
"TPI" => 698.30,
"FORt" => 234.38,
"ACONTb" => 159.74,
"GLNabc" => 233.80,
"RPE" => 1772.485,
"ACKr" => 554.61,
"THD2" => 24.73,
"PFL" => 96.56,
"RPI" => 51.77,
"D_LACt2" => 233.51,
"TALA" => 109.05,
"PPCK" => 218.42,
"PGL" => 2120.42,
"NADTRHD" => 186.99,
"PGK" => 57.64,
"LDH_D" => 31.11,
"ME1" => 487.01,
"PIt2r" => 233.86,
"ATPS4r" => 71.42,
"GLCpts" => 233.90,
"GLUDy" => 105.32,
"CYTBD" => 153.18,
"FUMt2_2" => 234.37,
"FRUpts2" => 234.19,
"GAPD" => 128.76,
"PPC" => 165.52,
"NADH16" => 971.74,
"PFK" => 1000.46,
"MDH" => 25.93,
"PGI" => 468.11,
"ME2" => 443.09,
"GND" => 240.12,
"SUCCt2_2" => 234.18,
"GLUN" => 44.76,
"ADK1" => 111.64,
"SUCDi" => 680.31,
"ENO" => 209.35,
"MALS" => 252.75,
"GLUt2r" => 234.22,
"PPS" => 706.14,
"FUM" => 1576.83,
)Dict{String, Float64} with 62 entries:
"ACALD" => 568.11
"PTAr" => 1171.97
"PGL" => 2120.42
"NADTRHD" => 186.99
"ALCD2x" => 75.95
"PGK" => 57.64
"PDH" => 529.76
"LDH_D" => 31.11
"ME1" => 487.01
"PIt2r" => 233.86
"MALt2_2" => 234.03
"CS" => 113.29
"PGM" => 681.4
"TKT1" => 311.16
"ATPS4r" => 71.42
"ACONTa" => 191.02
"GLNS" => 89.83
"GLCpts" => 233.9
"GLUDy" => 105.32
⋮ => ⋮We have these here:
ecoli_core_reaction_kcats # units = 1/sDict{String, Float64} with 62 entries:
"ACALD" => 568.11
"PTAr" => 1171.97
"PGL" => 2120.42
"NADTRHD" => 186.99
"ALCD2x" => 75.95
"PGK" => 57.64
"PDH" => 529.76
"LDH_D" => 31.11
"ME1" => 487.01
"PIt2r" => 233.86
"MALt2_2" => 234.03
"CS" => 113.29
"PGM" => 681.4
"TKT1" => 311.16
"ATPS4r" => 71.42
"ACONTa" => 191.02
"GLNS" => 89.83
"GLCpts" => 233.9
"GLUDy" => 105.32
⋮ => ⋮Each reaction in a constraint-based model usually has gene reaction rules associated with it. These typically take the form of, possibly multiple, isozymes that can catalyze a reaction. A turnover number needs to be assigned to each isozyme, as shown below. Additionally, some enzymes are composed of multiple subunits, which differ in subunit stoichiometry. This also needs to be accounted for. Assuming a stoichiometry of 1 for everything tends to work just right OK if there is no better information available.
reaction_isozymes = Dict{String,Dict{String,Isozyme}}() # a mapping from reaction IDs to isozyme IDs to isozyme structs.
for rid in A.reactions(model)
grrs = A.reaction_gene_association_dnf(model, rid)
isnothing(grrs) && continue # skip if no grr available
haskey(ecoli_core_reaction_kcats, rid) || continue # skip if no kcat data available
for (i, grr) in enumerate(grrs)
d = get!(reaction_isozymes, rid, Dict{String,Isozyme}())
d["isozyme_"*string(i)] = Isozyme( # each isozyme gets a unique name
gene_product_stoichiometry = Dict(grr .=> fill(1.0, size(grr))), # assume subunit stoichiometry of 1 for all isozymes
kcat_forward = ecoli_core_reaction_kcats[rid] * 3.6, # forward reaction turnover number units = 1/h
kcat_reverse = ecoli_core_reaction_kcats[rid] * 3.6, # reverse reaction turnover number units = 1/h
)
end
endTake care with the units of the turnover numbers. In literature they are usually reported in 1/s. However, flux units are typically mmol/gDW/h, suggesting to rescale the turnover numbers to 1/h in order to use the conventional flux units.
Enzyme molar masses
We also require the mass of each enzyme, to properly weight the contribution of each flux/isozyme in the capacity bound(s). These data can typically be found in uniprot.
Gene product masses
This data is downloaded from Uniprot for E. coli K12, gene mass in kDa. To obtain these data manually, go to Uniprot and search using these terms: reviewed:yes AND organism:"Escherichia coli (strain K12) [83333]".
const ecoli_core_gene_product_masses = Dict(
"b4301" => 23.214,
"b1602" => 48.723,
"b4154" => 65.972,
"b3236" => 32.337,
"b1621" => 56.627,
"b1779" => 35.532,
"b3951" => 85.96,
"b1676" => 50.729,
"b3114" => 85.936,
"b1241" => 96.127,
"b2276" => 52.044,
"b1761" => 48.581,
"b3925" => 35.852,
"b3493" => 53.389,
"b3733" => 31.577,
"b2926" => 41.118,
"b0979" => 42.424,
"b4015" => 47.522,
"b2296" => 43.29,
"b4232" => 36.834,
"b3732" => 50.325,
"b2282" => 36.219,
"b2283" => 100.299,
"b0451" => 44.515,
"b2463" => 82.417,
"b0734" => 42.453,
"b3738" => 30.303,
"b3386" => 24.554,
"b3603" => 59.168,
"b2416" => 63.562,
"b0729" => 29.777,
"b0767" => 36.308,
"b3734" => 55.222,
"b4122" => 60.105,
"b2987" => 53.809,
"b2579" => 14.284,
"b0809" => 26.731,
"b1524" => 33.516,
"b3612" => 56.194,
"b3735" => 19.332,
"b3731" => 15.068,
"b1817" => 35.048,
"b1603" => 54.623,
"b1773" => 30.81,
"b4090" => 16.073,
"b0114" => 99.668,
"b3962" => 51.56,
"b2464" => 35.659,
"b2976" => 80.489,
"b1818" => 27.636,
"b2285" => 18.59,
"b1702" => 87.435,
"b1849" => 42.434,
"b1812" => 50.97,
"b0902" => 28.204,
"b3403" => 59.643,
"b1612" => 60.299,
"b1854" => 51.357,
"b0811" => 27.19,
"b0721" => 14.299,
"b2914" => 22.86,
"b1297" => 53.177,
"b0723" => 64.422,
"b3919" => 26.972,
"b3115" => 43.384,
"b4077" => 47.159,
"b3528" => 45.436,
"b0351" => 33.442,
"b2029" => 51.481,
"b1819" => 30.955,
"b0728" => 41.393,
"b2935" => 72.212,
"b2415" => 9.119,
"b0727" => 44.011,
"b0116" => 50.688,
"b0485" => 32.903,
"b3736" => 17.264,
"b0008" => 35.219,
"b3212" => 163.297,
"b3870" => 51.904,
"b4014" => 60.274,
"b2280" => 19.875,
"b2133" => 64.612,
"b2278" => 66.438,
"b0118" => 93.498,
"b2288" => 16.457,
"b3739" => 13.632,
"b3916" => 34.842,
"b3952" => 32.43,
"b2925" => 39.147,
"b2465" => 73.043,
"b2297" => 77.172,
"b2417" => 18.251,
"b4395" => 24.065,
"b3956" => 99.063,
"b0722" => 12.868,
"b2779" => 45.655,
"b0115" => 66.096,
"b0733" => 58.205,
"b1478" => 35.38,
"b2492" => 30.565,
"b0724" => 26.77,
"b0755" => 28.556,
"b1136" => 45.757,
"b2286" => 68.236,
"b0978" => 57.92,
"b1852" => 55.704,
"b2281" => 20.538,
"b2587" => 47.052,
"b2458" => 36.067,
"b0904" => 30.991,
"b1101" => 50.677,
"b0875" => 23.703,
"b3213" => 52.015,
"b2975" => 58.92,
"b0720" => 48.015,
"b0903" => 85.357,
"b1723" => 32.456,
"b2097" => 38.109,
"b3737" => 8.256,
"b0810" => 24.364,
"b4025" => 61.53,
"b1380" => 36.535,
"b0356" => 39.359,
"b2277" => 56.525,
"b1276" => 97.677,
"b4152" => 15.015,
"b1479" => 63.197,
"b4153" => 27.123,
"b4151" => 13.107,
"b2287" => 25.056,
"b0474" => 23.586,
"b2284" => 49.292,
"b1611" => 50.489,
"b0726" => 105.062,
"b2279" => 10.845,
"s0001" => 0.0,
)Dict{String, Float64} with 137 entries:
"b4301" => 23.214
"b1602" => 48.723
"b4154" => 65.972
"b3236" => 32.337
"b1621" => 56.627
"b1779" => 35.532
"b3951" => 85.96
"b1676" => 50.729
"b3114" => 85.936
"b1241" => 96.127
"b2276" => 52.044
"b1761" => 48.581
"b3925" => 35.852
"b3493" => 53.389
"b3733" => 31.577
"b2926" => 41.118
"b0979" => 42.424
"b4015" => 47.522
"b2296" => 43.29
⋮ => ⋮We have the molar masses here:
ecoli_core_gene_product_masses # unit kDa = kg/molDict{String, Float64} with 137 entries:
"b4301" => 23.214
"b1602" => 48.723
"b4154" => 65.972
"b3236" => 32.337
"b1621" => 56.627
"b1779" => 35.532
"b3951" => 85.96
"b1676" => 50.729
"b3114" => 85.936
"b1241" => 96.127
"b2276" => 52.044
"b1761" => 48.581
"b3925" => 35.852
"b3493" => 53.389
"b3733" => 31.577
"b2926" => 41.118
"b0979" => 42.424
"b4015" => 47.522
"b2296" => 43.29
⋮ => ⋮Just as with the turnover numbers, take extreme care about the units of the molar masses. In literature they are usually reported in Da or kDa (g/mol). However, as noted above, flux units are typically mmol/gDW/h. Since the enzyme kinetic equation is v = k * e (where k is the turnover number) it suggests that the enzyme variable will have units of mmol/gDW. The molar masses come into play when setting the capacity limitations, e.g. usually a sum over all enzymes weighted by their molar masses as e * M. Thus, if the capacity limitation has units of g/gDW, then the molar masses must have units of g/mmol (i.e., kDa).
Capacity limitation
The capacity limitation usually denotes an upper bound of protein available to the cell. Multiple capacity bounds can be used (cytosol, membrane, etc).
total_enzyme_capacity = 50.0 # mg of enzyme/gDW50.0Running a basic enzyme constrained model
With all the parameters specified, we can directly use the enzyme constrained convenience function to run enzyme constrained FBA in one shot:
ec_solution = enzyme_constrained_flux_balance_analysis(
model;
reaction_isozymes,
gene_product_molar_masses = ecoli_core_gene_product_masses,
capacity = total_enzyme_capacity,
optimizer = HiGHS.Optimizer,
)ConstraintTrees.Tree{Float64} with 12 elements:
:coupling => ConstraintTrees.Tree{Float64}(#= 0 elements …
:flux_stoichiometry => ConstraintTrees.Tree{Float64}(#= 72 elements…
:fluxes => ConstraintTrees.Tree{Float64}(#= 95 elements…
:fluxes_forward => ConstraintTrees.Tree{Float64}(#= 95 elements…
:fluxes_reverse => ConstraintTrees.Tree{Float64}(#= 95 elements…
:gene_product_amounts => ConstraintTrees.Tree{Float64}(#= 130 element…
:gene_product_capacity => ConstraintTrees.Tree{Float64}(#= 1 element =…
:isozyme_flux_forward_balance => ConstraintTrees.Tree{Float64}(#= 62 elements…
:isozyme_flux_reverse_balance => ConstraintTrees.Tree{Float64}(#= 62 elements…
:isozyme_forward_amounts => ConstraintTrees.Tree{Float64}(#= 62 elements…
:isozyme_reverse_amounts => ConstraintTrees.Tree{Float64}(#= 62 elements…
:objective => 0.706993We can notice that the objective function is a little lower than with unconstrained E. coli core:
ec_solution.objective0.7069933828572321One can also observe many interesting thing, e.g. the amount of gene product material required for the system to run. Importantly, the units of these values depend on the units used to set the turnover numbers and protein molar masses.
ec_solution.gene_product_amountsConstraintTrees.Tree{Float64} with 130 elements:
:b0008 => 0.00482419
:b0114 => 0.00550415
:b0115 => 0.00550415
:b0116 => 0.00550415
:b0118 => 0.00243563
:b0351 => 0.0
:b0356 => 0.0
:b0474 => 0.0
:b0485 => 0.0
:b0720 => 0.00187026
:b0721 => 0.0
:b0722 => 0.0
:b0723 => 0.0
:b0724 => 0.0
:b0726 => 0.0
:b0727 => 0.0
:b0728 => 0.0
:b0729 => 0.0
:b0733 => 0.0
⋮ => ⋮The total amount of required gene product mass is, by default, present as total_capacity:
ec_solution.gene_product_capacityConstraintTrees.Tree{Float64} with 1 element:
:total_capacity => 50.0Simplified models
Because most active reactions typically only use a single isozyme, we may also use a simplified representation of the problem where this fact is reflected, saving the variable allocation for the isozymes.
simplified_enzyme_constrained_flux_balance_analysis takes similar arguments as the enzyme_constrained_flux_balance_analysis, but automatically chooses the "fastest" reaction isozyme for each reaction direction and builds the model with that.
simplified_ec_solution = simplified_enzyme_constrained_flux_balance_analysis(
model;
reaction_isozymes,
gene_product_molar_masses = ecoli_core_gene_product_masses,
capacity = total_enzyme_capacity,
optimizer = HiGHS.Optimizer,
)ConstraintTrees.Tree{Float64} with 8 elements:
:capacity_limits => ConstraintTrees.Tree{Float64}(#= 1 element =#)
:coupling => ConstraintTrees.Tree{Float64}(#= 0 elements =#)
:flux_stoichiometry => ConstraintTrees.Tree{Float64}(#= 72 elements =#)
:fluxes => ConstraintTrees.Tree{Float64}(#= 95 elements =#)
:fluxes_forward => ConstraintTrees.Tree{Float64}(#= 95 elements =#)
:fluxes_reverse => ConstraintTrees.Tree{Float64}(#= 95 elements =#)
:gene_product_amounts => ConstraintTrees.Tree{Float64}(#= 97 elements =#)
:objective => 0.706993In this case, the result is the same as with the full analysis:
simplified_ec_solution.capacity_limits.total_capacity50.0Gene product amounts are not present in the model but are reconstructed nevertheless (they are uniquely determined by the flux):
simplified_ec_solution.gene_product_amountsConstraintTrees.Tree{Float64} with 97 elements:
:b0008 => 0.00482419
:b0114 => 0.00550415
:b0115 => 0.00550415
:b0116 => 0.00550415
:b0118 => 0.00243563
:b0351 => 0.0
:b0474 => 0.0
:b0485 => 0.0
:b0720 => 0.00187026
:b0721 => 0.0
:b0722 => 0.0
:b0723 => 0.0
:b0724 => 0.0
:b0726 => 0.0
:b0727 => 0.0
:b0728 => 0.0
:b0729 => 0.0
:b0767 => 0.000794011
:b0809 => 0.0
⋮ => ⋮Variability analysis with enzyme constraints
Enzyme-constrained variability analysis can be executed on a model by combining enzyme_constrained_flux_balance_constraints (or simplified_enzyme_constrained_flux_balance_constraints) with constraints_variability (or any other analysis function):
ec_system = enzyme_constrained_flux_balance_constraints(
model;
reaction_isozymes,
gene_product_molar_masses = ecoli_core_gene_product_masses,
capacity = total_enzyme_capacity,
)ConstraintTrees.Tree{ConstraintTrees.Constraint} with 12 elements:
:coupling => ConstraintTrees.Tree{ConstraintTrees.Constra…
:flux_stoichiometry => ConstraintTrees.Tree{ConstraintTrees.Constra…
:fluxes => ConstraintTrees.Tree{ConstraintTrees.Constra…
:fluxes_forward => ConstraintTrees.Tree{ConstraintTrees.Constra…
:fluxes_reverse => ConstraintTrees.Tree{ConstraintTrees.Constra…
:gene_product_amounts => ConstraintTrees.Tree{ConstraintTrees.Constra…
:gene_product_capacity => ConstraintTrees.Tree{ConstraintTrees.Constra…
:isozyme_flux_forward_balance => ConstraintTrees.Tree{ConstraintTrees.Constra…
:isozyme_flux_reverse_balance => ConstraintTrees.Tree{ConstraintTrees.Constra…
:isozyme_forward_amounts => ConstraintTrees.Tree{ConstraintTrees.Constra…
:isozyme_reverse_amounts => ConstraintTrees.Tree{ConstraintTrees.Constra…
:objective => ConstraintTrees.Constraint(ConstraintTrees.L…Here, we can do the FVA "manually", first solving the system:
ec_optimum = optimized_values(
ec_system,
output = ec_system.objective,
objective = ec_system.objective.value,
optimizer = HiGHS.Optimizer,
)0.7069933828572321...then creating a system constrained to near-optimal growth:
import ConstraintTrees as C
ec_system.objective.bound = C.Between(0.99 * ec_optimum, Inf)ConstraintTrees.Between(0.6999234490286598, Inf)...and finally, finding the extremes of the near-optimal part of the feasible space:
ec_variabilities =
constraints_variability(ec_system, ec_system, optimizer = HiGHS.Optimizer)ConstraintTrees.Tree{Tuple{Union{Nothing, Float64}, Union{Nothing, Float64}}} with 12 elements:
:coupling => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:flux_stoichiometry => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:fluxes => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:fluxes_forward => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:fluxes_reverse => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:gene_product_amounts => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:gene_product_capacity => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:isozyme_flux_forward_balance => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:isozyme_flux_reverse_balance => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:isozyme_forward_amounts => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:isozyme_reverse_amounts => ConstraintTrees.Tree{Tuple{Union{Nothing, Fl…
:objective => (0.699923, 0.706993)By default, the result computes variabilities of all possible values in the model. (I.e., it also computes variabilities for the variable combinations that are present in the tree!) As usual, the results can be observed in the original constraint tree structure, giving us the variabilities for reaction fluxes:
ec_variabilities.fluxesConstraintTrees.Tree{Tuple{Union{Nothing, Float64}, Union{Nothing, Float64}}} with 95 elements:
:ACALD => (-0.582453, 0.0)
:ACALDt => (-0.582453, 0.0)
:ACKr => (-7.49528, -6.65899)
:ACONTa => (0.755147, 0.955009)
:ACONTb => (0.755147, 0.955009)
:ACt2r => (-7.49528, -6.65899)
:ADK1 => (-0.0, 0.550397)
:AKGDH => (-0.0, 0.19941)
:AKGt2r => (-0.180894, -4.55705e-13)
:ALCD2x => (-0.46972, 0.0)
:ATPM => (8.39, 8.99477)
:ATPS4r => (31.2442, 32.3197)
:BIOMASS_Ecoli_core_w_GAM => (0.699923, 0.706993)
:CO2t => (-15.7459, -14.6315)
:CS => (0.755147, 0.955009)
:CYTBD => (28.67, 29.8735)
:D_LACt2 => (-0.419985, 0.0)
:ENO => (14.6876, 15.2254)
:ETOHt2r => (-0.46972, 0.0)
⋮ => ⋮...as well as for gene product requirements:
ec_variabilities.gene_product_amountsConstraintTrees.Tree{Tuple{Union{Nothing, Float64}, Union{Nothing, Float64}}} with 130 elements:
:b0008 => (-0.0, 0.020956)
:b0114 => (0.00499882, 0.00571416)
:b0115 => (0.00499882, 0.00571416)
:b0116 => (0.00499882, 0.00578451)
:b0118 => (-0.0, 0.00850601)
:b0351 => (-0.0, 0.0170398)
:b0356 => (-0.0, 0.0144781)
:b0474 => (-0.0, 0.0241603)
:b0485 => (-0.0, 0.00270586)
:b0720 => (0.00185156, 0.0023416)
:b0721 => (-0.0, 0.000551851)
:b0722 => (-0.0, 0.000551851)
:b0723 => (-0.0, 0.000551851)
:b0724 => (-0.0, 0.000551851)
:b0726 => (-0.0, 0.000209436)
:b0727 => (-0.0, 0.000209436)
:b0728 => (-0.0, 0.00800682)
:b0729 => (-0.0, 0.00800682)
:b0733 => (1.41735e-16, 0.0541366)
⋮ => ⋮...and for the individual directional isozymes:
ec_variabilities.isozyme_forward_amounts.PGMConstraintTrees.Tree{Tuple{Union{Nothing, Float64}, Union{Nothing, Float64}}} with 3 elements:
:isozyme_1 => (-0.0, 0.0108292)
:isozyme_2 => (-0.0, 0.00710008)
:isozyme_3 => (-0.0, 0.0118397)If we do not need to compute all these values, it is often more efficient to only ask for the part of the output that is required:
ec_gp_amount_variabilities = constraints_variability(
ec_system,
ec_system.gene_product_amounts,
optimizer = HiGHS.Optimizer,
)ConstraintTrees.Tree{Tuple{Union{Nothing, Float64}, Union{Nothing, Float64}}} with 130 elements:
:b0008 => (-0.0, 0.020956)
:b0114 => (0.00499882, 0.00571416)
:b0115 => (0.00499882, 0.00571416)
:b0116 => (0.00499882, 0.00578451)
:b0118 => (-0.0, 0.00850601)
:b0351 => (-0.0, 0.0170398)
:b0356 => (-0.0, 0.0144781)
:b0474 => (-0.0, 0.0241603)
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⋮ => ⋮This page was generated using Literate.jl.