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3.9 Manual curation

A draft GEM always requires manual curation to become a high-quality model. Curation can mean correcting gene associations, adding or removing reactions, and moving reactions between compartments — all aiming for a more accurate description of the in vivo network. (For comparison, the S. cerevisiae model has been continuously curated since 2003.)

Replace template genes with target homologs

Gap-filling adds reactions from the templates without searching for homology of the associated genes, so the draft contains S. cerevisiae and R. toruloides genes that should be replaced by H. polymorpha homologs. List the reactions still carrying an R. toruloides gene:

rhtoRxns = find(contains(model.grRules, 'RHTO'));
model.rxnNames(rhtoRxns);
model.grRules(rhtoRxns);

By literature search and BLAST, gene RHTO_03911 (oleoyl-CoA desaturase, reaction y300009) was found to have the homolog Hanpo2_15704. Curate the gene association:

model = changeGrRules(model, 'y300009', 'Hanpo2_15704', true);
model = deleteUnusedGenes(model);

Add methanol assimilation

H. polymorpha is a methylotroph able to assimilate methanol, but the draft lacks the required pathway. Five reactions are needed; reactions 3–5 (formate dehydrogenase, peroxisomal catalase, dihydroxyacetone kinase) are already present, so two are added manually:

  1. methanol oxidase — methanol[p] + oxygen[p] => formaldehyde[p] + hydrogen peroxide[p]
  2. dihydroxyacetone synthase — formaldehyde[p] + D-xylulose 5-phosphate[p] => glyceraldehyde 3-phosphate[p] + glycerone[p]

A rxnsToAdd structure collects all the information for the new reactions, which are then added with addRxns:

rxnsToAdd.rxns      = {'MOX', 'DAS'};
rxnsToAdd.equations = {'methanol[p] + oxygen[p] => formaldehyde[p] + hydrogen peroxide[p]', ...
                       'formaldehyde[p] + D-xylulose 5-phosphate[p] => glyceraldehyde 3-phosphate[p] + glycerone[p]'};
rxnsToAdd.rxnNames  = {'methanol oxidase', 'dihydroxyacetone synthase'};
rxnsToAdd.lb        = [0, 0];
rxnsToAdd.ub        = [1000, 1000];
rxnsToAdd.eccodes   = {'1.1.3.13', '2.2.1.3'};
rxnsToAdd.grRules   = {'Hanpo2_76277', 'Hanpo2_95557'};
rxnsToAdd.rxnNotes  = {'Methanol metabolism reaction added by manual curation', ...
                       'Methanol metabolism reaction added by manual curation'};
model = addRxns(model, rxnsToAdd, 3, '', true, true);

A few transport and exchange reactions complete the pathway: methanol (and glucose) exchange, peroxisomal diffusion of O₂ and H₂O, and transport of the intermediates between cytoplasm and peroxisome:

model = addRxnsGenesMets(model, modelSce, {'r_4494', 'r_4391', 'r_1714', 'r_1980', 'r_2098'});
model = addTransport(model, 'c', 'p', {'D-xylulose 5-phosphate' 'methanol' ...
    'glycerone' 'glyceraldehyde 3-phosphate'}, true, false, 't_');

Confirm growth on methanol

Block other carbon sources, allow methanol uptake, and verify growth:

model = setParam(model, 'eq', {'r_1808', 'r_1714'}, 0);   % no glycerol/glucose
model = setParam(model, 'lb', 'r_4494', -1);              % allow methanol uptake
sol = solveLP(model, 1);
printFluxes(model, sol.x);

newCommit(model);

The draft model is now methylotrophic, in agreement with the literature.

See Anticipated results for what the finished draft should look like, and the full runnable script in code/reconstructionProtocol.m.