Manual curation#

A draft GEM needs manual curation to become a high-quality model. Curation corrects gene associations, adds or removes reactions, and moves reactions between compartments, all to describe the in vivo network more accurately. For comparison, the S. cerevisiae model is curated continuously.

Replacing template genes with target homologs#

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

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

BLAST identified that gene RHTO_03911 (oleoyl-CoA desaturase, reaction y300009) has the homolog Hanpo2_15704. Curate the gene association:

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

Adding methanol assimilation#

H. polymorpha is a methylotroph and assimilates methanol, and the draft lacks the required pathway. The pathway has five reactions. Reactions 3 to 5 (formate dehydrogenase, peroxisomal catalase and dihydroxyacetone kinase) are present already, so two reactions 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 the information of the new reactions, and addRxns adds them:

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: the exchange of methanol (and glucose), the peroxisomal diffusion of O₂ and H₂O, and the 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_');

Confirming growth on methanol#

Block the 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);

After these additions the draft model is methylotrophic, in agreement with the literature.

code/reconstructionProtocol.m is the complete runnable script of the protocol.