A comprehensive genome-scale model for Rhodosporidium toruloides IFO0880 accounting for functional genomics and phenotypic data

Abstract

Rhodosporidium toruloides is a red, basidiomycetes yeast that can accumulate a large amount of lipids and produce carotenoids. To better assess this non-model yeast's metabolic capabilities, we reconstructed a genome-scale model of R. toruloides IFO0880's metabolic network (iRhto1108) accounting for 2204 reactions, 1985 metabolites and 1108 genes. In this work, we integrated and supplemented the current knowledge with in-house generated biomass composition and experimental measurements pertaining to the organism's metabolic capabilities. Predictions of genotype-phenotype relations were improved through manual curation of gene-protein-reaction rules for 543 reactions leading to correct recapitulations of 84.5% of gene essentiality data (sensitivity of 94.3% and specificity of 53.8%). Organism-specific macromolecular composition and ATP maintenance requirements were experimentally measured for two separate growth conditions: (i) carbon and (ii) nitrogen limitations. Overall, iRhto1108 reproduced R. toruloides's utilization capabilities for 18 alternate substrates, matched measured wild-type growth yield, and recapitulated the viability of 772 out of 819 deletion mutants. As a demonstration to the model's fidelity in guiding engineering interventions, the OptForce procedure was applied on iRhto1108 for triacylglycerol overproduction. Suggested interventions recapitulated many of the previous successful implementations of genetic modifications and put forth a few new ones.

Fig. 1

Classifications of genes in iRhto1108. Eukaryotic orthologous groups (KOG) annotations are provided in the genome annotation and used for classifying genes to the corresponding functions. Group abbreviations are in the parentheses. A gene with multiple KOG groups assignments were added to all the groups. A gene without KOG annotation was manually assigned to a KOG group. Other groups include A, B, D, J, K, L, N, O, S, T, U, V, W, Y, and Z (see https://genome.jgi.doe.gov/Tutorial/tutorial/kog.html).

Fig. 2

Phenotype phase planes of TAG production (column A) and maximal growth yield (column B) in nutrient (i.e., ammonium, phosphate, sulphate) and oxygen limited conditions. Values on the figure are percentage of maximal allowed flux for nutrients uptake and maximal yield for TAG production and growth rate. Determined by the model, upper bounds of uptake values are minimal amounts required to sustain maximal growth (oxygen 12.78, ammonium 2.43, phosphate 0.20, and sulphate 0.03 mmol.gDW⁻¹.hr⁻¹). Maximal TAG production is 0.31 g/g glucose and maximal growth rate is 0.38 hr⁻¹.

Fig. 3

Visualization of triacylglycerol production pathway. Interventions identified by OptForce and implemented in vivo were annotated. Reaction abbreviations are listed in Table 4 and detailed in Supplementary Materials 1. Metabolite abbreviations: DHA – dihydroxyacetone, DHAP – DHA phosphate, Ficyt – ferricytochrome, Focyt – ferrocytochrome, Pyr – pyruvate, Mal – malate, AcCoa – acetyl-CoA, Oaa – oxaloacetate, Cit – citrate, Acon – aconitate, Akg – alpha-ketoglutarate, Sucdhl – S(8)-succinyldihydrolipoamide, SucCoA – succinyl-CoA, TAG – triacylglycerol.