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.

Genome-scale metabolic reconstruction of the non-model yeast Issatchenkia orientalis SD108 and its application to organic acids production

“…Seven new inorganic ions (i.e., calcium, copper, iron, manganese, magnesium, potassium and zinc) were also included in the biomass reaction following the measurements for S. cerevisiae ( Lange and Heijnen, 2001 ) as these ions are known to be essential (see Supplementary Materials 3). In the absence of direct measurement, the total inorganic ions composition were ported from S. cerevisiae ( Lange and Heijnen, 2001 ), which was the approach earlier considered for another non-model yeast, R. toruloides ( Dinh et al., 2019 ). The cofactor and prosthetic group fractions in the biomass reaction are set to small values (i.e., 10 −4 each) which imposes a biosynthesis requirement on the model but keeps their total amount to only 0.06% of the total biomass by weight.…”

“…For growth predictions involving rich media, supplementary compound uptake rates were set to 0.165 ​mmol gDW −1 hr −1 (i.e., 5% of default substrate uptake rate of 3.3 ​mmol gDW −1 hr −1 ). Simulating rich media components has been described previously ( Dinh et al., 2019 ). In general, the supplementary nutrients present in YNB included thiamine, riboflavin, nicotinate, pyridoxin, folate, (R)-pantothenate, 4-aminobenzoate, and myo-inositol.…”

“…Curated genome-scale reconstructions for Candida glabrata ( Xu et al., 2013 ), Pichia pastoris ( Chung et al., 2010 ; Sohn et al., 2010 ; Caspeta et al., 2012 ; Tomas-Gamisans et al., 2016 ), Scheffersomyces stipites ( Caspeta et al., 2012 ; Balagurunathan et al., 2012 ; Liu et al., 2012 ), S chizosaccharomyces pombe ( Sohn et al., 2012 ), Yarrowia lipolytica ( Loira et al., 2012 ; Pan and Hua, 2012 ; Kavscek et al., 2015 ; Kerkhoven et al., 2016 ), and Rhodosporidium toruloides ( Tiukova et al., 2019 ; Dinh et al., 2019 ) are already available. These models offer templates for reconstructing metabolic models for other yeast species.…”

Recent advances in domesticating non‐model microorganisms

“…129 In fact, omics studies and GSMs have been developed for a few of the non-model microorganisms including P. pastoris, 130,131 S. stipitis, 132 A. niger, 133cyanobacteria, 134,135 and R. toruloides. 113,136 To facilitate strain development from benchtop to industrial scale, bioprocess optimization also plays a critical role, which includes optimization of medium, culture conditions, and downstream processing. With the development of more efficient synthetic biology tools and increased knowledge on cell metabolism and physiology, more non-model microorganisms will be exploited for production of a wide variety of industrially important chemicals, fuels, and materials.…”

Xylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in Rhodotorula toruloides: Insights for Future Biorefinery

“…GEMs are constructed based on the available genome sequence of a specific organism, thus providing a summary of the metabolic network (Kerkhoven et al, 2015). Regarding R. toruloides, a number of metabolic models are available to assess lipid production (Bommareddy et al, 2015;Castañeda et al, 2018), and two reports of GEMs are available (Dinh et al, 2019;Tiukova et al, 2019a). Lopes et al (2020a) reported the first study that combined data from cultivations of R. toruloides under different carbon sources with the GEM.…”