Development of a genome-scale metabolic model for the lager hybrid yeast S. pastorianus to understand the evolution of metabolic pathways in industrial settings

Abstract

In silico tools such as genome-scale metabolic models have shown to be powerful for metabolic engineering of microorganisms. Saccharomyces pastorianus is a complex aneuploid hybrid between the mesophilic Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. This species is of biotechnological importance because it is the primary yeast used in lager beer fermentation and is also a key model for studying the evolution of hybrid genomes, including expression pattern of ortholog genes, composition of protein complexes, and phenotypic plasticity. Here, we created the iSP_1513 GSMM for S. pastorianus CBS1513 to allow top-down computational approaches to predict the evolution of metabolic pathways and to aid strain optimization in production processes. The iSP_1513 comprises 4,062 reactions, 1,808 alleles, and 2,747 metabolites, and takes into account the functional redundancy in the gene-protein-reaction rule caused by the presence of orthologous genes. Moreover, a universal algorithm to constrain GSMM reactions using transcriptome data was developed as a python library and enabled the integration of temperature as parameter. Essentiality data sets, growth data on various carbohydrates and volatile metabolites secretion were used to validate the model and showed the potential of media engineering to improve specific flavor compounds. The iSP_1513 also highlighted the different contributions of the parental sub-genomes to the oxidative and non-oxidative parts of the pentose phosphate pathway. Overall, the iSP_1513 GSMM represent an important step toward understanding the metabolic capabilities, evolutionary trajectories, and adaptation potential of S. pastorianus in different industrial settings.

Importance: Genome-scale metabolic models (GSMM) have been successfully applied to predict cellular behavior and design cell factories in several model organisms, but no models to date are currently available for hybrid species due to their more complex genetics and general lack of molecular data. In this study, we generated a bespoke GSMM, iSP_1513, for this industrial aneuploid hybrid Saccharomyces pastorianus, which takes into account the aneuploidy and functional redundancy from orthologous parental alleles. This model will (i) help understand the metabolic capabilities and adaptive potential of S. pastorianus (domestication processes), (ii) aid top-down predictions for strain development (industrial biotechnology), and (iii) allow predictions of evolutionary trajectories of metabolic pathways in aneuploid hybrids (evolutionary genetics).

Keywords: genome-scale metabolic model; yeast hybrid.

Fig 1

Venn diagrams showing the proportion of S. cerevisiae-like (purple) and S. eubayanus-like (light blue) genes found in the Yeast8 genome-scale model. The intersection represents the proportion of genes that have both parental copies in S. pastorianus.

Fig 2

Predicted biomass (millimoles per gram of dry cell weight per hour) according to the sugar uptake. A sugar import of 20 represents the total consumption of the sugar present in the SD medium. Panel A, predictions using Yeast8; panel B, predictions using iSP_1513.

Fig 3

S. pastorianus CBS 1513 metabolite production in SD medium, SD without leucine, and SD with an additional 100 mg/L leucine measured by GC-MS for the metabolites: panel A, 2-phenyl ester; panel B, ethyl octanoate; panel C, ethyl decanoate; panel D, isoamyl acetate. Symbols: *, P-value ≤ 0.05, **, P-value ≤ 0.01; ***, P-value ≤ 0.001; ns, nonsignificant.

Fig 4

S. pastorianus CBS 1513 glycerol production in SD medium as predicted by the iSP_1513 model constrained using the following: panel A, the transcriptome data obtained in SD medium at 13 °C, 22°C, and 30°C; panel B, as measured experimentally with HPLC in SD medium at 13°C and 30°C. Symbols: ****, P-value ≤ 0.0001.

Fig 5

Carbon central metabolism in S. pastorianus. Panel A, proportion of S. eubayanus-like and S. cerevisiae-like genes present in the genome supporting each reaction. Panel B, proportion of S. eubayanus-like and S. cerevisiae-like genes expressed at 22 °C in SD medium. S. eubayanus-like and S. cerevisiae-like genes are represented as blue and red bars, respectively.