Search

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)., Competing Interests: The authors declare no conflict of interest.

Kazachstania bulderi is a non-conventional yeast species able to grow efficiently on glucose and δ-gluconolactone at low pH. These unique traits make K. bulderi an ideal candidate for use in sustainable biotechnology processes including low pH fermentations and the production of green chemicals including organic acids. To accelerate strain development with this species, detailed information of its genetics is needed. Here, by employing long read sequencing we report a high-quality phased genome assembly for three strains of K. bulderi species, including the type strain. The sequences were assembled into 12 chromosomes with a total length of 14 Mb, and the genome was fully annotated at structural and functional levels, including allelic and structural variants, ribosomal array and mating type locus. This high-quality reference genome provides a resource to advance our fundamental knowledge of biotechnologically relevant non-conventional yeasts and to support the development of genetic tools for manipulating such strains towards their use as production hosts in biotechnological processes., (© 2023. Springer Nature Limited.)

The quantification of low abundant membrane-binding proteins such as transcriptional factors and chaperones has proven difficult, even with the most sophisticated analytical technologies. Here, we exploit and optimise the non-invasive Fluorescence Correlation Spectroscopy (FCS) for the quantitation of low abundance proteins, and as proof of principle, we choose two interacting proteins involved in the fission of mitochondria in yeast, Fis1p and Mdv1p. In Saccharomyces cerevisiae , the recruitment of Fis1p and Mdv1p to mitochondria is essential for the scission of the organelles and the retention of functional mitochondrial structures in the cell. We use FCS in single GFP-labelled live yeast cells to quantify the protein abundance in homozygote and heterozygote cells and to investigate the impact of the environments on protein copy number, bound/unbound protein state and mobility kinetics. Both proteins were observed to localise predominantly at mitochondrial structures, with the Mdv1p bound state increasing significantly in a strictly respiratory environment. Moreover, a compensatory mechanism that controls Fis1p abundance upon deletion of one allele was observed in Fis1p but not in Mdv1p, suggesting differential regulation of Fis1p and Mdv1p protein expression.

Saccharomyces pastorianus is a natural yeast evolved from different hybridization events between the mesophilic S. cerevisiae and the cold-tolerant S. eubayanus. This complex aneuploid hybrid carries multiple copies of the parental alleles alongside specific hybrid genes and encodes for multiple protein isoforms which impart novel phenotypes, such as the strong ability to ferment at low temperature. These characteristics lead to agonistic competition for substrates and a plethora of biochemical activities, resulting in a unique cellular metabolism. Here, we investigated the transcriptional signature of the different orthologous alleles in S. pastorianus during temperature shifts. We identified temperature-dependent media-independent genes and showed that 35% has their regulation dependent on extracellular leucine uptake, suggesting an interplay between leucine metabolism and temperature response. The analysis of the expression of ortholog parental alleles unveiled that the majority of the genes expresses preferentially one parental allele over the other and that S. eubayanus-like alleles are significantly over-represented among the genes involved in the cold acclimatization. The presence of functionally redundant parental alleles may impact on the nature of protein complexes established in the hybrid, where both parental alleles are competing. Our expression data indicate that the majority of the protein complexes investigated in the hybrid are likely to be either exclusively chimeric or unispecific and that the redundancy is discouraged, a scenario that fits well with the gene balance hypothesis. This study offers the first overview of the transcriptional pattern of S. pastorianus and provides a rationalization for its unique industrial traits at the expression level., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Non-coding RNAs (ncRNAs), including the more recently identified Stable Unannotated Transcripts (SUTs) and Cryptic Unstable Transcripts (CUTs), are increasingly being shown to play pivotal roles in the transcriptional and post-transcriptional regulation of genes in eukaryotes. Here, we carried out a large-scale screening of ncRNAs in Saccharomyces cerevisiae, and provide evidence for SUT and CUT function. Phenotypic data on 372 ncRNA deletion strains in 23 different growth conditions were collected, identifying ncRNAs responsible for significant cellular fitness changes. Transcriptome profiles were assembled for 18 haploid ncRNA deletion mutants and 2 essential ncRNA heterozygous deletants. Guided by the resulting RNA-seq data we analysed the genome-wide dysregulation of protein coding genes and non-coding transcripts. Novel functional ncRNAs, SUT125, SUT126, SUT035 and SUT532 that act in trans by modulating transcription factors were identified. Furthermore, we described the impact of SUTs and CUTs in modulating coding gene expression in response to different environmental conditions, regulating important biological process such as respiration (SUT125, SUT126, SUT035, SUT432), steroid biosynthesis (CUT494, SUT053, SUT468) or rRNA processing (SUT075 and snR30). Overall, these data capture and integrate the regulatory and phenotypic network of ncRNAs and protein-coding genes, providing genome-wide evidence of the impact of ncRNAs on cellular homeostasis., Competing Interests: The authors have declared that no competing interests exist.