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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).

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Strigolactones (SLs) are bioactive carotenoid derivatives which function as signaling molecules to regulate plant architecture, nutrient absorption and communication with other organisms. The α/ß-fold hydrolase, D14, hydrolyzes SLs, and the hydrolysis product activates D14 to bind to downstream signaling partners, including an E3 ubiquitin ligase MAX2 and SMXL6/7/8 proteins. What was not known was whether binding with one downstream partner would alter the affinity of D14 for other binding partners. Here, we developed an efficient yeast four-hybrid (Y4H) detection system and demonstrate that SL induces the interaction of D14 with both SMXL7 and MAX2 in a dose-dependent manner. Moreover, using our newly established yeast four-hybrid system, we found that the SL-induced D14 interaction with SMXL7 was strengthened by MAX2 while SMXL7 weakened the SL-induced D14 interaction with MAX2. Our findings provide novel insights into the regulatory effects of these signaling components and shed light on the molecular mechanism controlling the core SL signaling pathway. Furthermore, the heterologous yeast platform used for investigating SL complex formation has great potential to explore dynamic interactions in other signaling pathways or elucidate the unknown complex formation for biosynthesis of the parent carotenoids of SLs.

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MAIN CONCLUSION: High-throughput sequencing and yeast one and two-hybrid library screening reveal that DKGA2ox1 and miR171f_3 are involved in the regulation of scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks. Diospyros kaki Thunb. cv. Nan-tong-xiao-fang-shi ('Nan-tong-xiao-fang-shi') interstocks play a critical role in the scion dwarfing. However, the understanding of the molecular signaling pathways that regulate the scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks remain unclear. In this work, the regulatory network in the scion dwarfing with 'Nan-tong-xiao-fang-shi' as interstocks was identified. Using a yeast one-hybrid library screening, luciferase activity analysis, luciferase complementation imaging assays and GFP signal detection, a SPL transcription factor named Diospyros kaki SPL (DKSPL), potentially functioning as a transcriptional activator of the Diospyros kaki GA2ox1 (DKGA2ox1) gene, was identified as a key stimulating factor in the persimmon growth and development. The DKSPL was found in the nucleus, and might play a role in the transcriptional regulation system. A microRNA named miR171f_3 was identified, which might act as a negative regulator of Diospyros kaki SCR (DKSCR) in persimmon. The interactions between DKSCR and seven proteins were experimentally validated with a yeast two-hybrid library screening. Compared to the non-grafted wildtype persimmon, the tissue section of graft union healed well due to the increased expression of cinnamyl-alcohol dehydrogenase. These results indicate that DKGA2ox1 and miR171f_3 may co-promote the scion dwarfing by plant hormone signal transduction pathways.

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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.

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The formation of interspecific hybrids results in the coexistence of two diverged genomes within the same nucleus. It has been hypothesized that negative epistatic interactions and regulatory interferences between the two sub-genomes may elicit a so-called genomic shock involving, among other alterations, broad transcriptional changes. To assess the magnitude of this shock in hybrid yeasts, we investigated the transcriptomic differences between a newly formed Saccharomyces cerevisiae × Saccharomyces uvarum diploid hybrid and its diploid parentals, which diverged ∼20 mya. RNA sequencing (RNA-Seq) based allele-specific expression (ASE) analysis indicated that gene expression changes in the hybrid genome are limited, with only ∼1-2% of genes significantly altering their expression with respect to a non-hybrid context. In comparison, a thermal shock altered six times more genes. Furthermore, differences in the expression between orthologous genes in the two parental species tended to be diminished for the corresponding homeologous genes in the hybrid. Finally, and consistent with the RNA-Seq results, we show a limited impact of hybridization on chromatin accessibility patterns, as assessed with assay for transposase-accessible chromatin using sequencing (ATAC-Seq). Overall, our results suggest a limited genomic shock in a newly formed yeast hybrid, which may explain the high frequency of successful hybridization in these organisms.

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Yeast hybrid systems have been widely used due to their convenience and low cost. Based on these systems, many methods have been developed to analyze protein-protein, protein-DNA and protein-RNA interactions. In this paper, we are reviewing these different yeast hybrid systems. According to the number of hybrid proteins, yeast hybrid systems can be divided into three categories, yeast one-hybrid, yeast two-hybrid and yeast three-hybrid systems. Alternatively, yeast hybrid systems can be categorized according to the subcellular localization of the protein interaction process in the cell into nuclear protein-protein interactions, cytosol protein-protein interactions and membrane protein-protein interactions. Throughout the review, we focus on the progress and limitations of each yeast hybrid system over the recent years.

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BACKGROUND: The rapid increase in the incidence rate of colorectal cancer has led to the search and identification of biomarkers that can predict risk for and future behavior of this malignancy and management. To study the biological role of the phosphorylated Fas associated death domain (pFADD) gene in colorectal cancer, we performed a GAL4-based yeast two-hybrid screening of a human heart cDNA library. METHODS: A series of two yeast hybrid method was used to identification of protein-protein interaction. It was confirmed by glutathione S-transferase (GST) pull down assay and co-immunoprecipitation (co-IP). Three channeled fluorescence microscopy further confirmed the interaction in cellular level. Xenograft in vivo model was developed and knockdown relevant genes by RNAi techniques and confirmed the relationship which leads to colorectal cancer. RESULTS: Using the FADD cDNA as bait, we identified six putative clones as associated proteins. The interaction of pFADD and metallothionein 2A (MT2A) was confirmed by GST pull-down assays in vitro and co-IP experiments in vivo. FADD co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by three channel fluorescence microscopy. Co-transfection of pFADD with MT2A gene inhibited cell apoptosis and induced cell proliferation in colorectal cancer cells compared with control groups. When we used antisense MT2A and pFADD which is serine 194 in the C terminal of FADD gene that has been reported to be phosphorylated to interdict the effect of respective genes the inhibition of cell proliferation and induction of apoptosis were significantly enhanced in animal model. CONCLUSIONS: Further in this study we identify non-canonical nuclear factor-κB (NF-κB) signaling up regulated and it was directly linked with the tumor necrosis with MT2A and pFADD genes. pFADD with MT2A can inhibit the apoptosis and promote proliferation, of colorectal cancer cells, and antisense sequence of MT2A and pFADD approaches which might swell the combination of deregulated proliferation and suppressed apoptosis.

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