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Complete LEU2 ORF deletion by PCR-based gene targeting in H . uvarum . Hanseniaspora uvarum WT, WT/ ( LEU2 / leu2 :: hygXL ; G373), and two / strains ( leu2 :: hygXL / leu2 :: clnXL ; G374 and G375) were streaked out on plates containing (A) YPD, (B) YPD supplemented with 200 μg/mL of hygromycin, (C) YPD supplemented with 50 μg/mL of nourseothricin, and (D) CSM-Leu medium.
Source publication
Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL a...
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Citations
... The latter work attributed the lower fermentative capacity of H. uvarum as compared to S. cerevisiae to a reduced pyruvate kinase activity as the final step of glycolysis. Based on the genome sequence methods for the introduction of DNA and the construction of deletion mutants have been successfully implemented only recently [34,35]. The construction of autonomously replicating plasmids and homozygous auxotrophic deletion mutants in the diploid-type strain (i.e., Huade2, Huhis3, Huleu2, and Huura3) provided a set of essential tools for future genetic manipulations of this yeast [36]. ...
Hanseniaspora uvarum is the predominant yeast species in the majority of wine fermentations, which has only recently become amenable to directed genetic manipulation. The genetics and metabolism of H. uvarum have been poorly studied as compared to other yeasts of biotechnological importance. This work describes the construction and characterization of homozygous deletion mutants in the HuZWF1 gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), which provides the entrance into the oxidative part of the pentose phosphate pathway (PPP) and serves as a major source of NADPH for anabolic reactions and oxidative stress response. Huzwf1 deletion mutants grow more slowly on glucose medium than wild-type and are hypersensitive both to hydrogen peroxide and potassium bisulfite, indicating that G6PDH activity is required to cope with these stresses. The mutant also requires methionine for growth. Enzyme activity can be restored by the expression of heterologous G6PDH genes from other yeasts and humans under the control of a strong endogenous promoter. These findings provide the basis for a better adaptation of H. uvarum to conditions used in wine fermentations, as well as its use for other biotechnological purposes and as an expression organism for studying G6PDH functions in patients with hemolytic anemia.
Core histone genes display a remarkable diversity of cis-regulatory mechanisms despite their protein sequence conservation. However, the dynamics and significance of this regulatory turnover are not well understood. Here we describe the evolutionary history of core histone gene regulation across 400 million years in budding yeasts. We find that canonical mode of core histone regulation – mediated by the trans-regulator Spt10 – is ancient, likely emerging between 320-380 million years ago and is fixed in the majority of extant species. Unexpectedly, we uncovered the emergence of a novel core histone regulatory mode in the Hanseniaspora genus, from its fast-evolving lineage (FEL), which coincided with the loss of one copy of its paralogous core histones genes. We show that the ancestral Spt10 histone regulatory mode was replaced, via cis-regulatory changes in the histone control regions, by a derived Mcm1 histone regulatory mode and that this rewiring event occurred with no changes to the trans-regulator, Mcm1, itself. Finally, we studied the growth dynamics of the cell cycle and histone synthesis in genetically modified Hanseniaspora uvarum. We find that H. uvarum divides rapidly, with most cells completing a cell cycle within 60 minutes. Interestingly, we observed that the regulatory coupling between histone and DNA synthesis was lost in H. uvarum. Our results demonstrate that core histone gene regulation was fixed anciently in budding yeasts, however it has greatly diverged in the Hanseniaspora FEL.
