FIND-IT: Accelerated trait development for a green evolution.

Improved agricultural and industrial production organisms are required to meet the future global food demands and minimize the effects of climate change. A new resource for crop and microbe improvement, designated FIND-IT (Fast Identification of Nucleotide variants by droplet DigITal PCR), provides ultrafast identification and isolation of predetermined, targeted genetic variants in a screening cycle of less than 10 days. Using large-scale sample pooling in combination with droplet digital PCR (ddPCR) greatly increases the size of low-mutation density and screenable variant libraries and the probability of identifying the variant of interest. The method is validated by screening variant libraries totaling 500,000 barley (Hordeum vulgare) individuals and isolating more than 125 targeted barley gene knockout lines and miRNA or promoter variants enabling functional gene analysis. FIND-IT variants are directly applicable to elite breeding pipelines and minimize time-consuming technical steps to accelerate the evolution of germplasm.

Assessing Population Diversity of Brettanomyces Yeast Species and Identification of Strains for Brewing Applications.

Brettanomyces yeasts have gained popularity in many sectors of the biotechnological industry, specifically in the field of beer production, but also in wine and ethanol production. Their unique properties enable Brettanomyces to outcompete conventional brewer's yeast in industrially relevant traits such as production of ethanol and pleasant flavors. Recent advances in next-generation sequencing (NGS) and high-throughput screening techniques have facilitated large population studies allowing the selection of appropriate yeast strains with improved traits. In order to get a better understanding of Brettanomyces species and its potential for beer production, we sequenced the whole genome of 84 strains, which we make available to the scientific community and carried out several in vitro assays for brewing-relevant properties. The collection includes isolates from different substrates and geographical origin. Additionally, we have included two of the oldest Carlsberg Research Laboratory isolates. In this study, we reveal the phylogenetic pattern of Brettanomyces species by comparing the predicted proteomes of each strain. Furthermore, we show that the Brettanomyces collection is well described using similarity in genomic organization, and that there is a direct correlation between genomic background and phenotypic characteristics. Particularly, genomic patterns affecting flavor production, maltose assimilation, beta-glucosidase activity, and phenolic off-flavor (POF) production are reported. This knowledge yields new insights into Brettanomyces population survival strategies, artificial selection pressure, and loss of carbon assimilation traits. On a species-specific level, we have identified for the first time a POF negative Brettanomyces anomalus strain, without the main spoilage character of Brettanomyces species. This strain (CRL-90) has lost DaPAD1, making it incapable of converting ferulic acid to 4-ethylguaiacol (4-EG) and 4-ethylphenol (4-EP). This loss of function makes CRL-90 a good candidate for the production of characteristic Brettanomyces flavors in beverages, without the contaminant increase in POF. Overall, this study displays the potential of exploring Brettanomyces yeast species biodiversity to find strains with relevant properties applicable to the brewing industry.

Interaction with Cfd1 increases the kinetic lability of FeS on the Nbp35 scaffold.

P-loop NTPases of the ApbC/Nbp35 family are involved in FeS protein maturation in nearly all organisms and are proposed to function as scaffolds for initial FeS cluster assembly. In yeast and animals, Cfd1 and Nbp35 are homologous P-loop NTPases that form a heterotetrameric complex essential for FeS protein maturation through the cytosolic FeS cluster assembly (CIA) pathway. Cfd1 is conserved in animals, fungi, and several archaeal species, but in many organisms, only Nbp35 is present, raising the question of the unique roles played by Cfd1 and Nbp35. To begin to investigate this issue, we examined Cfd1 and Nbp35 function in budding yeast. About half of each protein was detected in a heterocomplex in logarithmically growing yeast. Nbp35 readily bound (55)Fe when fed to cells, whereas (55)Fe binding by free Cfd1 could not be detected. Rapid (55)Fe binding to and release from Nbp35 was impaired by Cfd1 deficiency. A Cfd1 mutation that caused a defect in heterocomplex stability supported iron binding to Nbp35 but impaired iron release. Our results suggest a model in which Cfd1-Nbp35 interaction increases the lability of assembled FeS on the Nbp35 scaffold for transfer to target apo-FeS proteins.

Sulfite action in glycolytic inhibition: in vivo real-time observation by hyperpolarized (13)C NMR spectroscopy.

Detecting the molecular targets of xenobiotic substances in vivo poses a considerable analytical challenge. Here, we describe the use of an NMR-based tracer methodology for the instantaneous in vivo observation of sulfur(IV) action on cellular metabolism. Specifically, we find that glycolytic flux is directed towards sulfite adducts of dihydroxyacetone phosphate and pyruvate as off-pathway intermediates that obstruct glycolytic flux. In particular, the pyruvate-sulfite association hinders the formation of downstream metabolites. The apparent in vivo association constant of pyruvate and sulfite agrees with the apparent inhibition constant of CO(2) formation, thus supporting the importance of pyruvate interception in disturbing central metabolism and inhibiting NAD regeneration.

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance.

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18° Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent.

A novel eukaryotic factor for cytosolic Fe-S cluster assembly.

Iron regulatory protein 1 (IRP1) is regulated through the assembly/disassembly of a [4Fe-4S] cluster, which interconverts IRP1 with cytosolic aconitase. A genetic screen to isolate Saccharomyces cerevisiae strains bearing mutations in genes required for the conversion of IRP1 to c-aconitase led to the identification of a previously uncharacterized, essential gene, which we call CFD1 (cytosolic Fe-S cluster deficient). CFD1 encodes a highly conserved, putative P-loop ATPase. A non-lethal mutation of CFD1 (cfd1-1) reduced c-aconitase specific activity in IRP1-transformed yeast by >90%, although IRP1 in these cells could be readily converted to c-aconitase in vitro upon incubation with iron alone. IRP1-transformed cfd1-1 yeast lacked EPR-detectable Fe-S clusters in c-aconitase, pointing to a defect in Fe-S cluster assembly. The specific activity of another cytosolic Fe-S protein, Leu1p, was also inhibited by >90% in cfd1-1 yeast, whereas activity of mitochondrial Fe-S proteins was not inhibited. Consistent with a cytosolic site of activity, Cfd1p was localized in the cytoplasm. To our knowledge, Cfd1p is the first cytoplasmic Fe-S cluster assembly factor described in eukaryotes.