Modifying flavor profiles of Saccharomyces spp. for industrial brewing using FIND-IT, a non-GMO approach for metabolic engineering of yeast.

Species of Saccharomyces genus have played an irreplaceable role in alcoholic beverage and baking industry for centuries. S. cerevisiae has also become an organism of choice for industrial production of alcohol and other valuable chemicals and a model organism shaping the rise of modern genetics and genomics in the past few decades. Today´s brewing industry faces challenges of decreasing consumption of traditional beer styles and increasing consumer demand for new styles, flavors and aromas. The number of currently used brewer's strains and their genetic diversity is yet limited and implementation of more genetic and phenotypic variation is seen as a solution to cope with the market challenges. This requires modification of current production strains or introduction of novel strains from other settings, e.g. industrial or wild habitats into the brewing industry. Due to legal regulation in many countries and negative customer perception of GMO organisms, the production of food and beverages requires non-GMO production organisms, whose development can be difficult and time-consuming. Here, we apply FIND-IT (Fast Identification of Nucleotide variants by DigITal PCR), an ultrafast genome-mining method, for isolation of novel yeast variants with varying flavor profiles. The FIND-IT method uses combination of random mutagenesis, droplet digital PCR with probes that target a specific desired mutation and a sub-isolation of the mutant clone. Such an approach allows the targeted identification and isolation of specific mutant strains with eliminated production of certain flavor and off-flavors and/or changes in the strain metabolism. We demonstrate that the technology is useful for the identification of loss-of function or gain of function mutations in unrelated industrial and wild strains differing in ploidy. Where no other phenotypic selection exists, this technology serves together with standard breeding techniques as a modern tool facilitating a modification of (brewer's) yeast strains leading to diversification of the product portfolio.

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.

Never Change a Brewing Yeast? Why Not, There Are Plenty to Choose From.

Fermented foods and particularly beer have accompanied the development of human civilization for thousands of years. Saccharomyces cerevisiae, the dominant yeast in the production of alcoholic beverages, probably co-evolved with human activity. Considering that alcoholic fermentations emerged worldwide, the number of strains used in beer production nowadays is surprisingly low. Thus, the genetic diversity is often limited. This is among others related to the switch from a household brewing style to a more artisan brewing regime during the sixteenth century and latterly the development of single yeast isolation techniques at the Carlsberg Research Laboratory in 1883, resulting in process optimizations in the brewing industry. However, due to fierce competition within the beer market and the increasing demand for novel beer styles, diversification is becoming increasingly important. Moreover, the emergence of craft brewing has influenced big breweries to rediscover yeast as a significant contributor to a beer's aroma profile and realize that there is still room for innovation in the fermentation process. Here, we aim at giving a brief overview on how currently used S. cerevisiae brewing yeasts emerged and comment on the rationale behind replacing them with novel strains. We will present potential sources of yeasts that have not only been used in beer brewing before, including natural sources and sources linked to human activity but also an overlooked source, such as yeast culture collections. We will briefly comment on common yeast isolation techniques and finally touch on additional challenges for the brewing industry in replacing their current brewer's yeasts.

The 5' Untranslated Region of the EFG1 Transcript Promotes Its Translation To Regulate Hyphal Morphogenesis in Candida albicans.

Extensive 5' untranslated regions (UTR) are a hallmark of transcripts determining hyphal morphogenesis in Candida albicans The major transcripts of the EFG1 gene, which are responsible for cellular morphogenesis and metabolism, contain a 5' UTR of up to 1,170 nucleotides (nt). Deletion analyses of the 5' UTR revealed a 218-nt sequence that is required for production of the Efg1 protein and its functions in filamentation, without lowering the level and integrity of the EFG1 transcript. Polysomal analyses revealed that the 218-nt 5' UTR sequence is required for efficient translation of the Efg1 protein. Replacement of the EFG1 open reading frame (ORF) by the heterologous reporter gene CaCBGluc confirmed the positive regulatory importance of the identified 5' UTR sequence. In contrast to other reported transcripts containing extensive 5' UTR sequences, these results indicate the positive translational function of the 5' UTR sequence in the EFG1 transcript, which is observed in the context of the native EFG1 promoter. It is proposed that the 5' UTR recruits regulatory factors, possibly during emergence of the native transcript, which aid in translation of the EFG1 transcript.IMPORTANCE Many of the virulence traits that make Candida albicans an important human fungal pathogen are regulated on a transcriptional level. Here, we report an important regulatory contribution of translation, which is exerted by the extensive 5' untranslated regulatory sequence (5' UTR) of the transcript for the protein Efg1, which determines growth, metabolism, and filamentation in the fungus. The presence of the 5' UTR is required for efficient translation of Efg1, to promote filamentation. Because transcripts for many relevant regulators contain extensive 5' UTR sequences, it appears that the virulence of C. albicans depends on the combination of transcriptional and translational regulatory mechanisms.

An Arf-GAP promotes endocytosis and hyphal growth of Ashbya gossypii.

The ADP-ribosylation factor (ARF) family of GTPases are highly conserved from yeast to human and regulate vesicle budding. Sec7 domain containing proteins stimulate the guanine nucleotide exchange on Arf proteins, while ARF-GTPase activating proteins stimulate the hydrolysis of GTP. Since vesicle trafficking is important for hyphal growth, we studied the Ashbya gossypii homolog of Saccharomyces cerevisiae ARF3 along with its putative GEF and GTPase-activating protein (GAP) encoded by YEL1 and GTS1, respectively. Deletion of YEL1 had no discernible phenotype and deletion of ARF3 had only a minor defect in vacuolar fusion. In contrast, deletion of GTS1 severely impaired hyphal growth, and mutants showed defects in the maintenance of polarity and the localization of cortical actin patches. The uptake of the lipophilic dye FM4-64 was delayed in gts1 hyphae, indicating a defect in endocytosis. Gts1 has several protein domains, of which the Arf-GAP domain is required for complementation of the gts1 mutant phenotype. GFP-tagged GTS1 under control of its endogenous promoter localized to the plasma membrane but was enriched at hyphal tips and septal sites corresponding to a role in polarized vesicle trafficking. Our results indicate that this ARF-GTPase module plays an important role for filamentous hyphal growth.

Differential stress response of Saccharomyces hybrids revealed by monitoring Hsp104 aggregation and disaggregation.

Proteotoxic stress may occur upon exposure of yeast cells to different stress conditions. The induction of stress response mechanisms is important for cells to adapt to changes in the environment and ensure survival. For example, during exposure to elevated temperatures the expression of heat shock proteins such as Hsp104 is induced in yeast. Hsp104 extracts misfolded proteins from aggregates to promote their refolding. We used an Hsp104-GFP reporter to analyze the stress profiles of Saccharomyces species hybrids. To this end a haploid S. cerevisiae strain, harboring a chromosomal HSP104-GFP under control of its endogenous promoter, was mated with stable haploids of S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. uvarum. Stress response behaviors in these hybrids were followed over time by monitoring the appearance and dissolution of Hsp104-GFP foci upon heat shock. General stress tolerance of these hybrids was related to the growth rate detected during exposure to e.g. ethanol and oxidizing agents. We observed that hybrids were generally more resistant to high temperature and ethanol stress compared to their parental strains. Amongst the hybrids differential responses regarding the appearance of Hsp104-foci and the time required for dissolving these aggregates were observed. The S. cerevisiae/S. paradoxus hybrid, combining the two most closely related strains, performed best under these conditions.

Developmental Growth Control Exerted via the Protein A Kinase Tpk2 in Ashbya gossypii.

Sporulation in Ashbya gossypii is induced by nutrient-limited conditions and leads to the formation of haploid spores. Using RNA-seq, we have determined a gene set induced upon sporulation, which bears considerable overlap with that of Saccharomyces cerevisiae but also contains A. gossypii-specific genes. Addition of cyclic AMP (cAMP) to nutrient-limited media blocks sporulation and represses the induction of sporulation specific genes. Deletion of the protein kinase A (PKA) catalytic subunits encoded by TPK1 and TPK2 showed reduced growth in tpk1 but enhanced growth in the tpk2 strain; however, both mutants sporulated well. Sporulation can be blocked by cAMP in tpk1 but not in tpk2 strains. Similarly, TPK2 acts at a second developmental switch promoting the break in spore dormancy. In S. cerevisiae, PKA phosphorylates and inhibits Msn2/4. The transcript profiles of the tpk1 and msn2/4 mutants were very similar to that of the wild type under sporulation conditions. However, deletion of the single A. gossypii MSN2/4 homolog generated a specific sporulation defect. We identified a set of genes involved in spore wall assembly that was downregulated in the msn2/4 mutant, particularly DIT2, suggesting that poor spore viability may be due to lysis of spores. Our results reveal specific functional differences between the two catalytic PKA subunits in A. gossypii and identified Tpk2 as the key A kinase that transduces developmental decisions of growth. Our data also suggest that Msn2/4 is involved only at a late step of sporulation in A. gossypii and is not a major regulator of IME1.

Fungal model systems and the elucidation of pathogenicity determinants.

Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity.

Molecular determinants of sporulation in Ashbya gossypii.

Regulation of development and entry into sporulation is critical for fungi to ensure survival of unfavorable environmental conditions. Here we present an analysis of gene sets regulating sporulation in the homothallic ascomycete Ashbya gossypii. Deletion of components of the conserved pheromone/starvation MAP kinase cascades, e.g., STE11 and STE7, results in increased sporulation. In kar3 mutants sporulation is severely reduced, while deletion of KAR4 as well as of homologs of central Saccharomyces cerevisiae regulators of sporulation, IME1, IME2, IME4, and NDT80, abolishes sporulation in A. gossypii. Comparison of RNAseq transcript profiles of sporulation-deficient mutants identified a set of 67 down-regulated genes, most of which were up-regulated in the oversporulating ste12 mutant. One of these differentially expressed genes is an endoglucanase encoded by ENG2. We found that Eng2p promotes hyphal fragmentation as part of the developmental program of sporulation, which generates single-celled sporangia. Sporulation-deficient strains are arrested in their development but form sporangia. Supply of new nutrients enabled sporangia to return to hyphal growth, indicating that these cells are not locked in meiosis. Double-strand break (DSB) formation by Spo11 is apparently not required for sporulation; however, the absence of DMC1, which repairs DSBs in S. cerevisiae, results in very poor sporulation in A. gossypii. We present a comprehensive analysis of the gene repertoire governing sporulation in A. gossypii and suggest an altered regulation of IME1 expression compared to S. cerevisiae.

Analysis of the cell wall integrity pathway of Ashbya gossypii.

Fungal cells are exposed to rapidly changing environmental conditions, in particular with regard to the osmotic potential. This requires constant remodeling of the cell wall and, therefore, the cell wall integrity (CWI) MAP-kinase pathway plays a major role in shaping the fungal cell wall to protect from adverse external stresses. To provide a comprehensive functional analysis of the Ashbya gossypii CWI pathway we generated a set of ten deletion mutants in conserved components including the cell surface sensors AgWSC1 and AgMID2, a putative Rho1-guanine nucleotide exchange factor, AgTUS1, the protein kinase C, AgPKC1, the MAP-kinases AgBCK1, AgMKK1 and AgMPK1, and transcription factors known to be involved in CWI signaling AgRLM1, AgSWI4 and AgSWI6. Deletion of AgPKC1 shows a severe growth defect with frequent tip cell lysis. Deletion of components of the MAP-kinase module generates a pronounced colony lysis phenotype in older regions of the mycelium. Cytoplasmic leakage was assayed using alkaline phosphatase and ß-galactosidase release assays. This indicated that the lysis phenotypes of CWI pathway mutants may be useful to facilitate the isolation of riboflavin from A. gossypii. Remarkably, the Agwsc1 mutant showed a strong (up to 8-fold) increase of riboflavin in the growth medium compared to the parental strain.

Characterization of the PMT gene family in Cryptococcus neoformans.

BACKGROUND: Protein-O-mannosyltransferases (Pmt's) catalyze the initial step of protein-O-glycosylation, the addition of mannose residues to serine or threonine residues of target proteins. METHODOLOGY/PRINCIPAL FINDINGS: Based on protein similarities, this highly conserved protein family can be divided into three subfamilies: the Pmt1 sub-family, the Pmt2 sub-family and the Pmt4 sub-family. In contrast to Saccharomyces cerevisiae and Candida albicans, but similar to filamentous fungi, three putative PMT genes (PMT1, PMT2, and PMT4) were identified in the genome of the human fungal pathogen Cryptococcus neoformans. Similar to Schizosaccharomyces pombe and C. albicans, C. neoformans PMT2 is an essential gene. In contrast, the pmt1 and pmt4 single mutants are viable; however, the pmt1/pmt4 deletions are synthetically lethal. Mutation of PMT1 and PMT4 resulted in distinct defects in cell morphology and cell integrity. The pmt1 mutant was more susceptible to SDS medium than wild-type strains and the mutant cells were enlarged. The pmt4 mutant grew poorly on high salt medium and demonstrated abnormal septum formation and defects in cell separation. Interestingly, the pmt1 and pmt4 mutants demonstrated variety-specific differences in the levels of susceptibility to osmotic and cell wall stress. Delayed melanin production in the pmt4 mutant was the only alteration of classical virulence-associated phenotypes. However, the pmt1 and pmt4 mutants showed attenuated virulence in a murine inhalation model of cryptococcosis. CONCLUSION/SIGNIFICANCE: These findings suggest that C. neoformans protein-O-mannosyltransferases play a crucial role in maintaining cell morphology, and that reduced protein-O-glycosylation leads to alterations in stress resistance, cell wall composition, cell integrity, and survival within the host.

Functional mapping of the Candida albicans Efg1 regulator.

Efg1p is a key transcriptional regulator in Candida albicans which controls various aspects of morphogenesis and metabolism in this organism. Efg1p contains a central basic helix-loop-helix (bHLH) domain, flanked by sequences highly conserved in fungal APSES proteins, as well as polyglutamine stretches at the N- and C-terminal ends. A systematic deletion approach to specify functional domains of Efg1p revealed that the APSES domain is essential for morphogenesis of the normal yeast and true hyphal cell forms and that bHLH flanking sequences are needed for Efg1p stability. Additional C-terminal sequences were required for hyphal formation on some inducing media, and most Efg1p sequences were needed for chlamydospore morphogenesis. Overexpression of EFG1 led to pseudohypha formation only if a functional APSES domain was present, while a switch from the opaque to the white cell type in addition depended on the presence of certain N- and C-terminal segments. Yeast two-hybrid experiments revealed that binding of Efg1p to its antagonist Czf1p required two regions outside of the APSES domain, which did not coincide with Efg1p sequences needed for its transcriptional repressor activity. Binding of the Flo8 transcription factor to Efg1p did not require the APSES domain but appeared to occur at two or more redundant domains. In contrast, DNA binding of Efg1p to an MluI cell cycle box (MCB) element solely required the APSES domain. Overall, these results suggest that functional domains of Efg1p are spread throughout most of its sequences, including the central APSES domain involved in DNA binding, as well as flanking regions required for various protein interactions and regulatory activities.

The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its approximately 20-megabase genome, which contains approximately 6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.

Convergent evolution of chromosomal sex-determining regions in the animal and fungal kingdoms.

Sexual identity is governed by sex chromosomes in plants and animals, and by mating type (MAT) loci in fungi. Comparative analysis of the MAT locus from a species cluster of the human fungal pathogen Cryptococcus revealed sequential evolutionary events that fashioned this large, highly unusual region. We hypothesize that MAT evolved via four main steps, beginning with acquisition of genes into two unlinked sex-determining regions, forming independent gene clusters that then fused via chromosomal translocation. A transitional tripolar intermediate state then converted to a bipolar system via gene conversion or recombination between the linked and unlinked sex-determining regions. MAT was subsequently subjected to intra- and interallelic gene conversion and inversions that suppress recombination. These events resemble those that shaped mammalian sex chromosomes, illustrating convergent evolution in sex-determining structures in the animal and fungal kingdoms.

Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes.

The sexual development and virulence of the fungal pathogen Cryptococcus neoformans is controlled by a bipolar mating system determined by a single locus that exists in two alleles, alpha and a. The alpha and a mating-type alleles from two divergent varieties were cloned and sequenced. The C. neoformans mating-type locus is unique, spans >100 kb, and contains more than 20 genes. MAT-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The alpha and a alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The C. neoformans mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.

Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving fusion of haploid MATalpha and MATa cells. Virulence has been linked to the mating type, and MATalpha cells are more virulent than congenic MATa cells. To study the link between the mating type and virulence, we functionally analyzed three genes encoding homologs of the p21-activated protein kinase family: STE20alpha, STE20a, and PAK1. In contrast to the STE20 genes that were previously shown to be in the mating-type locus, the PAK1 gene is unlinked to the mating type. The STE20alpha, STE20a, and PAK1 genes were disrupted in serotype A and D strains of C. neoformans, revealing central but distinct roles in mating, differentiation, cytokinesis, and virulence. ste20alpha pak1 and ste20a pak1 double mutants were synthetically lethal, indicating that these related kinases share an essential function. In summary, our studies identify an association between the STE20alpha gene, the MATalpha locus, and virulence in a serotype A clinical isolate and provide evidence that PAK kinases function in a MAP kinase signaling cascade controlling the mating, differentiation, and virulence of this fungal pathogen.

Physical maps for genome analysis of serotype A and D strains of the fungal pathogen Cryptococcus neoformans.

The basidiomycete fungus Cryptococcus neoformans is an important opportunistic pathogen of humans that poses a significant threat to immunocompromised individuals. Isolates of C. neoformans are classified into serotypes (A, B, C, D, and AD) based on antigenic differences in the polysaccharide capsule that surrounds the fungal cells. Genomic and EST sequencing projects are underway for the serotype D strain JEC21 and the serotype A strain H99. As part of a genomics program for C. neoformans, we have constructed fingerprinted bacterial artificial chromosome (BAC) clone physical maps for strains H99 and JEC21 to support the genomic sequencing efforts and to provide an initial comparison of the two genomes. The BAC clones represented an estimated 10-fold redundant coverage of the genomes of each serotype and allowed the assembly of 20 contigs each for H99 and JEC21. We found that the genomes of the two strains are sufficiently distinct to prevent coassembly of the two maps when combined fingerprint data are used to construct contigs. Hybridization experiments placed 82 markers on the JEC21 map and 102 markers on the H99 map, enabling contigs to be linked with specific chromosomes identified by electrophoretic karyotyping. These markers revealed both extensive similarity in gene order (conservation of synteny) between JEC21 and H99 as well as examples of chromosomal rearrangements including inversions and translocations. Sequencing reads were generated from the ends of the BAC clones to allow correlation of genomic shotgun sequence data with physical map contigs. The BAC maps therefore represent a valuable resource for the generation, assembly, and finishing of the genomic sequence of both JEC21 and H99. The physical maps also serve as a link between map-based and sequence-based data, providing a powerful resource for continued genomic studies