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.

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.

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.

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.

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.

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.

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