Jørgen Hansen

Saccharomyces carlsbergensis contains two functional MET2 alleles similar to homologues from S. cerevisiae and S. monacensis.

The brewing yeast, Saccharomyces, carlsbergensis, is allopolyploid, derived from two diverged genomes. To obtain information about the possible origin of this yeast, we cloned two different S. carlsbergensis MET2 genes (encoding homoserine acetyltransferase). One has a nucleotide (nt) sequence identical or very similar to MET2 of Saccharomyces cerevisiae. The other has a different sequence, but was functional in S. cerevisiae. This allele was sequenced and revealed a coding region of 486 amino acids (aa). The nt sequence of the coding region showed 82% homology to S. cerevisiae MET2, while the derived aa sequences were 94% identical. Hybridization experiments to genomic DNA of different yeast strains revealed that the divergent MET2 gene had higher sequence homology to segments from type strains of S. monacensis, S. bayanus and S. uvarum than to MET2 from S. cerevisiae. Sequencing of 330 bp of a PCR-amplified fragment of MET2 from these organisms shows that the non-S. cerevisiae-like sequence from S. carlsbergensis is identical to the corresponding sequence in S. monacensis, while it is 93% homologous with S. bayanus and S. uvarum. Our results are consistent with the proposal that S. carlsbergensis originated as a hybrid between S. monacensis and S. cerevisiae. The complete identity of the MET2 fragments from S. monacensis and the S. carlsbergensis-specific MET2 allele suggests that the hybridization must have been a quite recent event.

A natural chimeric yeast containing genetic material from three species

The Saccharomyces sp. CID1 isolate (CBS 8614) and several other Saccharomyces sensu stricto yeasts were analysed for their mitochondrial and nuclear genes. The data show that Saccharomyces sp. CID1, found so far only in one location in Europe, is a natural hybrid between three different Saccharomyces yeast species. Two of them, Saccharomyces cerevisiae-like and Saccharomyces bayanus-like, are ubiquitous and contributed parts of the nuclear genome; the third, Saccharomyces sp. IFO 1802-like, which has been found only in Japan, contributed the mitochondrial DNA molecule. These data suggest that the yeast cell is able to accommodate, express and propagate genetic material that originates from different species, and the very existence of the resulting natural hybrids indicates that such hybrids are well adapted to their habitats.

Modification of biochemical pathways in industrial yeasts.

Yeasts have many applications in industrial food and beverage production. While these uses are often of ancient origin, modern demands for control of the amounts of process-derived compounds and for cost-effective processing can make it desirable to modify metabolic pathways of production yeasts. While the genetics of standard laboratory strains of Saccharomyces cerevisiae are well described, industrial strains and species are less characterized, and many of them have a complicated genetic constitution. Nevertheless, their biochemical pathways can be modified, and the knowledge becoming available on the physiology of genetic reference strains of S. cerevisiae is a great help in directing the modifications of the industrial yeasts towards practical goals.

Cysteine uptake by Saccharomyces cerevisiae is accomplished by multiple permeases.

Abstract Uptake by Saccharomyces cerevisiae of the sulphur-containing amino acid L-cysteine was found to be non-saturable under various conditions, and uptake kinetics suggested the existence of two or more transport systems in addition to the general amino-acid permease, Gap1p. Overexpression studies identified BAP2, BAP3, AGP1 and GNP1 as genes encoding transporters of cysteine. Uptake studies with disruption mutants confirmed this, and identified two additional genes for transporters of cysteine, TAT1 and TAT2, both very homologous to BAP2, BAP3, AGP1 and GNP1. While Gap1p and Agp1p appear to be the main cysteine transporters on the non-repressing nitrogen source proline, Bap2p, Bap3p, Tat1p, Tat2p, Agp1p and Gnp1p are all important for cysteine uptake on ammonium-based medium. Furthermore, whereas Bap2p, Bap3p, Tat1p and Tat2p seem most important under amino acid-rich conditions, Agp1p contributes significantly when only ammonium is present, and Gnp1p only contributes under the latter condition.

Inactivation of MET2 in brewer's yeast increases the level of sulfite in beer

Brewers yeasts sometimes produce inadequate or excessive amounts of sulfite, an antioxidant and flavour stabilizer, so means of controlling the sulfite production are desired. Understanding the physiology and regulation of the sulfur assimilation pathway of Saccharomyces yeasts is the key to change sulfite production. The MET2 gene of Saccharomyces yeasts encodes homoserine O-acetyl transferase, which catalyzes the conversion of homoserine to O-acetyl homoserine which in turn combines with hydrogen sulfide to form homocysteine, the immediate precursor of methionine. We expected that inactivation of MET2 would lead to accumulation of sulfide and derepression of the entire sulfur assimilation pathway and, therefore, possibly also to sulfite accumulation. Brewers yeasts were constructed in which several of the four MET2 gene copies were inactivated. Sulfite production was increased in strains with one remaining MET2 gene and even more so when no active MET2 was present. In both cases, hydrogen sulfide production was also increased. To the extent that excess sulfide can be removed, this strategy may be applied to control sulfite accumulation by brewers yeast in beer production.

GAP1, a novel selection and counter-selection marker for multiple gene disruptions in Saccharomyces cerevisiae

An Erratum has been published for this article in Yeast 18 (4) 2001, 389.

Assessment of the Total Inflammatory Potential of Bioaerosols by Using a Granulocyte Assay

ABSTRACT Occupational health symptoms related to bioaerosol exposure have been observed in a variety of working environments. Bioaerosols contain microorganisms and microbial components. The aim of this study was to estimate the total inflammatory potential (TIP) of bioaerosols using an in vitro assay based on granulocyte-like cells. A total of 129 bioaerosol samples were collected in the breathing zone of workers during their daily working routine at 22 biofuel plants. The samples were analyzed by traditional assays for dust, endotoxin, fungal spores, (1→3)-β-d-glucan, total number of bacteria, the enzyme N-acetyl-β-d-glucosaminidase (NAGase; primarily originating from fungi), Aspergillus fumigatus, and mesophilic and thermophilic actinomycetes; the samples were also assayed for TIP. In a multilinear regression four factors were significant for the TIP values obtained: endotoxin (P < 0.0001), fungal spores (P < 0.0001), (1→3)-β-d-glucan (P = 0.0005), and mesophilic actinomycetes (P = 0.0063). Using this model to estimate TIP values on the basis of microbial composition, the correlation to the measured values was r = 0.91. When TIP values obtained in the granulocyte assay were related to the primary working area, we found that bioaerosol samples from personnel working in straw storage facilities showed high TIP values (≈50 times the TIP of unstimulated controls). In contrast, bioaerosol samples from personnel with work functions in offices or laboratories showed low TIP values (≈5 times the TIP of the unstimulated control). This indicates, as expected, that these areas were less contaminated. In conclusion, the granulocyte assay reacts to multiple contaminants in the environmental samples and can be used to obtain a measurement of TIP. Therefore, potential occupational health effects related to inflammation of the airways in a working environment can be estimated using this assay.

Further development of the cassette-based pYC plasmid system by incorporation of the dominant hph, nat and AUR1-C gene markers and the lacZ reporter system

Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance. We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also in an industrial strain of S. carlsbergensis (syn. of S. pastorianus) brewing yeast as well as in Saccharomyces kluyveri. As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option. Furthermore, the lacZ system for analyzing gene expression was included in the system.

Brewer’s yeast: genetic structure and targets for improvement

The art of beer brewing is ancient, and Saccharomyces yeast probably played a pivotal role from the beginning. Production of beer from the barley grain consists of multiple steps, of which only the last few involve the yeast. Nevertheless, the behaviour of the yeast is highly decisive for both speed and outcome of the whole process, and to a large extent influences the total beer flavour profile. Long known fermentation-related problems as well as the occasional evolution of certain offflavours can be blamed on the yeast. Great efforts have therefore gone into studies of the genetics of brewing yeast, starting with the pure cultivation of lager brewing yeast more than 100 years ago and continuing with today’s detailed description of the brewing yeast genome, as well as ventures into genetic modification to solve brewing problems.

Fungi, β-Glucan, and Bacteria in Nasal Lavage of Greenhouse Workers and Their Relation to Occupational Exposure

The nose and mouth are the first regions of the respiratory tract in contact with airborne microorganisms. Occupational exposures to airborne microorganisms are associated with inflammation and different symptoms of the airways. The purpose of this study is to investigate the relation between occupational exposure to fungi, β-glucan, and bacteria and contents of fungi, β-glucan, and bacteria in nasal lavage (NAL) of greenhouse workers. We also studied whether contents of microorganisms in NAL were related to gender, time of the work week, and runny nose. NAL samples (n = 135) were taken Monday morning and Thursday at noon and personal exposure to inhalable bioaerosols was measured during a working day. The content of fungi and β-glucan in NAL of men was affected by their exposure to fungi and β-glucan. The content of fungi, β-glucan, and bacteria in NAL was higher Thursday at noon than Monday morning. The ratios of fungi in NAL between Thursday at noon and Monday morning were 14 (median value) for men and 3.5 for women. Gender had no effect on the exposure level but had a significant effect on the content of fungi, β-glucan, and bacteria in NAL, with the highest contents in NAL of men. On Thursdays, the median content of fungi in NAL samples of men without runny noses was 9408 cfu per NAL sample, whereas the same content for women was 595 cfu per NAL sample. Workers with runny noses had fewer fungi in NAL than workers without runny noses. A higher content of β-glucan per fungal spore was found in NAL than in the air. This indicates that mainly the larger fungal spores or pollen grains deposit in the nose. The difference between genders and the fact that the content of fungi in NAL was significantly affected by the exposure indicate that the two genders are affected by the same exposure level differently.