APPLICATION OF PULSED FIELD CHROMOSOME ELECTROPHORESIS IN THE STUDY OF CHROMOSOME XIII AND THE ELECTROPHORETIC KARYOTYPE OF INDUSTRIAL STRAINS OF SACCHAROMYCES YEASTS

Pulsed field chromosome electrophoresis is a powerful new technique in yeast genetics which permits the resolution of intact yeast chromosomes in an agarose gel matrix. We utilized contour-clamped homogeneous electric field electrophoresis (CHEF) to survey representative strains of Saccharomyces yeasts from the brewing, baking, distilling, sake and wine industries for their electrophoretic karyotypes. All of the strains tested were found to have a unique chromosomal profile, indicating the potential of this technology for “fingerprinting” prototrophic strains of Saccharomyces yeasts. By employing an ILV2 gene probe specific for chromosome XIII, we determined that all of the industrial strains of Saccharomyces yeasts possessed a chromosome XIII which migrated in an identical fashion to chromosome XIII from a reference haploid strain of Saccharomyces cerevisiae. While one lager yeast strain, Saccharomyces carlsbergensis M244, was found to contain two alleles of ILV2 when digested genomic DNA was probed with ILV2, the presence of a novel independently migrating chromosome XIII could not be detected. A homeologous chromosome XIII in this yeast will therefore have to be determined by genetic analysis. Pulsed field chromosome electrophoresis is concluded to be a technology with immediate application to Quality Control and Research and Development programs in industries using Saccharomyces yeasts.     

EXPRESSION OF THE STA2 GLUCOAMYLASE GENE OF SACCHAROMYCES CEREVISIAE IN BREWERS' YEAST

Two fragments of DNA containing the Saccharomyces cerevisiae STA2 glucoamylase gene, with differing lengths of 5î non-coding DNA, were separately subcloned into a yeast centromeric plasmid. Of these two subclones, only the shorter one (containing 127 base-pairs of 5î non-coding DNA) was able to confer glucoamylase production on a standard laboratory strain of S. cerevisiae. The longer subclone (containing 465 bp of 5î non-coding DNA) did, however, confer glucoamylase production on a strain of S. cerevisiae lacking a functional STA10 gene (which encodes a repressor of STA2 gene expression). All-yeast plasmids lacking bacterial DNA were constructed from the two STA2 subclones for the transformation of a lager brewing yeast. Only the shorter STA2 subclone conferred glucoamylase activity on this yeast. The level of enzyme activity was comparable to that produced by the same yeast strain containing STA2 expressed from the PGK1 (that is, PGK1) promoter.     

RESTRICTION ANALYSIS OF 2 μm DNA FROM DIVERSE STRAINS OF YEAST

2 μm plasmids from an ale yeast, a lager yeast and a strain of Saccharomyces diastaticus have been characterised by restriction analysis. Plasmids were similar to each other and conformed to a pattern which has been reported for well-characterised genetic stocks. The structure of 2 μm DNA, therefore, has been conserved in diverse strains of Saccharomyces. Preparative procedures used here are likely to be applicable to the detection and characterisation of plasmid DNA from a range of yeast genera. The apparent yield of 2 μm was increased when strains were grown at elevated temperature.     

CONTENTS OF CYTOCHROMES IN YEAST

The contents of cytochromes in yeast were determined quantitatively from the absorption spectra, using a solid cell paste of intact yeast. During the industrial production of baker's yeast, the contents of the cytochromes, particularly of cytochrome aa₃ at successive stages, increased gradually with increasing aeration. In semi-aerobically grown baker's yeast, the contents of cytochromes aa₃, b and c were 0·9, 2·9 and 2·9 × 10^?5 moles/litre of fresh yeast (total amount 6·7 × 10^?5 moles/litre), while in vigorously aerated commercial baker's yeast the respective values were 2·3, 4·8 and 5·2 × 10^?5 moles/litre (total amount 12·3 × 10^?5 moles/litre). In brewer's yeasts separated after the brewing process, the contents of cytochromes were markedly lower than in baker's yeast grown with limited aeration, whereas in top-fermenting yeast the total cytochrome content, aa₃ + b + c, was in some samples markedly higher, 7·1 × 10^?5 moles/litre, than in bottom-fermenting brewer's yeast, 2·4 × 10^?5 moles/litre. When brewer's bottom yeast was grown on a laboratory scale under increasing aeration, a maximum appeared in the cytochrome contents when aeration was moderate, and increased aeration inhibited the formation of cytochromes. The cytochrome contents in brewer's bottom yeast may exceed the amounts found in commercial baker's yeast. In addition to aeration, the type of metabolism influences the amounts of cytochromes in yeast.     

CINNAMIC ACID AS THE BASIS OF A MEDIUM FOR THE DETECTION OF WILD YEASTS

Cinnamic acid (100 μg ml^?1) incorporated in a solid medium was found to inhibit the growth of brewing strains (Pof^?) of yeast while permitting the growth of Pof+ wild yeast contaminants. Typically, colonies of Saccharomyces cerevisiae var. diastaticus (Pof+) mixed with brewing yeast (S. cerevisiae NCYC 240) were visible after 5d incubation at 25°C. The incubation time required to detect a selection of brewery wild yeast isolates was found to vary from 3–12 d.     

KINETIC STUDIES OF THE DECONTAMINATION OF YEAST SLURRIES WITH PHOSPHORIC ACID AND ACIDIFIED AMMONIUM PERSULPHATE AND A METHOD FOR THE DETECTION OF SURVIVING BACTERIA INVOLVING SOLID MEDIUM REPAIR IN THE PRESENCE OF CATALASE

Destruction of Obesumbacterium proteus in pitching yeast slurries is more efficiently accomplished using an acidified ammonium persulphate wash (0·75% w/v; pH 2·8) than by using phosphoric acid at pH 2·1. The effectiveness of both methods against the lactic acid bacteria, acetic acid bacteria and Enterobacter agglomerans is similar. Material cost savings of up to 60% can be achieved through the use of the acidified ammonium persulphatewash. Economy in the use of materials and optimum process efficiency is achieved by determination of the quantity of acid required to adjust the pH of the yeast slurry before washing. Bacteria surviving the yeast wash can be detected reliably using a plating technique involving solid medium repair in the presence of catalase. A buffered diluent must be used to prepare the inoculum.     

THE STRUCTURAL AND STORAGE CARBOHYDRATES OF SACCHAROMYCES CEREVISIAE: CHANGES DURING FERMENTATION OF WORT AND A ROLE FOR GLYCOGEN CATABOLISM IN LIPID BIOSYNTHESIS

Detailed analysis of the structural and storage carbohydrates of Saccharomyces cerevisiae (NCYC 240) during wort fermentation showed that there were no significant changes in the amounts of trehalose or alkali-soluble glycogen. However, glucan and mannan individually increased from ca4% of the yeast dry weight at pitching to ca6% during the first 3–17 h of fermentation, butthen declined to the former level. In the first 2 h of fermentation, prior to yeast multiplication, acid-soluble glycogen was rapidly dissimilated from ca 40% to ca 6% of the yeast dry weight. During this period of oxygen uptake, wort sugars were not removed by the yeast. Glycogen, therefore, was the sole source of metabolic energy for lipid resynthesis and hexose transport appeared to require the formation of a component membrane. During the latter phase of fermentation when the yeast was not growing but expending energy for maintenance of cellular functions, glycogen reserves were slowly depleted; after a period of prolonged anaerobic storage, the content of glycogen fell well below that which was initially present in the pitching yeast.     

SOME CONSIDERATIONS OF THE FLOCCULATION CHARACTERISTICS OF ALE AND LAGER YEAST STRAINS*

While some ale yeast strains are able to flocculate when cultured in a defined medium of glucose, ammonium salts, vitamins and ions, others require the presence of a nitrogen-containing inducer in the growth medium. On the other hand, all flocculent lager strains examined to date are able to flocculate after being cultured in a defined medium and do not appear to require the addition of inducer material to the growth medium. The inducer material present in wort has been identified as peptide. By the use of ion exchange chromatography the peptide fraction that induces flocculation has been found to contain a high level of acidic amino acid residues with a very similar structure to that reported for the α-factor involved in sexual agglutination of haploid α and a cells of Sacch. cerevisiae. Studies on the adsorption of Ca⁺⁺ ion by the cell wall failed to reveal any significant differences in total uptake between flocculent and non-flocculent cultures. It would appear that Ca⁺⁺ ions are bound less tightly by non-flocculent cells than by flocculent cells. The contribution of calcium to flocculation is not the absolute amount of this ion adsorbed by the yeast cell wall but rather the stereo-specific manner by which it is bound, i.e., its position relative to the three-dimensional structure of the yeast cell wall.     

STUDY OF THE PHENOMENON OF AGGLOMERATION IN THE YEAST SACCHAROMYCES CEREVISIAE

Agglomeration or “grittiness” is detrimental to bakers' yeast quality. Gritty yeast only partially resuspends when mixed in water, most of it remaining as macroscopic cell aggregates. A macroscopic sedimentation test was developed for measuring agglomeration intensity. Expression of the gritty phenotype was investigated in two strains (N176 and GB1) of Saccharomyces cerevisiae grown on a 14-liter scale by varying fermentation conditions of agitation and aeration. Results show that yeast agglomeration is different from yeast flocculation, and is determined by both strain genetic background and environmental factors. The gritty phenotype was expressed in the strain prone to agglomeration (N176) when dissolved oxygen was limiting in the fermenter. Gritty cells had a lower phosphorus and lipid concentration and a higher protein concentration at the surface of the cell, and a higher amount of whole cell and cell wall proteins and calcium than non-gritty cells. Some proteins were also extracted from gritty cells with sodium hydroxide or mercaptoethanol, that were not present in non-gritty cells. Agglomeration did not result in major differences in the structure or composition of the structural cell wall mannoprotein (CWMP). A model for agglomeration is proposed: proteins (cognors) activated by Ca²⁺ (cofactors) to increase their binding capacity bind the mannans (cognons) of adjoining cells; binding is facilitated by the lower phosphorus and lipid concentration at the surface of gritty cells.     

PREDICTION OF THE FERMENTATION PERFORMANCE OF BREWING YEAST WITH THE ACIDIFICATION POWER TEST

Fermentation performance (defined as the time required, in hours, to attenuate an all malt wort from OG 1040° to 1020°) can be accurately predicted for Saccharomyces cerevisiae NCYC 1681 using the Acidification Power test (r =0.943, p = <0.001: 37 samples). The results obtained with four other yeasts suggest that the test has wide applicability. The test measures the ability of a population of yeast cells to maintain different intracellular and extracellular hydrogen ion concentrations when placed in distilled water. It also measures the change in these concentrations induced by the presence of glucose. The method is reproducible (standard deviation of the mean =0.61 %) and is unaffected by variations of ±11 % of yeast weight in the assay, this makes the Acidification Power test a ‘stand alone’ test, no secondary analyses (such as dry weight determination) are necessary. The test can be performed using common laboratory equipment and can be applied routinely in breweries for yeast selection based on the use of Reject Quality Limits (RQL). In addition, the technique may find application in the evaluation of yeast handling protocols, in the assessment of new or novel yeast strains, and in the assessment of yeast condition prior to acid washing.     

KAR1-MEDIATED TRANSFORMATION OF BREWING YEAST

Transfer of plasmid between nuclei has been observed in heterokaryons obtained from kar1 × KAR1 crosses³. On this basis, brewing yeast strain CC45 was forced to mate with K5-5A, a kar1 laboratory strain bearing plasmid YEpCR21. This multicopy plasmid carries the gene cyh2 and determines a dominant resistance to cycloheximide . Heteroplasmons having a CC45 nucleus and hosting YEpCR21 were obtained. Plasmid stability and brewing performance have been studied. The utility of kar1-mediated plasmid transference as an alternative method to transform brewing yeast is discussed. Advantages of YEpCR21 as a vector cloning for brewing yeast are considered as well .     

INDUCTIVE EFFECTS OF OXYGEN ON YEAST FERMENTATION IN GLUCOSE- AND MALTOSE-MEDIA

The findings presented demonstrate that both growth yield and fermentation activity of yeast cells cultured in maltose-media are highly affected by pretreatment of the pitching yeast or by oxygen induction in the medium and by the presence of ergosterol and Tween 80. Variable responses to oxygen are reported from three different strains of brewer's yeast (Saccharomyces carlsbergensis). Yeast growth and fermentation were significantly reduced in media containing glucose above a certain threshold value. Previous reports concerning glucose repression are discussed. It is suggested that sucrose has a repressive effect similar to that of glucose. When nitrogen is a limiting factor, the fermentation velocity in the presence of maltose is reduced.     

EXTRACELLULAR PROTEASE ACTIVITY IN A STRAIN OF SACCHAROMYCES CEREVISIAE

The isolation and analysis of a strain of laboratory yeast with extracellular protease activity is described. The proteolytic activity found in culture supernatants exceeded the parental strain by at least an order of magnitude and apparently was due neither to cell lysis nor to increased cell wall permeability. The extracellular proteases were heterogeneous in composition, consisting of possibly 3 aberrantly secreted intracellular proteinases. This extracellular protease activity was conferred by a single recessive mutation (epr1.1) in a gene displaying classical Mendelian inheritance. EPR1 was tentatively assigned to chromosome XV with loose linkage to the HIS3 gene. Strains carrying the mutant epr1.1) allele also possessed increased levels of secreted invertase activity and it is proposed that EPR1 is closely involved in the intracellular protein translocation pathway of yeast.     

DETERMINATION OF DNA AND OF OTHER NUCLEIC ACID FRACTIONS IN SACCHAROMYCES CEREVISIAE

After extraction overnight of Saccharomyces cerevisiae (baker's yeast) with dilute alkali, to remove nucleotide pools and RNA, acidification gave an insoluble residue from which DNA guanine and adenine were extracted, in the free form, by 1M perchloric acid at room temperature, and determined by cation-exchange chromatography. Percentage DNA in the yeast was computed as 0.062 × μmol/g of guanine + adenine. Preliminary extraction of nucleotides was unnecessary, and gave low results if acid reagents (perchloric or trichloroacetic acids) were used. Several reagents were tested for their ability to extract the nucleotide pool from intact yeast without significant effect on cell RNA content. The content of guanine + adenine in pool-free yeast was taken as giving the sum of RNA + DNA purines, RNA purines being found by difference.     

FORMATION OF N-PROPYL ALCOHOL BY CELL-FREE EXTRACTS OF SACCHAROMYCES CEREVISIAE*

n-Propyl alcohol formation from α-ketobutyric acid was obtained in cell-free extracts of Saccharomyces cerevisiae var. ellipsoideus and the optimal conditions for the formation were determined. In the presence of NAD? the concentration of n-propyl alcohol formed was the same with extracts of yeast grown either aerobically or anaerobically, but in the presence of NADP more n-propyl alcohol was formed with extracts of aerobic yeast. There was no evidence for an increase in an NADP-specific alcohol dehydrogenase, and the increased formation of n-propyl alcohol by extracts of aerobic yeast with added NADP was attributed to a greater reductive capacity of the extracts of aerobic cells in the presence of NADP. No system of enzymes for the formation of n-propyl alcohol was found other than those involved in ethyl alcohol formation. However, alcohol dehydrogenase from aerobic cells had a greater specificity for n-propyl alcohol, compared with ethyl alcohol, than the enzyme from anaerobically grown cells. This difference may be part of the explanation for greater n-propyl alcohol formation in aerobic cultures.     

DIFFERENTIATION OF BREWERY YEAST STRAINS BY RESTRICTION ENDONUCLEASE ANALYSIS OF THEIR MITOCHONDRIAL DNA

Mitochondrial DNA (mtDNA) was isolated from different strains of brewery yeast and digested with various restriction endonucleases. The digestion products were separated by electrophoresis in agarose gels. Of the twenty restriction endonucleases used, only two—Aval and Haelli—produced different restriction fragment patterns when applied to the mtDNA from two strains of Saccharomyces uvarum. The restriction fragment patterns produced by the other eighteen enzymes were identical. Analysis of mtDNA from a strain of Saccharomyces cerevisiae with the same twenty restriction endonucleases revealed several differences with respect to Saccharomyces uvarum. Taken together, these results indicate that restriction endonuclease fragmentation patterns of mtDNA are useful as diagnostic tools for distinguishing strains of ale and larger yeast.     

DETECTION OF WILD YEASTS IN THE BREWERY

The potential of an established culture medium, Wallerstein Laboratories' nutrient agar, for the detection of wild yeasts has been evaluated and recommended for microbiological quality control in brewery laboratories. Wild yeast strains, including Saccharomyces species, can be differentiated from strains of Saccharomyces cerevisiae by the colour, form and rate of growth of their colonies on this medium within 2–3 days. The sensitivity of the method is such that one wild yeast can be detected in a Saccharomyces cerevisiae population of the order of 10° cells.     

FACTORS INFLUENCING THE FORMATION OF A YEAST HEAD BY BREWING YEASTS

Yeast head production is dependent upon the relative rates of (a) arrival of yeast at the surface of the fermenting liquid, (b) its incorporation into a foam and (c) the inherent rate of foam collapse. Under (a), strains of Saccharomyces cerevislae and S. carlsbergensis vary in their ability to adhere to carbon dioxide bubbles which transport them to the wort surface. With regard to (b), the incorporation of yeast into a wort foam increases its stability; the variation in foam stabilizing properties between different strains of S. cerevislae and S. carlsbergensis is dependent on the degree to which they are transported to the surface and thus incorported into the foam. The importance of a wort with good foam stabilizing properties for satisfactory yeast head production during fermentation is emphasized.     

DIFFERENTIATION OF BREWERY YEAST STRAINS BY DNA FINGERPRINTING

A reliable and reproducible non-radioactive DNA fingerprinting technique has been used to differentiate between twelve production brewery yeast strains. The method comprises total DNA preparation from the yeast strains, enzymatic digestion of the DNA and analysis of the DNA fragments by “probing” with known DNA labelled with the plant steroid digoxigenin. A range of probes and several enzymes have been investigated. A successful combination is the restriction endonuclease Hind III and a probe derived from Ty1 (a yeast transposable element). The main advantages of this method are its unambiguity, reproducibility and suitability for routine brewery QC use.     

THE IMMUNOFLUORESCENT STAINING TECHNIQUE FOR THE DETECTION OF WILD YEASTS - PRACTICAL PROBLEMS

An apparent heavy wild yeast infection in pitching yeast has been detected using the immunofluorescent detection method. This infection could not be detected by conventional liquid forcing or plating techniques. The yeasts responsible were isolated and identified as Saccharomyces cerevisiae. The yeasts were very similar to the pitching yeast but varied in a number of respects associated with the cell wall such as flocculation character and giant colony morphology. The results suggest that the immunofluorescent-positive (IP) yeasts are variants of the culture yeast. As a result of this work it is felt that although immunofluorescence is of value for the rapid detection of infection, it must always be used in association with more conventional microbiological techniques.