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.     

2μm DNA PLASMID IN BREWERY YEASTS

Using an agarose gel screening procedure, 2 μm DNA plasmid was detected in all of 10 brewing strains of Saccharomyces yeast examined and in both of two non-brewing, dextrin-utilising strains. Plasmid DNA was identified in yeast grown with access to air in MYGP medium or in hopped wort, and in yeast harvested from 3-day wort fermentations. The yeast plasmid is a suitable self-cloning vector for the genetic manipulation of brewing yeasts by transformation.     

INTERSTRAIN TRANSFER OF THE 2μm DNA PLASMID OF SACCHAROMYCES BY CYTODUCTION

Respiratory-sufficient, [rho+], cytoductants were obtained from matings where the respiratory-deficient, [rho^?], parent lacked 2μm DNA. In these crosses, the cytoduction frequency (no. cytoductants × 100/no. cytoductants plus no. diploids) was ca. 20% or less, even though the [rho+] parent was defective in nuclear fusion. This apparent suppression of the kar defect was specific to matings where the kar nucleus was derived from a [rho+] cytoplasm, but was not a consequence of the lack of 2μm DNA in the recipient strains. Of 16 cytoductants examined from such a cross, only 3 contained 2μm DNA. This confirmed an anomalous mode of cytoplasmic inheritance for the plasmid but demonstrated that the frequency of transmission during cytoduction can be somewhat lower than has been reported previously. The plasmid, in those [cir+] cytoductants which received it, was stably maintained over more than 120 cell doublings. From a preliminary comparison of [cir+] and [cir^o] cytoductants, no evidence for a plasmid-specific phenotype was obtained.     

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.     

SPHEROPLAST FUSION OF BREWER'S YEAST STRAINS

Spheroplasts of brewing polyploid yeast strains have been successfully fused with spheroplasts of haploid yeast strains. After regeneration of the cell wall, stable fusion recombinants were isolated. Genetic analysis of these recombinants revealed that they contained the genotype of both parents, sporulated well with each ascus containing four spores and were indeed diploid. Spheroplast fusion thus affords a means to genetically analyse brewing yeast strains, such an analysis having been difficult if not impossible by conventional hybridization techniques.     

APPLICATION OF THE POLYMERASE CHAIN REACTION TO THE RAPID ANALYSIS OF BREWERY YEAST STRAINS

The rapid discrimination of closely-related Saccharomyces cerevisiae strains can pose a significant problem to breweries, in particular where closely related strains are being used simultaneously to manufacture different products. In this study, two PCR approaches have been examined to assess their usefulness for the discrimination of brewery ale and lager yeast strains. PCR using arbitrary primers (RAPD PCR) was found unsuitable for such an application since the DNA profiles generated from brewery strains were generally found to be identical, due presumably to the close genetic relatedness of these yeasts. In contrast, PCR using δ sequence primers could rapidly differentiate between many ale and lager strains and characteristic profiles for these were generated. This method could also be applied directly to yeasts isolated from brewery worts or from active dried yeast preparations. Results of such analyses were available within the working day.     

CAMBRIDGE PRIZE LECTURE DEVELOPING NEW STRAINS OF YEAST*

Genetic engineering or recombinant DNA technology is now routinely applied to the construction and development of new strains of brewing yeast. This is a direct consequence of the power of the technology which facilitates the modification, introduction and stable maintenance of specific genes in brewing yeast, without compromising the intrinsic brewing properties of the yeast itself. The way in which gene technology has been applied to the development of new strains of Bass yeast is briefly illustrated by the provision of plasmid-based systems for ensuring the stable maintenance of recombinant genes, the construction of amylolytic and β-glucanolytic yeast and the design and development of genetic systems for enhancing the value of waste brewers yeast. Commercial and regulatory issues are discussed.     

PRODUCTION OF ESTERS BY DIFFERENT YEAST STRAINS IN SUGAR FERMENTATIONS

The production of the acetates of isoamyl alcohol and phenethyl alcohol and the ethyl esters of the C₆-C₁₀ fatty acids was investigated in semiaerobic sugar fermentations by 56 strains of Saccharomyces cerevisiae and 3 strains of S. uvarum. The S. cerevisiae yeasts generally produced more esters than the S. uvarum yeasts. Isoamyl acetate was the main component in the ester fractions examined and others in decreasing order, were ethyl caprylate, ethyl caproate, ethyl caprate and phenethyl acetate.     

THE GENETIC MANIPULATION OF KILLER CHARACTER INTO BREWING YEAST

A procedure is described whereby the cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae is transferred to a brewing yeast strain. Neither preparation of protoplasts of the brewing yeast nor mutation of its nuclear genes are required for this process. The brewing yeast killer strains produced have the advantages over their parent brewing cell that they kill sensitive yeasts and are immune to the killing action of certain killer yeasts. The method described offers significant advantages over the process of transformation as a means of genetically manipulating commercial yeasts.     

DNA FINGERPRINTING OF BREWERS' YEAST: CURRENT PERSPECTIVES

A recent application of molecular biology has been the development of DNA fingerprinting techniques, which have met with considerable success in the field of forensic science. Within the brewing industry, DNA fingerprinting has been proposed as a fresh approach to the problem of differentiating brewing strains with similar properties. As progress in molecular biology continues, so it seems likely that DNA fingerprinting methods will become more rapid and widespread. This paper reviews the information that has been gained so far from DNA fingerprinting, compares the existing methods and discusses the likely directions for future development.     

REVISED NOMENCLATURE OF GENES THAT CONTROL YEAST FLOCCULATION

It is proposed that the flocculation genes in Saccharomyces spp. hitherto referred to as FLO 1, FLO 2, and FLO 4 are allelic and that they be consolidated into a single gene locus, to be known henceforth as FLO 1.     

A RAPID,SIMPLE AND RELIABLE METHOD OF DIFFERENTIATING BREWING YEAST STRAINS BASED ON DNA RESTRICTION PATTERNS

DNA was isolated from polyploid brewing ale and lager yeast strains using a simple and rapid procedure which was a modification of a previously described method of Seehaus et al.¹⁴ The isolated DNA was cut with a number of restriction enzymes and subjected to agarose gel electrophoresis. Significant differences in banding patterns were observed between a Saccharomyces cerevisiae ale strain DNA and Saccharomyces uvarum (carlsbergensis) lager strain DNA, particularly with the enzyme Hpal. Differences were also observed between the banding patterns of digests from two ale strains, and from two lager strains. Use of this technique with appropriate restriction enzymes should prove useful for the rapid differentiation of brewing yeast strains.     

DETERMINATION OF BREWING YEAST PLOIDY BY DNA MEASUREMENT

A method has been derived which permits the determination of the degree of ploidy in industrial strains of yeast. The method involves measuring the DNA content per cell of the strains, which is achieved by careful counting of the cells using a haemocytometer and a determination of the amount of DNA by a colorimetric method. Using this technique it was determined that the two industrial lager strains under investigation are tetraploid. * 1 These results have been briefly described at the meeting of the EBC Subgroup on Yeast Genetics, Copenhagen 1982.
     

CHARACTERIZATION OF BREWING YEAST STRAINS BY NUMERICAL ANALYSIS OF TOTAL SOLUBLE CELL PROTEIN PATTERNS

Total soluble cell proteins from 33 yeast strains from the brewing industry were extracted and subjected to polyacrylamide gel electrophoresis. Yeast strains were grouped by computerized numerical analysis of protein banding patterns. Three clusters were obtained at r>0.90. Cluster I contained 21 Saccharomyces cerevisiae lager beer strains. Cluster II comprised two strains isolated from beer with a phenolic off flavour and a third strain used for lager beer brewing. Cluster III consisted of two bottom ale yeasts. Protein patterns of yeast strains within each cluster corresponded closely or were identical. However, the intensity of certain bands often varied and the number of peaks recorded was not identical. These minor differences were reproducible and regarded as characteristic for the specific strains. Protein patterns can therefore be used to characterize or fingerprint individual yeast strains.     

OXYGEN REQUIREMENTS OF BREWING STRAINS OF SACCHAROMYCES UVARUM (CARLSBERGENSIS)—BOTTOM FERMENTATION YEAST

The requirement of oxygen by brewing strains of Saccharomyces uvarum (carlsbergensis) varies with the strain. For a given strain, the requirement can be assessed quantitatively from an equation developed on the basis of model fermentations carried out at four different oxygen levels under a set of standardized experimental conditions. Using this equation, the strains are subdivided into four groups, differing significantly in their oxygen requirements. Among several other factors, access to oxygen during propagation of pitching yeast determines the magnitude of the oxygen requirement during subsequent fermentation. In this regard, the strains referred to the groups having a high demand for oxygen are the most sensitive.     

DIFFERENTIATION OF BREWING YEAST

Current methods for the differentiation of brewing yeasts are described and their performance critically discussed. Although these methods are widely used they are non-specific and require confirmation by other tests. Emergent approaches such as pyrolysis gas chromatography or the use of gene cloning techniques are considered and their application reviewed. It is concluded that development of such methods could offer a unique ‘fingerprint’ for each brewing yeast based on a single test.     

THE GENETICS OF YEAST FLOCCULATION

A short review of the development of yeast genetics in general, and with respect to flocculation in particular, is presented. At least three genes, two dominant and one recessive, confer flocculence, only one of these genes requiring to be present. The spontaneous gene mutation or mitotic segregation rates from flocculence to non-flocculence are high and are much higher than those rates in the reverse direction. Attempts were made to estimate the ploidy of some commercial strains of Saccharomyces cerevisiae by measurement of cell volume and DNA content.     

CONSTRUCTION OF YEAST STRAINS FOR THE PRODUCTION OF LOW-CARBOHYDRATE BEER

By way of somatic hybridization of a lager brewing strain with Saccharomyces diastaticus, a hybrid was constructed, which is suitable for the production of low carbohydrate beer. The hybrid character of the fusion product was confirmed by physiological tests and segregation analyses, as well as, on a molecular basis, by OFAGE (TAFE) and Ty-hybridization.     

STRAINS OF YEAST LETHAL TO BREWERY YEASTS

Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.     

Sequences of Saccharomyces cerevisiae 2 μm DNA improving plasmid partitioning in Hansenula polymorpha

Insertion of the HindIII-PstI fragment of Saccharomyces cerevisiae 2 μm DNA into the Hansenula polymorpha replicative plasmids decreases plasmid copy number and ensures their distribution to daughter cells at both mitotic and meiotic cell divisions. This suggests that the stabilization effect is caused by the improvement of plasmid partitioning. Deletion analysis revealed that the region of 2 μm DNA sequence responsible for the increase of mitotic stability of H. polymorpha plasmids involves the 2 μm STB locus and adjoining region. Further analysis demonstrated that the stabilization effect may depend on the number of 24–28 bp imperfect repeats which were found in several copies in the STB locus and adjoining region. © 1998 John Wiley & Sons, Ltd.