Construction and analysis of recombinant glucanolytic brewer's yeast strains

Summary Four different types of endo--1,4-glucanase active bottom-fermenting brewer's yeast strains were constructed using recombinant DNA technology. To study the effects of different promoters, copy numbers and integration sites, the egl1 gene of the filamentous fungus Trichoderma reesei was inserted between the promoter and terminator regions of either the PGK1 or ADH1 gene of yeast. The egl1 gene was transferred to the industrial brewer's yeast on a multicopy plasmid or alternatively integrated into the LEU2, PGK1 or ADH1 locus of the yeast. Integration into the PGK1 or ADH1 locus did not affect the brewing properties of the yeast or the quality of the finished beer. Integration into the LEU2 locus, however, decreased the metabolic activity of yeast and prolonged fermentation was needed. In pilot brewing conditions the PGK1 promoter was stronger than that of ADH1. Even a single copy of the egl1 gene in the PGK1 integrant strains gave rise to sufficient enzyme activity for the hydrolysis even of unusually high total amounts of -glucans in worts. Offprint requests to: M.-L. Suihko     

Transformation of commercial baker's yeast strains by electroporation

Summary We developed an electroporation protocol for transformation which was particularly optimized for commercial baker's yeast strains. The protocol is based on the standard BIORAD GENE PULSER/PULSE CONTROLLER machine. It works efficiently both for the introduction of standard multicopy plasmids (ARS and 2m based) and for integrative transformation. In particular we were able to transform genuine prototrophic baker's yeast strains with a 2m-based multicopy plasmid, carrying the dominant sulfometuron methyl resistance marker. For plasmids requiring the introduction of more than one copy for complementation, the transformation frequency was considerably lower. This suggests that transformation by the electroporation method introduces on average only one or a few copies of the transforming plasmid per cell.     

Recombinant brewer's yeast strains suitable for accelerated brewing.

Four brewer's yeast strains carrying the alpha-ald gene of Klebsiella terrigena (ex. Aerobacter aerogenes) or of Enterobacter aerogenes on autonomously replicating plasmids were constructed. The alpha-ald genes were linked either to the ADC1 promoter or to the PGK1 promoter of yeast Saccharomyces cerevisiae. In pilot scale brewing (50 l) with three of these recombinant yeasts the formation of diacetyl in beer was so low during fermentation that lagering was not required. All other brewing properties of the strains were unaffected and the quality of finished beers was as good as that of finished beer prepared with the control strain. The total process time of beer production could therefore be reduced to 2 weeks, in contrast to about 5 weeks required in the conventional process.     

Fermentation of maltotriose by brewer's and baker's yeasts

Two brewer's yeasts and one baker's yeast grew with 95% (w/w) pure maltotriose as carbon source in the presence of antimycin A to block respiration. Biomass yields (0.15 and 0.24 g dry yeast g^–1 sugar, respectively, with and without antimycin A) were similar for growth on maltose and maltotriose, and yields of ethanol were 80% of stoichiometric. Yeasts harvested during growth on glucose and containing low maltose transport activity did not begin to use maltotriose in the presence of antimycin A until after a long lag phase (up to 50 h), but yeast harvested during growth on maltose, and containing high maltose transport activity, began to use maltotriose after about 25 h. Much shorter lags were observed before growth started in the absence of antimycin A.     

啤酒酵母工业菌株单倍体的诱导、分离和鉴定

【目的】探索适宜的方法进行啤酒酵母工业菌株单倍体的诱导、分离和鉴定,为啤酒酵母改良和遗传学研究提供便利。【方法】首先,选择产孢效果最好的培养基进行产孢诱导,诱导产生的孢子在YPD培养基上形成菌落后,用流式细胞技术检测其DNA含量,进而判断其倍性;单倍体菌株的交配型通过MAT-PCR和杂交实验确定。【结果】啤酒酵母工业菌G-03通过产孢诱导和孢子分离、富集后得到26株菌,最终通过流式细胞技术确定了其中4株为单倍体,MATa和MATα型各2株。通过扫描电镜法观察4株单倍体菌株及出发菌G-03的细胞形态,发现单倍体菌株的形态和出发菌有较大区别,单倍体菌株长期培养没有假丝生长的现象发生。【结论】啤酒酵母工业菌单倍体育种较为困难,严格的单倍体筛选、鉴定尤其具有挑战性。     

Comparison of melibiose utilizing baker's yeast strains produced by genetic engineering and classical breeding

Yeast strains currently used in the baking industry cannot fully utilize the trisaccharide raffinose found in beet molasses due to the absence of melibiase (alpha-galactosidase) activity. To overcome this deficiency, the MEL1 gene encoding melibiase enzyme was introduced into baker's yeast by both classical breeding and recombinant DNA technology. Both types of yeast strains were capable of vigorous fermentation in the presence of high levels of sucrose, making them suitable for the rapidly developing Asian markets where high levels of sugar are used in bread manufacture. Melibiase expression appeared to be dosage-dependent, with relatively low expression sufficient for complete melibiose utilization in a model fermentation system.     

Computer-aided baker's yeast fermentations.

The economics of yeast production depend heavily upon the cellular yield coefficient on the carbon source and the volumetric productivity of the process. The application of an on-line computer to maximize these two terms during the fermentation requires a continuous method of measuring cell density and growth rate. Unfortunately, a direct sensor for biomass concentration suitable for use in industrial fermentations is not available. Material balancing, with the aid of on-line computer monitoring, offers an indirect method of measurement. Laboratory results from baker's yeast production in a 14-liter fermentor (with a PDP-11/10 computer for on-line analyses) show this indirect measurement technique to be a viable alternative. From the oxygen uptake and carbon dioxide production data, gas flow rate, and ammonia addition rate, the cell density during the fermentation has been estimated and found to compare well with actual fermentation data.     

Construction of industrial baker's yeast strains able to assimilate maltose under catabolite repression conditions

Spore progeny from an industrial baker's yeast strain were mutagenized with UV and mutants resistant to 2-deoxyglucose isolated. One of these mutants (10a12–13) showed high levels of maltase (-glucosidase) and external invertase, and assimilated maltose when growing under catabolite repression conditions. This mutant was not allelic to any of the catabolite repression mutants tested cat4, cat80, cid1, cyc8, hex2, hxk2 and tup1. Mutant 10a12–13 was crossed with appropriate strains to construct hybrids that were also able to assimilate maltose in the presence of glucose. These hybrids may be useful in fermentation processes where both glucose and maltose are present.     

Mutarotase in galactose-induced baker's yeast

Cell-free extracts of baker's yeast possess mutarotase activity only after induction of cells in the presence of galactose. The mutarotase activity appears 1 h after transfer to a galactose-containing medium and rises in synchrony with the utilization of galactose. Cycloheximide blocks the induction completely at a concentration of 100 μg/ml. InSaccharomyces fragilis the mutarotase is constitutive but its activity is strikingly increased after growth on galactose. The yeast mutarotase resembles in some respects analogous enzymes from other cells (pH dependence, substrate specificity, heat lability). Its affinity ford-galactose is substantially greater than ford-glucose. There may exist a coupling between mutarotase activity and the anomer-specific galactokinase.     

Wort sugar uptake by brewer's yeast

Summary A culture of brewer's yeast,Saccharomyces cerevisiae (NCYC 240), maintained on Wickerham's MYGP medium, utilized the principal wort sugars sequentially in the order glucose-maltose-maltotriose, when inoculated into brewer's wort. A culture of the same strain maintained on brewer's wort utilized these three sugars simultaneously. Simultaneous utilization could be induced in MYGP-maintained cultures by successive sub-culture in brewer's wort, and appears to be the general rule of sugar uptake during wort fermentation under brewery conditions.