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      The temperature dependence of maltose transport in ale and lager strains of brewer's yeast

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          Abstract

          Lager beers are traditionally made at lower temperatures (6–14 °C) than ales (15–25 °C). At low temperatures, lager strains ( Saccharomyces pastorianus) ferment faster than ale strains ( Saccharomyces cerevisiae). Two lager and two ale strains had similar maltose transport activities at 20 °C, but at 0 °C the lager strains had fivefold greater activity. AGT1, MTT1 and MALx1 are major maltose transporter genes. In nine tested lager strains, the AGT1 genes contained premature stop codons. None of five tested ale strains had this defect. All tested lager strains, but no ale strain, contained MTT1 genes. When functional AGT1 from an ale strain was expressed in a lager strain, the resultant maltose transport activity had the high temperature dependence characteristic of ale yeasts. Lager yeast MTT1 and MALx1 genes were expressed in a maltose-negative laboratory strain of S. cerevisiae. The resultant Mtt1 transport activity had low temperature dependence and the Malx1 activity had high temperature dependence. Faster fermentation at low temperature by lager strains than ale strains may result from their different maltose transporters. The loss of Agt1 transporters during the evolution of lager strains may have provided plasma membrane space for the Mtt1 transporters that perform better at a low temperature.

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          Improved method for high efficiency transformation of intact yeast cells.

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            New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae.

            We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading-frame of the E. coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading-frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4). The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.
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              New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites.

              We describe the production of new alleles of the LEU2, URA3 and TRP1 genes of Saccharomyces cerevisiae by in vitro mutagenesis. Each new allele, which lacks restriction enzyme recognition sequences found in the pUC19 multicloning site, was used to construct a unique series of yeast-Escherichia coli shuttle vectors derived from the plasmid pUC19. For each gene a 2 mu vector (YEplac), an ARS1 CEN4 vector (YCplac) and an integrative vector (YIplac) was constructed. The features of these vectors include (i) small size; (ii) unique recognition site for each restriction enzyme found in the pUC19 multicloning site; (iii) screening for plasmids containing inserts by color assay; (iv) high plasmid yield; (v) efficient transformation of S. cerevisiae. These vectors should allow greater flexibility with regard to DNA restriction fragment manipulation and subcloning.
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                Author and article information

                Journal
                FEMS Yeast Res
                fyr
                Fems Yeast Research
                Blackwell Publishing Ltd
                1567-1356
                1567-1364
                June 2010
                09 April 2010
                : 10
                : 4
                : 402-411
                Affiliations
                simpleVTT Technical Research Centre of Finland Espoo, Finland
                Author notes
                Correspondence: John Londesborough, VTT Technical Research Centre of Finland, PO Box 1000, Espoo, FIN-02044, Finland. Tel.: +358 20 722 5996; fax: +358 20 722 7071; e-mail: john.londesborough@ 123456vtt.fi
                Article
                10.1111/j.1567-1364.2010.00627.x
                2878602
                20402791
                d9f0a34d-c5c3-488a-a535-c856d002680f
                © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved

                Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

                History
                : 20 January 2010
                : 22 February 2010
                : 25 February 2010
                Categories
                Research Articles

                Molecular biology
                brewer's yeast strains,evolution,α-glucoside transporters,temperature dependence of fermentation,temperature dependence of transport

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