Helsinki, Finland
Helsinki, Finland

Time filter

Source Type

Ekberg J.,VTT Technical Research Center of Finland | Ekberg J.,Aalto University | Rautio J.,PlexPress | Mattinen L.,PlexPress | And 3 more authors.
FEMS Yeast Research | Year: 2013

An adaptive evolution method to obtain stable Saccharomyces pastorianus brewing yeast variants with improved fermentation capacity is described. The procedure involved selection for rapid growth resumption at high osmotic strength. It was applied to a lager strain and to a previously isolated ethanol-tolerant strain. Fermentation performance of strains was compared at 15 °P wort strength. A selected osmotolerant variant of the ethanol-tolerant strain showed significantly shorter fermentation time than the parent strain, producing 6.45% alcohol by volume beer in 4-5 days with mostly similar organoleptic properties to the original strain. Diacetyl and pentanedione contents were 50-75% and 3-methylbutyl acetate and 2-phenylethyl acetate 50% higher than with the original strain, leading to a small flavour change. The variant contained significantly less intracellular trehalose and glycogen than the parent. Transcriptional analysis of selected genes at 24 h revealed reduced transcription of hexose transport genes and increased transcription of the MALx1 and MALx2 genes, responsible for α-glucoside uptake and metabolism. It is suggested that an attenuated stress response contributes to the improved fermentation performance. Results show that sequential selection for both ethanol tolerance and rapid growth at high osmotic strength can provide strains with enhanced fermentation speed with acceptable product quality. © 2013 Federation of European Microbiological Societies.


Nisamedtinov I.,Tallinn University of Technology | Nisamedtinov I.,Lallemand Inc. | Orumets K.,Tallinn University of Technology | Rautio J.J.,PlexPress | Paalme T.,Tallinn University of Technology
Applied Microbiology and Biotechnology | Year: 2010

Shot-wise supplementation of cysteine to a yeast culture is a common means of promoting glutathione (GSH) production. In the present work, we study the accumulation kinetics of cysteine, γ-glutamylcysteine, and GSH and the expression of genes involved in GSH and sulfur metabolism in ethanol-stat fed-batch cultures as a result of switching to a medium enriched with cysteine and glycine. Supplementation in this fashion resulted in a rapid but short-term increase in the rate of GSH synthesis, while the expression of GSH1 decreased. Expression of GSH1 and GSH synthesis rate were observed to revert close to the base level after a few hours. These results indicate that, under such conditions, the control of GSH synthesis at higher concentrations occurred at the enzymatic, rather than the transcriptional level. The incorporation of cysteine into GSH was limited to 40% of the theoretical yield, due to its requirement as a source of sulfur for protein synthesis under conditions whereby the sulfate assimilation pathway is down-regulated. This was supported by the expression profiles of genes involved in cysteine and homocysteine interconversion. © 2010 Springer-Verlag.


Gibson B.,VTT Technical Research Center of Finland | Krogerus K.,VTT Technical Research Center of Finland | Ekberg J.,VTT Technical Research Center of Finland | Monroux A.,Agro ParisTech | And 3 more authors.
Yeast | Year: 2015

A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer's wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid (S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling α-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form. © 2014 John Wiley & Sons, Ltd.


Mattinen L.,PlexPress | Kublbeck J.,University of Eastern Finland | Rechardt O.,PlexPress | Honkakoski P.,University of Eastern Finland | Rautio J.,PlexPress
Drug Metabolism Letters | Year: 2014

Induction of cytochrome P450 (CYP) enzymes is commonly analyzed in cultured human primary hepatocytes (HPHs) by measuring CYP1A2, CYP2B6 and CYP3A4/3A5 activities after exposure to test and reference compounds. Because chemicals can both inhibit and induce CYP enzymes, this traditional approach fails to distinguish such simultaneous effects. Regulatory authorities have therefore suggested that measurement of CYP expression levels should complement activity measurements. We aimed to compare a hybridization and bead-based assay termed transcript analysis w ith the aid of affinity capture (TRAC) with the routinely used quantitative real-time PCR (qRT-PCR) assay and to study its suitability for CYP induction studies on mRNA level. HPHs from three donors were treated with vehicle, reference substances omeprazole, phenobarbital and rifampicin and six test compounds on 48-well plates. The mRNA expression of ten CYP isoforms important for drug metabolism was determined by TRAC and qRT-PCR methods in order to validate the novel TRAC method. The fold-increases of CYP mRNA levels showed a good correlation between the assays. With TRAC, the marker CYP mRNAs for induction could be easily detected from about 10 000 hepatocytes per sample, with a coefficient of variation below 10% between triplicates. Time spent for TRAC analysis was significantly shorter. Thus, TRAC is a sensitive and reproducible high-throughput assay, which enables accurate and direct detection of multiple mRNA targets simultaneously from large number of samples without enzymatic reactions inherent to qRT-PCR. It is a valuable method to study CYP induction and expandable to other genes relevant for drug metabolism and toxicity. © 2014 Bentham Science Publishers.


Vidgren V.,VTT Technical Research Center of Finland | Viljanen K.,VTT Technical Research Center of Finland | Mattinen L.,PlexPress | Rautio J.,PlexPress | Londesborough J.,VTT Technical Research Center of Finland
FEMS Yeast Research | Year: 2014

Zero-trans rates of maltose transport by brewer's yeasts exert strong control over fermentation rates and are strongly temperature-dependent over the temperature range (20-0 °C) of brewery fermentations. Three α-glucoside transporters, ScAgt1(A60) (a Saccharomyces cerevisiae version of Agt1 from an ale strain), ScAgt1-A548V (a variant of ScAgt1(A60) with a single amino acid change in a transmembrane domain), and SbAgt1 (a Saccharomyces (eu)bayanus version from a lager strain), were compared. When expressed in the same laboratory yeast, grown at 24 °C and assayed at 0, 10, and 20 °C, SbAgt1 had the lowest absolute maltose uptake activity at 20 °C but smallest temperature dependence, ScAgt1-A548V had the highest activity but greatest temperature dependence, and ScAgt1(A60) had intermediate properties. ScAgt1(A60) exhibited higher absolute rates and smaller temperature dependencies when expressed in laboratory rather than brewer's strains. Absolute rates closely reflected the amounts of GFP-tagged ScAgt1(A60) transporter in each host's plasma membrane. Growth at 15 °C instead of 24 °C decreased the absolute activities of strains expressing ScAgt1(A60) by two- to threefold. Evidently, the kinetic characteristics of at least ScAgt1(A60) depended on the nature of the host plasma membrane. However, no consistent correlation was observed between transport activities and fatty acid or ergosterol compositions. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.


Gibson B.R.,VTT Technical Research Center of Finland | Londesborough J.,VTT Technical Research Center of Finland | Rautio J.,PlexPress | Mattinen L.,PlexPress | Vidgren V.,VTT Technical Research Center of Finland
Journal of the Institute of Brewing | Year: 2013

The hybrid lager yeast Saccharomyces pastorianus (S. cerevisiae×S. eubayanus) contains several genes encoding proteins responsible for the uptake and metabolism of maltose and maltotriose. In many cases the genes occur as orthologues, that is, the S. cerevisiae gene exists along with the S. eubayanus gene. Prior to formation of the hybrid, these genes existed in organisms, which had been separated for tens of millions of years and were expected to show some level of genetic and functional differentiation. In this study, oligonucleotide probes were designed for TRAC analysis of transcription of the S. cerevisiae and S. eubayanus orthologues of AGT1, MALx1, MALx2 and MALx3 as well as the S. cerevisiae-derived MPH2/3 genes within the S. pastorianus genome. Specificity of probes was validated using mRNA from S. cerevisiae and from S. eubayanus. Probes were used to analyse gene expression during 15°P wort fermentations conducted at different temperatures (10-20°C). As well as differential expression of different genes, differential expression of orthologues was also observed during fermentation. The differences suggest that, where two forms of the gene exist, either one will dominate (as with AGT1) or expression will be staggered (MALx2), possibly to maximize transport and for efficient degradation of sugars. © 2013 The Institute of Brewing & Distilling.


Rautio J.J.,PlexPress
Methods in molecular biology (Clifton, N.J.) | Year: 2010

For an increasing number of microorganisms of scientific and industrial interest, the genome sequences have become available, which in turn has enabled genome-wide microarray studies. Global level transcriptomic analysis has flooded the research community with gene expression data from diverse biological states. One of the key aspects of this research is that in many cases the analysis of thousands of genes leads to the discovery of significantly smaller sets of genes, from a few to a few hundred, which provide the essential information about biological systems of interest. As a consequence, the requirement for technologies enabling rapid, cost-effective and quantitative detection of specific gene transcripts has increased. A method named TRAC (Transcript analysis with aid of affinity capture) is a novel solution hybridization and bead-based assay enabling multiplex mRNA target detection simultaneously from large sample numbers. Functionality of TRAC has been shown in a number of applications including microbial quantification and gene expression-based monitoring of biotechnical processes as well as cell-based cancer marker gene screening and siRNA validation.


Arvas M.,VTT Technical Research Center of Finland | Pakula T.,VTT Technical Research Center of Finland | Smit B.,NIZO food research | Rautio J.,PlexPress | And 6 more authors.
BMC Genomics | Year: 2011

Background: Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype.Results: We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response.Conclusions: Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR). © 2011 Arvas et al; licensee BioMed Central Ltd.


PubMed | PlexPress
Type: Comparative Study | Journal: Drug metabolism letters | Year: 2015

Induction of cytochrome P450 (CYP) enzymes is commonly analyzed in cultured human primary hepatocytes (HPHs) by measuring CYP1A2, CYP2B6 and CYP3A4/3A5 activities after exposure to test and reference compounds. Because chemicals can both inhibit and induce CYP enzymes, this traditional approach fails to distinguish such simultaneous effects. Regulatory authorities have therefore suggested that measurement of CYP expression levels should complement activity measurements. We aimed to compare a hybridization and bead-based assay termed transcript analysis with the aid of affinity capture (TRAC) with the routinely used quantitative real-time PCR (qRT-PCR) assay and to study its suitability for CYP induction studies on mRNA level. HPHs from three donors were treated with vehicle, reference substances omeprazole, phenobarbital and rifampicin and six test compounds on 48-well plates. The mRNA expression of ten CYP isoforms important for drug metabolism was determined by TRAC and qRT-PCR methods in order to validate the novel TRAC method. The fold-increases of CYP mRNA levels showed a good correlation between the assays. With TRAC, the marker CYP mRNAs for induction could be easily detected from about 10 000 hepatocytes per sample, with a coefficient of variation below 10% between triplicates. Time spent for TRAC analysis was significantly shorter. Thus, TRAC is a sensitive and reproducible high-throughput assay, which enables accurate and direct detection of multiple mRNA targets simultaneously from large number of samples without enzymatic reactions inherent to qRT-PCR. It is a valuable method to study CYP induction and expandable to other genes relevant for drug metabolism and toxicity.


Loading PlexPress collaborators
Loading PlexPress collaborators