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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.

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.

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.

PlexPress | Date: 2009-05-14

Analysis kit for laboratory use, which kit contains reagents, namely, buffers, biomolecules, indicators, catalysts, enzymes, diluents and magnetic particles used in genomic study. Analysis kit for laboratory use, which kit contains reagents, namely, buffers, biomolecules, indicators, catalysts, enzymes, diluents and magnetic particles used in the diagnosis of genetic disorders in humans. Software used for the analysis of results provided by analysis kits for laboratory use.

Arvas M.,VTT Technical Research Center of Finland | Haiminen N.,University of Helsinki | Smit B.,FrieslandCampina Innovation Europe | Rautio J.,PlexPress | And 11 more authors.
Gene | Year: 2010

Species-specific genes play an important role in defining the phenotype of an organism. However, current gene prediction methods can only efficiently find genes that share features such as sequence similarity or general sequence characteristics with previously known genes. Novel sequencing methods and tiling arrays can be used to find genes without prior information and they have demonstrated that novel genes can still be found from extensively studied model organisms. Unfortunately, these methods are expensive and thus are not easily applicable, e.g., to finding genes that are expressed only in very specific conditions.We demonstrate a method for finding novel genes with sparse arrays, applying it on the 33.9. Mb genome of the filamentous fungus Trichoderma reesei. Our computational method does not require normalisations between arrays and it takes into account the multiple-testing problem typical for analysis of microarray data. In contrast to tiling arrays, that use overlapping probes, only one 25mer microarray oligonucleotide probe was used for every 100. b. Thus, only relatively little space on a microarray slide was required to cover the intergenic regions of a genome. The analysis was done as a by-product of a conventional microarray experiment with no additional costs. We found at least 23 good candidates for novel transcripts that could code for proteins and all of which were expressed at high levels. Candidate genes were found to neighbour ire1 and cre1 and many other regulatory genes. Our simple, low-cost method can easily be applied to finding novel species-specific genes without prior knowledge of their sequence properties. © 2010 Elsevier B.V.

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