Rapid RNA expression profiling of the fungus Trichoderma reesei in production conditions

Microorganisms used for various types of bioprocesses are subjected to constantly changing environmental conditions to which they adapt by adjusting their cellular physiology. Changes in the genomic expression program are the first signs of adaptation to chancing conditions or to potential process disturbances. However, tools suitable for high-throughput expression monitoring of process-relevant genes are scarce. Moving to postgenomic era with a growing number of organisms has increased the interest in functional genomics, and the need for fast and reliable transcriptional profiling methods is thus growing. We have developed a rapid method for transcriptional profiling of microbial cultivations based on a novel technique called TRAC[1] "transcriptional profiling with the aid of affinity capture". This method allows fast gene expression analysis for sets of mRNAs by solution hybridisation with a pool of target-specific oligonucleotide probes of distinct sizes that are identified and quantified by capillary electrophoresis. The assay procedure has been semi-automated for simultaneous treatment of 96 samples using magnetic bead particle processor. To further enhance the robustness of the method it was set up to work with crude cell lysates. TRAC has been shown to produce results highly consistent with mRNA quantification by Northern hybridisation. Computational methods have been applied for design of target-specific oligonucleotide probes and to assign them into minimal number of pools[2]. The whole assay procedure can be performed in three hours, implying its usefulness in bioprocess monitoring and control. The developed TRAC method application has been used for monitoring the levels of a set of mRNAs in the filamentous fungus Trichoderma reesei in fermentation conditions. Chosen gene markers for bioprocess monitoring are involved in various cellular pathways including unfolded protein response, protection against various stress conditions, oxygen and nutrient limitation responses, protein synthesis and growth. Data collected from different types of fermentations shows the potential of the method for use in optimisation of production processes and provides novel information about regulation of various genes during different phases of long cultivations. [1] Söderlund, H., Kataja, K., Paloheimo, M., Ilmen, M. and Takkinen, K. (2003) Method and test kit for quantitative and/or coparative assessment of variations in polynucleotide amounts in cell or tissue samples. Finland FI20010041 / Int.pat.appl. No. PCT/FI02/00023 [2] Kivioja, T., Arvas, M., Kataja, K., Penttilä, M., Söderlund, H. and Ukkonen, E. (2002) Assigning probes into a small number of pools separable by electrophoresis. Bioinformatics, 18, S199-206.