2006

Rautio, Jari; Toivari, Mervi; Smit, Bart; Bailey, Michael; Söderlund, Hans; Saloheimo, Markku; Penttilä, Merja

Micro-organisms used in various types of biotechnical processes encounter constantly changing environmental conditions, to which they adapt by changing their cellular physiology. The performance of the used micro-organism has a major impact on the performance of the biotechnical process, and as a consequence, bioprocess monitoring and control strategies based on the physiological status of the cells culture have become more popular. Transcriptional analysis of selected marker genes is a potential way for robust monitoring of physiological events. However, tools suitable for high-throughput expression monitoring of process-relevant genes are scarce. We have applied a novel transcriptional analysis method called TRAC (TRanscript analysis with aid of Affinity Capture) for frequent expression analysis of a focused gene set in a wide range of fungal fermentations. TRAC method allows fast expression analysis of dozens of mRNAs from crude cell lysates 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 a magnetic bead particle processor. The whole assay procedure can be performed in few hours, implying its usefulness in bioprocess monitoring and control. We have used TRAC for instance to monitor the physiological state of the filamentous fungus Trichoderma during protein production processes and S. cerevisiae physiology in various chemostat cultures and brewing processes. By process relevant gene markers it is possible to evaluate the growth and extracellular protein production potential of the culture as well as physiological responses to various stress conditions such as lack of nutrients and oxygen or limitation of the protein folding capacity. TRAC also provides a quick and affordable means to monitor transient situations, and to address the quality and stability of e.g. chemostat cultures prior to selecting samples for more thorough and expensive systems-wide analyses.