TRAC offers multiplex gene expression analysis with high sample throughput, cost-efficiency and minimum hands-on-time. This makes it an excellent choice for use in industrial biotech settings, including for monitoring transcriptional regulation in yeast under protein production conditions.
In one example, Brigitte Gasser and colleagues at the University of Natural Resources and Applied Life Sciences in Vienna, Austria used TRAC to monitor the expression of more than 50 genes in four recombinant P. pastoris strains and the wild type, grown under various conditions.
Temperature has a large impact on the speed of microorganism growth, and can have unexpected effects on the efficiency of product protein generation. In this study, the reduction of cultivation temperature from 25 °C to 20 °C unexpectedly led to a 1.4-fold increase in product secretion rate, even though product transcription at the mRNA level was actually reduced at this lower temperature. This suggested that the greater yield in product formation was being driven by an increase in the efficiency of protein production and/or secretion rather than gene induction.
To probe this temperature response in more detail, TRAC was performed to detect changes in the expression levels of a panel of genes at 20 °C and 25 °C. These genes spanned a wide range of biological functions, from roles in amino acid synthesis, protein folding and secretion through to core metabolism, DNA repair and stress response.
As can be seen in Figure 1, among the results generated by the study was a list of genes that were expressed at significantly different levels when organisms were grown at different temperatures. This list only shows a subset of the results – the entire dataset can be obtained by downloading the full version of Gasser et al. from the BMC Genomics website, which provides full open access to the study.
Figure 1. Average gene expression between 20 °C and 25 °C in chemostat cultivation. Genes with ratios exceeding ± one standard deviation (SD) are marked in light blue, ± two SD in yellow and ± three SD in green. The p-value (χ2-test) is given for each individual marker gene. (a) *** significance level p ≤ 0.01; ** significance level p ≤ 0.05; * significance level p ≤ 0.1.
Among the transcripts upregulated at the lower temperature (i.e. higher protein production conditions) were components of the secretory pathway, suggesting a boost in secretion activity was occurring, which fits with what was seen at the protein level. In addition, a reduction in the expression of chaperones, such as those of the Hsp70 family, was also observed. This led the authors to speculate that at the lower temperature, a reduced amount of folding stress is imposed on the cells, thereby leading to an increase in the generation of correctly folded product. In combination, the gene expression data indicate why product manufacture may have increased at 20 °C, even though the gene expression of the recombinant product was lower.
Several other examples of TRAC being used to analyze yeast metabolism and protein production can be found by visiting the References section.