By Tiina Pakula
In this blog post we describe how we used a systems biology approach to discover new tools for strain improvement in Trichoderma reesei. As a saprophytic fungus by origin living on plant debris, it produces a large repertoire of enzyme activities degrading lignocellulosic material. Genomic analyses have revealed over 200 genes encoding carbohydrate active enzymes, over 60 of them predicted to encode cellulolytic or hemicellulolytic activities.
Based on literature, T. reesei is the most efficient organism for production of enzymes at industrial scale. Optimisation of culture conditions and systematic strain development has made it possible to produce secreted proteins even as much as over 100 g/l.
The enzyme industry exploits T. reesei widely in enzyme manufacturing for e.g. pulp and paper, food, feed, and textile applications. In the recent years the importance of this production host has increased greatly along with the rising trend of producing fuels and chemicals from lignocellulosic feedstocks in second generation biorefineries. We have long experience in developing T. reesei as a producer of lignocellulose degrading enzymes and recombinant proteins. This production platform is the flagship of VTT Expression Service providing custom recombinant protein production.
VTT’s Protein Production team
Understanding of regulatory mechanisms is the key for the enhancement of enzyme production
Production of extracellular enzymes by the fungus is modulated by a multitude of environmental and physiological conditions, the most important determinant being the carbon source available for the fungus. Hydrolysis of complex plant-derived material requires a synergistic action of different enzymatic activities, and therefore it is beneficial for the fungus to be able to adjust the enzyme production according to the need and to produce suitable combination of enzymes in the presence of the particular material.
Novel biotechnical applications aim at the utilisation of different types of bio-based raw materials in production of useful compounds, and tailor-made enzyme cocktails are needed for efficient utilisation of the material. Understanding of the regulatory mechanisms in production of the enzymes provides tools to enhance the overall production as well as to engineer enzyme cocktails for specific raw materials.
We have applied transcriptomics analysis to examine expression of genes encoding hydrolytic enzymes in the presence of different carbon sources, ranging from defined oligosaccharides to complex plant-derived raw materials such as bagasse, spruce and wheat straw. Groups of co-regulated genes encoding hydrolytic enzymes were identified on the different carbon source materials, suggesting a potentially important role for them in utilisation of the material. Furthermore, the analyses revealed novel candidate regulatory genes encoding transcription factors and other regulatory proteins that had a similar expression patterns with the genes encoding the hydrolytic enzymes (Fig. 1).
Figure 1. Heatmap presentation of expression patterns of selected genes in the presence of different lignocellulosic substrates. Red colour in the heatmap indicates induction of the gene by the specific substrates (shown on the bottom). Genes encoding hydrolytic enzymes are shown by green colour and candidate regulatory genes by red on the right.
A set of 28 such candidate regulatory genes were selected for further studies to reveal their possible role in regulation of enzyme production. These genes were first over-expressed in T. reesei with a constitutive promoter. Seven of the genes tested were shown to increase cellulase and/or xylanase production when overexpressed in the fungal cells (Fig. 2), and some genes appeared to have a negative effect.
Figure 2. Production of (A) xylanase and (B) cellulase activity by strains overexpressing the candidate regulatory genes as compared to the parental strain (parental level shown by red line).
A part of the genes had an effect on production of one particular enzyme activity whereas others appeared to have an effect on production of a broader set of activities. Further analysis of one of the genes, designated ace3, showed that it is essential for cellulase production by the fungus. Overexpression of ace3 (pMH15 in Figure 2) clearly enhanced cellulase and xylanase production, whereas deletion of the gene abolished cellulase production completely and reduced xylanase production significantly (Figure 3).
Figure 3. Production of cellulase and xylanase activity by strains deleted for ace3 and by the parental strain
The multitude of environmental factors affecting production of hydrolytic enzymes and the very large number of enzymes produced by T. reesei suggest that a complex regulatory and signalling network exists in order to maintain coordinated expression of the genes and production of the enzymes. Several regulator genes affecting the expression of cellulase and hemicellulase genes have been identified by us and others over the years, such as the positively acting xyr1 and ace2, and the negatively acting cre1 and ace1. Our results have brought new insights in the understanding of the regulation of the (hemi)cellulolytic system of T. reesei. Novel regulators have been identified, including essential positively acting regulators that can be utilised in further improvement of the production host organism.
Ongoing work focusing on the interplay of the known regulatory factors will lead to better understanding of the regulatory network involved in cellulase gene regulation. This offers tools for the generation of more efficient and robust, production strains of enzymes.
Dr. Tiina Pakula (Principal Scientist) has worked at VTT Technical Research Centre of Finland Ltd since 1994. Her current research topics are related to protein production in microbial host organisms, especially in the filamentous fungus Trichoderma reesei. Application of systems biology approaches in order to understand the cellular processes and to improve the properties of the organism are the main topics of interest. firstname.lastname@example.org
Additional information and reading:
Häkkinen, M., Arvas, M., Oja, M., Aro, N., Penttilä, M., Saloheimo, M., and Pakula, T. 2012. Re-annotation of the GAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates.Microbial Cell Factories 11, 134.
Häkkinen, M., Valkonen, M. J., Westerholm-Parvinen, A., Aro, N., Arvas, M., Vitikainen, M., Penttilä, M., Saloheimo, M. and Pakula, T. M. 2014. Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production. Biotechnology for Biofuels 7,14.
Häkkinen, M., Sivasiddhartan, D., Aro, N., Saloheimo, M. and Pakula, T. M. 2015. The effects of extracellular pH and of the transcriptional regulator PACI on the transcriptome of Trichoderma reesei. Microbial Cell Factories 14, 63.