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A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina).

ABSTRACT: BACKGROUND: The industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools for genetic engineering. RESULTS: In this study we present new genetic tools for T. reesei to further expand its use in industrial production. We have developed an expression platform where genes are inserted into a versatile expression vector via highly efficient uracil-excision cloning and subsequently inserted into a defined position in the T. reesei genome ensuring that enzyme production from different transformants can be directly compared. The ade2 locus was selected as integration site since ade2 mutants develop red pigment that facilitates easy and rapid detection of correctly targeted transformants. In addition, our system includes a tku70 disruption to increase gene targeting efficiency and a new bidirectional marker, pyr2, for iterative gene targeting. The dual selection system, color and prototrophism, ensures that correct transformants containing the desired gene inserted into the defined expression site can be selected with an efficiency approaching 100%. CONCLUSIONS: The new genetic tools we have developed are suitable for high-throughput integration of genes into the genome of T. reesei and can easily be combined with techniques for generation of defined mutants. Moreover, the usability of the novel expression system with ade2 as integration site was confirmed by expression of a Thermomyces lanuginosus lipase.

SUBMITTER: Jorgensen MS

PROVIDER: S-EPMC4015775 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina).

Jørgensen Mikael Skaanning MS Skovlund Dominique Aubert DA Johannesen Pia Francke PF Mortensen Uffe H UH

Microbial cell factories 20140306 1

<h4>Background</h4>The industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools for genetic engineering.<h4>Results</h4>In this study we present new genetic tools for T. reesei to further expand its use in industrial production. We have developed an expression platform where genes are ...[more]

PMID: 24602241

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Project description:Comparison of T. reesei grown on lactose fed chemostat cultivations in different growth rates and cell densities The analysis is further described in paper Correlation of gene expression and protein production rate - a system wide study. Mikko Arvas, Tiina Pakula, Bart Smit, Jari Rautio, Heini Koivistoinen, Paula Jouhten, Erno Lindfors, Marilyn Wiebe, Merja Penttilä and Markku Saloheimo, Submitted. Abstract: 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).

Dataset Information

A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina).

Publications

A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina).

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