Abstract
BackgroundThe filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its long-term application in industry and science. A prerequisite for successful heterologous gene expression is the ability to insert a corresponding expression cassette at suitable loci in the genome of T. reesei.ResultsIn this study, we test and demonstrate the applicability of the his1 gene [encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17), part of the histidine biosynthesis pathway] and locus for targeted gene insertion. Deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy. Reestablishment of the his1 locus restores prototrophy. We designed a matching plasmid that allows integration of an expression cassette at the his1 locus. This is demonstrated by the usage of the reporter EYFP (enhanced yellow fluorescence protein). Further, we describe a minimal effort and seamless marker recycling method. Finally, we test the influence of the integration site on the gene expression by comparing three strains bearing the same EYFP expression construct at different loci.ConclusionWith the establishment of his1 as integration locus and auxotrophic marker, we could expand the toolbox for strain design in T. reesei. This facilitates future strain constructions with the aim of heterologous gene expression.
Highlights
The filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its longterm application in industry and science
We describe the applicability of the his1 gene [TRIREDRAFT_80820, encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17)] as a suitable insertion locus and auxotrophic marker for gene integrations in T. reesei
Deletion of his1 leads to histidine auxotrophy in T. reesei First, we deleted a part of the his1 coding region and the native promoter using a homologous recombination strategy and the pyrG marker (Fig. 1A)
Summary
The filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its longterm application in industry and science. Please refer to our previous study for a detailed description of this strategy [10] This yields strains that are isogenic to the original parent strain except for the inserted gene. We test if and how the choice of the integration site effects the expression of the inserted gene. To this end, we determine the expression of the reporter EYFP (enhanced yellow fluorescence protein) in strains carrying the eyfp gene at the pyr, the asl, or the his locus by comparative transcript analysis and fluorescence measurements. We describe a minimal effort and seamless marker recycling strategy, and we construct a triple auxotrophic strain, which can be used for future multiple gene insertions
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