Abstract
Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6829-1) contains supplementary material, which is available to authorized users.
Highlights
Trichoderma reesei can secrete large amounts of extracellular protein, which makes it a paradigm host for homologous and heterologous protein production (Anderson et al 2013; Schuster and Schmoll 2010)
With the aim to generate I-SceI-mediated double-strand DNA breaks at a predetermined site in the T. reesei genome for targeted integration, we first wished to make sure that the expression of the S. cerevisiae gene encoding I-SceI results in an active protein
The rationale of the strategy is that active I-SceI expressed in a T. reesei strain containing this reporter construct would result in excision of the pyrG marker and repair of the double-strand break via the green fluorescent protein (GFP) repeats
Summary
Trichoderma reesei (teleomorph Hypocrea jecorina) can secrete large amounts of extracellular protein (up to 100 g/L), which makes it a paradigm host for homologous and heterologous protein production (Anderson et al 2013; Schuster and Schmoll 2010). Activity or other properties of industrially interesting enzymes, both random and systematic approaches to generate enzyme variants are employed (Adrio and Demain 2014; Turner 2009). To simplify comparison among enzyme variants or different homologues expressed in filamentous fungi and, in particular T. reesei, it is highly desirable that in order to ensure an identical genetic environment, the corresponding DNA constructs are integrated at a defined locus in the genome. Random integration of expression cassettes often leads to significant variation in production levels caused by differences in copy number and/or sites of integration. It has been attempted to increase the efficiency of gene targeting in T. reesei by several means, such as by increasing the size of the region homologous to the target locus (Catalano et al 2011) or by developing strains that are deficient in non-homologous end joining (NHEJ) (Catalano et al 2011; Guangtao et al 2009).
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