The increasing awareness of the limited availability of fossil fuels along with the environmental problems caused by their application initiated considerable research efforts towards clean and sustainable biofuels [1
]. Thereby, the cellulases required to degrade cellulosic plant materials into small building blocks, which can be metabolized by yeast or other microbes to ethanol or hydrocarbon biofuel precursors, respectively, are one major focus of investigation [4
]. Trichoderma reesei
) is currently the most efficient producer of enzyme mixtures for degradation of plant materials [5
]. The cellulases produced by this fungus are utilized for diverse industrial processes, from biobleaching of textiles, paper recycling to juice extraction and even as additives in animal feeds [6
The long-standing use of cellulase production by T. reesei
is paralleled by a thorough investigation of the cellulolytic enzyme system of this fungus and its regulation [9
]. With publication of the genomic sequence of T. reesei
], the progress in understanding the mechanisms of cellulase regulation was accelerated. Analysis of the genome indicated, that despite its production efficiency, T. reesei
has the lowest amount of cellulolytic enzymes among Sordariomycetes at its disposal.
In order to gain an understanding of gene regulation, manipulation of the genome of T. reesei
is indispensible. A transformation system for this fungus has been available for decades with amdS
] and hph
] being the most frequently used selection marker systems. Also pyrithiamine resistence [15
], benomyl resistence [16
] and hexokinase [17
] have proven useful for transformation of T. reesei
. Additionally, vector systems enabling excision of the marker gene cassette has facilitated multiple sequential genome modifications despite the limited availability of marker systems [18
]. Nevertheless, the typical efficiency of homologous integration is less than 10% using these methods.
One of the most important advancements in recent years, for improving the performance of research with T. reesei
was the development of strains deficient in non-homologous endjoining (NHEJ) [19
]. These strains strongly enhance the probability of homologous integration of DNA constructs for deletion or modification of genes. Up to 95% of transformants were the result of homologous integration events [20
]. While in most fungi little discernible phenotype is reported for this mutation, the respective strains are more sensitive to DNA damage [21
], the HOG-MAPkinase pathway is up-regulated [22
] and effects on genes involved in carbohydrate transport [23
] have been observed in some organisms. The major drawbacks of using strains deficient in non-homologous end joining, however, is that this mutation causes telomere shortening and defects in DNA repair [21
], both of which negatively influence genome stability and fitness of the respective strains.
Recently, a further tool for work with T. reesei
became available: After decades of research leading to the conclusion that T. reesei
is a clonal, asexual derivative of a previously sexual species [24
], the capability of this fungus for sexual development was discovered [25
]. Besides the physiological relevance, this finding also opens up a wide array of new possibilities for research with T. reesei
. Crossing can now be used for strain improvement and classical genetics. However, the fact that the parental strain of all T. reesei
strains used in research and industry, QM6a, is female sterile [25
], necessitates the use of a sexually competent wildtype isolate for crossing. Consequently, the genetic background introduced by this closely related but also phenotypically different isolate represents a serious drawback for use of sexual crossing in research.
Genome sequencing and high throughput analysis methods for transcriptomics, proteomics and metabolomics have had considerable impact on how research with fungi progresses [26
]. Despite considerable improvements in transformation techniques, the recent increase in available genomic sequences of fungi also caused the need to efficiently use these resources. However, despite the wealth of data created, in depth functional analysis of genes often lags behind and consequently many fungal genome databases still remain with their most precious treasures undiscovered. Mostly, this is due to the enormous effort necessary for creation of a gene knock out library, which necessitates thousands of experiments and subsequent screenings. Therefore the respective methods need to be streamlined and automated as much as possible. For the model organism, Neurospora crassa
, a community-wide effort recently led to the completion of a whole genome knock-out library [27
]. This resource is especially important for evaluation of transcriptome data, because screening of a large number of mutant strains considerably increases the knowledge and understanding to be gained from these studies [28
]. Additionally, the possibility to study a group of functionally related genes, such as transcription factors can provide intriguing insights into previously unexplored physiological processes [30
While elaborate molecular biological tools are available for other fungi, especially N. crassa, tools for efficient systematic analysis of gene function are relatively under-developed in T. reesei. Despite its widespread use in industry, no comprehensive gene knock out library is available for this fungus, nor are there currently efforts to start such an initiative. We therefore aimed to provide a dependable and easy to handle toolkit as a first step towards creation of larger sets of gene knock out strains in T. reesei.
In this study we explored strategies to enable high throughput gene-knockout along with efficient construction of multiple mutants. We applied yeast based recombination mediated vector construction, transformation by electroporation and subsequently removal of the NHEJ-deficient background by crossing with a sexually competent T. reesei strain derived from QM9414. Genome wide primer libraries for knock out vector construction using different marker systems complete this toolkit. These methods can serve as a basis for construction of large scale libraries of knock out strains for systematic functional genomic studies with T. reesei.