Development Of A Continuous Gene Targeting Toolbox And Identification Of A Three-gene Cluster Related To DNA Repair In Trichoderma Reesei

Filamentous fungi are widely used in biotechnology as cell factories for industry, agriculture and medicine. The ascomycete Hypocrea jecorina (teleomorph of Trichoderma reesei) is the most efficient industrial producer of cellulolytic and hemicellulolytic enzymes. With the rapid development of DNA sequencing techniques, the complete genome sequences of T. reesei are available in 2008. But the limitation lies in the fact that backward molecular biological technique and genetic and engineering. In order to get more thoroughly study on important functional genes, mechanism of high yield, protein secretion mechanisms and growth metabolism characteristics, we urgently need efficient gene manipulation methods to dig deeper into the molecular mechanisms of T. reesei.Genomic stability is very important for industrial strains. The DNA of any given cell is continually subjected to damage from both exogenous and endogenous sources. The DNA damage response is a protective mechanism that ensures the maintenance of genome integrity. The study of DNA repair mechanisms is far from deep understanding for T. reesei.The detail research content divided into two parts.In the first section, we have established an efficient and controllable gene targeting system.1. Carbon source inducible self-excisable gene disruption system LML2.0:LML2.0 was spliced two loxV sites, a Cre expression cassette and a selectable marker together to generate multiple fungal mutants through efficient one-step recycling of markers. We initially constructed five chimeric cre genes which contained predicted genes or introns from the T.reesei in cre coding sequence. The average self-excision efficiencies were about 80% when using the D-xylose as inducer. We also compared self-excision efficiencies under different carbon source induction. Our data showed that the induction efficiency of D-xylose was the best. These results proved that LML2.0 system can be stable in the prokaryotic and eukaryotic cell, and under the induction of xylose can produce stable self-excision efficiency.2. LML2.1, which employed the LE/RE mutant strategy using LE mutant I ox carrying mutations in the left-inverted repeat region and RE mutant lox carrying mutations in the right-inverted repeat. By using different mutant lox, we constructed 9 kinds of LML2.1. Combination of loxJT15 and loxJTZ17 was proved to be high self-excision efficiency (91%). Other combinations didn’t work well. Recombination of loxJTIS and loxJTZ17 sites results in a double-mutant lox32 site. The Cre recombinase cannot efficiently catalyze recombination between the double mutant lox sites. Therefore, chromosomal rearrangement rarely occurred between the double mutant lox sites in the genome during further rounds of marker rescue.3. Application of continuously gene targeting with LML2.1a marker free system:The tku70 deletion cassette including LML2.1a system was used to transform H. jecorina Qm6a. The Qm6aAtku70 strain showed high homologous integration rates. We achieved seven additional deletions in Qm6aAtku70 strain and third round deletion in the same strain. The results show that our systems were suitable for sequential gene modification, and too stable to induce recombination when carrying out further marker recycling processes. No self-excision was detected in Axyr1 strain for the general activator Xyrl as essential for xynl transcription.4. A blue light-inducible self-excisable gene disruption system LML3.0:approximately 40-70%of the transformed strains had successfully excised the LML 3.0 cassette. Light-inducible transcription system was independent of host species and carbon source induced mechanism. The LML 3.0 cassettes were widely used in more hosts than LML 2.0/2.1 due to artificial light-inducible control of self-excision.5. OFN 1.0 for the rapid and accurate on/off control of NHEJ pathway:the orientation of tku70 gene would continuously be converted by Cre recombinase catalyzing, because tku70 gene was flanked by two inverted loxP sites. By adding the Cre recombinase self-excision system, we can control the expression states of tku70 gene. We constructed 4 cassettes (OFN 1.0A-D) according to different loci of inverted TATA/ox or loxN sites. By analysis of relative transcription levels and sensitivity test of UV irradiation, we detected lower transcript levels of tkn70 in OFN 1.0A-C OFF states and no obviously transcription in OFN1.0D OFF state. Increased tolerance of UV irradiation showed that tkn70 were restored by inversion in OFN1.0D ON state.In the second section, Here we identified a three-gene cluster related to DNA repair, including a ferrochelatase gene tre78582 (hem8), tku70, and treJ08087, a downstream unknown gene oitku70.1. By photoinduction experiment, we compared the transcriptional level in the absence and presence of blue light via qRT-PCR. The transcriptional level of three genes was increased by 2-3-fold during 2 hours. We also found some specific motifs for light regulators binding in the promoter regions of the three genes. Therefore, these results showed that they were all induced by blue light.2. BLASTP analysis, gene knockout and complementation of tre78582 showed that tre78582, the upstream gene of tku70, encodes a putative ferrochelatase, an essential protein in T. reesei. We isolated Δtre78582 homokaryons from heterokaryons in malt extract agar with 500 mg/1 hematin. By adding different carbon source, we concluded that glucose or lactose addition induces porphyrin synthesis, while hematin inhibits it. hem8 can be a novel and highly distinct auxotrophic marker in T. reesei, because red auto-fluorescence of transformants can be detected under 365 nm light.3. In a similar way, this study identified ire108087 encoding a novel Zn(Ⅱ)2Cys6 family protein widely existed in fungi, whose function was not be reported previuously. We systematically studied the role of tku70 and its neighbor, treI08087, in DNA repair using different DNA damage treatments. The results indicated that tre108087 encodes a new family of DSBs repair protein that was never reported previously. The function of tre108087 might be involved in DNA biosynthesis or topoisomerases I activation and repair DSBs by a KU-independent NHEJ pathway.