## 2 Results and Discussion
### Creation and Verification of the _TaTS_ Mutants  
Recent advances in genome-mining-assisted synthetic biology and metabolic engineering have reinforced filamentous fungi as promising chassis cells to produce bioactive natural products [24]. In comparison to well-known engineering strains such _E. coli_, _Saccharomyces cerevisiae_, _Pichia pastoris_, and _Asppergillus_ spp., the development of _Trichoderma_ as a chassis strain has been limited by the scarcity of efficient genetic manipulation tools and application potentials. In the present study, hygromycin B and G418 were selected as the antibiotics for fungal sensitivity. It was found that 100 \(\mathrm{\SIUnitSymbolMicro}\)/mL hygromycin B and 400 \(\mathrm{\SIUnitSymbolMicro}\)/mL G418 completely inhibit the mycelial growth of ACCC 33109, respectively (Supplementary Figure S2). To prepare the protoplasts, fresh mycelia treated with lysing enzymes for 8 h yielded approximately 8.9 \(\times\) 10\({}^{7}\) mL\({}^{-1}\) protoplasts with a recovery rate of 20% on TB3 agar plates (Supplementary Figure S3). To generate the chassis strain _  
G418 (Figure 3b). To verify these mutants, genomic DNA was extracted and diagnostic PCR was performed using the designated primers. The correct mutants carrying the gene fragment of _neo_ showed a band of 1 kb and missed the gene fragment of _Talig4_ (Figure 3c,d). The results demonstrated that the mutant with the deletion of _Talig4_ in ACCC 33109 was successfully created.  
Mutant strains of four _TaTS_ gene deletions were then created using \(\Delta\)_Talig4:neo_ as the chassis strain following the protocol described above. As shown in Supplementary Figure S4, the recombinant fragments of _TaTS3/4/8/9_ upstream/downstream and _hph_ (_lph-TaTS-up_ and _TaTS-down-hph_) obtained by the fusion PCR reaction were successfully transformed into the protoplasts of \(\Delta\)_Talig4:neo_, which grew well on selective TB3 agar plates supplemented with 100 \(\,\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro}\)/mL hygromycin B and showed a _lph_ fragment of 600 bp on agarose gel. The knockout mutant strains were verified with the absence of _TaTS3/4/8/9_ on gel (Figure 4). In comparison to the very low recombination efficiency of ACCC 33109 (one out of three hundred transformantsants for _Talig4_ replacement with the _neo_ gene), the recombination efficiency of \(\Delta\)_Talig4:neo_ as the chassis strain increased a lot, which was 12-162 out of 300 transformantsants for _TaTS_ replacement with the _lph_ gene. These results indicate that the deletion of the _Talig4_ gene in ACCC 33109 significantly increased the frequency of the gene replacement, as reported in _T. hypoxylon_[25].  
Figure 3: Construction of the chassis strain \(\Delta\)_Talig4:neo_ of ACCC 33109. (**a**) Verification of the recombinant fragments upstream and downstream of the genes _Talig4_ and _neo_. Lanes: M, marker; 1 and 2, _neo-Lig-up_; 3 and 4, _Lig-down-neo_. (**b**) Colony morphology of two chassis strains. (**c**) Verification of the absence of _Talig4_ in the chassis strain. Lanes: M, marker; 1, _\(\Delta\)Talig4:neo_; 2, ACCC 33109. (**d**) Verification of the presence of _neo_ in the chassis strain. Lanes: M, marker; 1, _\(\Delta\)Talig4:neo_; 2, ACCC 33109; 3, the plasmid pKS666.