## 4 Conclusions  
In this study, we identified a key terpene synthase gene, _TaTS9_, in the biocontrol agent _T. afroharzianum_ ACCC 33109 through genome mining, qPCR analysis, gene knockout, and differential volatolome comparison. The deduced _TaTS9_ shares a low identity of 34.06% with the functionally characterized longiborneol synthase, and catalyzes the cyclization of FPP to produce \(\upbeta\)-cubenene. Furthermore, its knockout mutant strain is unable to synthesize 22 kinds of terpenoids (19 sesquiterpene, two diterpene, and one monoterene) and shows significantly decreased anti-fungal activity against _Foc_. The results indicate that _TaTS9_ is crucial to the biosynthesis of anti-fungal sesquiterpenes. This reveals the effectiveness of genome mining and omics in the identification of the key gene involved in the enzyme-catalyzed biosynthesis of bioactive natural products.  
**Supplementary Materials:** The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/catal15040341/s1](https://www.mdpi.com/article/10.3390/catal15040341/s1)]([https://www.mdpi.com/article/10.3390/catal15040341/s1](https://www.mdpi.com/article/10.3390/catal15040341/s1)), Table S1. Genome information of the ACCC 33109 and 6 other strains of _T. afroharzianum_ published in the NCBI database; Table S2. Putative gene clusters of _TaTS_ involved in the biosynthesis of terpenoids; Table S3. qPCR primers of the \(\upbeta\)-_tubulin_gene and 15 _TaTS_ genes; Table S4. Oligonucleotide primers for the construction and verification of the _TaTS_ gene knockout mutant strains; Figure S1. Phylogenetic analysis of the TPs from _Trichoderma_ spp. using the maximum similarity method with MEGA X. The genes from ACCC 33109 are indicated in bold and red; Figure S2. Sensitivity of ACCC 33109 against G408 and hygromycin B; Figure S3. Protoplast preparation of ACCC 33109 over 12 h lysis; Figure S4. Construction of the four _TaTS_ gene knockout mutant strains. (a) Verification of the recombinant fragments of the upstream and downstream of the genes _TaTS_ and _hph_. Lanes: M; marker; 1 & 2, _hph-TaTS3-up_; 3 & 4, _TaTS3-down-hph_; 5 & 6, _hph-TaTS4-up_; 7 & 8, _TaTS4-down-hph_; 9 & 10, _hph-TaTS8-up_; 11 & 12, _TaTS8-down-hph_; 13 & 14, _hph-TaTS9-up_; 15 & 16, _TaTS9-down-hph_. (b) Verification of the presence of _hph_ in the mutant strains. Lanes: M, marker; W, _A_T_alig4:_neo_; P, the plasmid PKH-KO; 1, _A_T_alig4:_neo_a_TaTS3:_hph_; 2-8, _A_T_alig4:_neo_a_TaTS8:_hph_; 9, _A_T_alig4:_neo_a_TaTS4:_hph_; 10-17, _A_T_alig4:_neo_a_TaTS9:_hph_. Figure S5. Colonies of _Foc_ with and without the sandwiched culture of different mutants of ACCC 33109. Figure, S6. GC-MS spectra of the VOCs of the chassis strain _A_T_alig4:_neo_ and two _TaTS_ gene knockout mutant strains _A_T_alig4:_neo_a_TaTS8:_hph_ and _A_T_alig4:_neo_a_TaTS9:_hph_ at 18-24 min. Figure S7. Structures and mass spectra of \(\beta\)-cubengene (A) and \(\gamma\)-amorphene (B).  
**Author Contributions:** F.Z., Y.H., Y.C. and Q.Z. conducted the experiments and data analysis. [PERSON] designed and supervised the project and wrote the manuscript. [PERSON] supervised the project and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This study was supported by the National Key Research and Development Program of China (2024 YED1700400).  
**Data Availability Statement:** Dataset available on request from the authors.  
**Acknowledgments:** The authors are grateful to [PERSON] and [PERSON] from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, for the kind sharing of the plasmids pKS666 and pKH-KO.  
**Conflicts of Interest:** The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.