Identification of Tr80437 as a Key Gene for Furfural Resistance and Enhanced Cellulase Production in Trichoderma reesei by Bulked Segregant Analysis and Cross-Species Screening

Trichoderma reesei is a primary fungal strain for industrial cellulase production but is strongly inhibited by furfural, a cytotoxic byproduct generated during lignocellulosic biomass pretreatment. To overcome this limitation, a furfural-tolerant T. reesei mutant was generated via atmospheric and room-temperature plasma (ARTP) mutagenesis and backcrossing. Bulked Segregant Analysis (BSA) of F1 progeny with contrasting tolerance phenotypes identified a quantitative trait locus on chromosome X containing 172 candidate genes. Comparative analysis with Neurospora crassa and functional screening using its knockout library identified a furfural tolerance-related gene, NCU04116. Disruption of Tr80437, the T. reesei orthologue of NCU04116, conferred enhanced furfural tolerance and increased cellulase activity under furfural stress compared with the wild-type strain. These findings identify Tr80437 as a promising genetic target for engineering robust T. reesei strains for industrial cellulase production.里氏木霉（Trichoderma reesei）是工业纤维素酶生产的核心真菌菌株，但其活性会被糠醛强烈抑制——糠醛是木质纤维素生物质预处理过程中产生的细胞毒性副产物。为破解这一产业瓶颈，研究人员通过常压室温等离子体（atmospheric and room-temperature plasma, ARTP）诱变结合回交技术，获得了耐糠醛的里氏木霉突变株。对表型差异显著的F1子代开展集群分离分析（Bulked Segregant Analysis, BSA）后，研究人员在X染色体上定位到一个包含172个候选基因的数量性状位点（quantitative trait locus, QTL）。通过与粗糙脉孢霉（Neurospora crassa）的比较基因组分析，并利用其敲除文库进行功能筛选，研究团队鉴定出一个与糠醛耐受相关的基因NCU04116。进一步实验显示，敲除NCU04116在里氏木霉中的同源基因Tr80437后，相较于野生型菌株，该突变株在糠醛胁迫下的糠醛耐受性显著提升，同时纤维素酶活性也得到增强。本研究证实Tr80437可作为极具应用前景的遗传靶点，用于工程改造适配工业纤维素酶生产的高稳定性里氏木霉菌株。