Laccase-Based Biosensors for Functional Screening of Transcription Factors in Trichoderma reesei

Efficient transcription factor (TF) screening is essential for the rational optimization of industrial microbial strains. Here, we present an absorbance-activated laccase-based biosensor (AALB) platform designed for rapid functional screening of TFs in Trichoderma reesei. By using laccase as a reporter, the AALB platform offers a robust and cost-effective screening system for TF functional evaluation. The secreted heterologous laccase also complements T. reesei's native cellulolytic machinery, expanding its enzymatic repertoire for lignocellulosic biomass deconstruction. The core of the AALB platform is a Pcbh1-regulated laccase expression cassette, enabling sensitive and efficient selection of TF variants based on absorbance readouts. To further improve sensitivity, we introduced a quadruple repeat of the cis-element-dense segment in the cbh1 promoter and integrated a high-activity heterologous laccase at the SLP1 site of the T. reesei genome. Following a miniaturized 2 day pre-culture in 24-well plates, the AALB platform enables TF variant ranking within 30 min of absorbance readout in 200 μL 96-well reactions, reducing the need for extended shake-flask fermentation and separate downstream enzymatic or molecular assays during the initial screen. Using AALB, we successfully identified TF variants with robust transcriptional regulation. The screening results were validated through transcript quantification of laccase and XYR1 and extracellular laccase activity assays. Notably, the XYR1 mutation XYR1_V821F, selected via AALB, significantly increased laccase transcript levels compared to PoLAC-SLP1 chassis control, with laccase activity reaching 17.74 ± 0.76 IU/mLa 2.7-fold improvement. Further characterization of the AALB-selected XYR1_V821F strain showed increased extracellular laccase activity together with increased xylanase and filter paper activities, with extracellular xylanase and filter paper activities increased by 2.7- and 1.9-fold, respectively, and enhanced rice straw hydrolysis under the tested fermentation and hydrolysis conditions. The AALB platform provides valuable insights into the synthetic biology and metabolic engineering of T. reesei, facilitating the development of industrial strains for lignocellulosic biomass processing and enzyme production.