Deploying Advanced Molecular & Cell Free Expression Tools to Accelerate Characterization of Fungal Cellulosomes
EMSL Project ID
60420
Abstract
This proposal seeks use of EMSL and JGI resources to determine the structure of unique, multi-enzyme cellulosomes made by anaerobic gut fungi. Cellulosomes enable synergistic breakdown of plant biomass into fermentable sugars, and are promising biotechnology tools to drive lignocellulose conversion. While cellulosomes sourced from fungi are known to host a wider diversity of enzymes compared to bacteria, this diversity hampers structure determination via methods like cryo-EM because the native complexes are too heterogenous to characterize. Here, we will pursue a two-pronged approach to enable structural characterization of fungal cellulosomes. First, we will develop advanced molecular tools to genetically engineer anaerobic fungi to produce homogeneous cellulosomes by uncovering key transcription factor binding sites and promoters that drive production of the major dockerin-fused enzymatic modules (via DAP-Seq). As a complementary approach, we will synthesize key cellulosome components for production in a cell-free expression system that allows for complete control of sample homogeneity. By either approach, we plan to produce milligram amounts of homogenous, simplified fungal cellulosomes to enable structure determination by cryo-TEM. Outcomes of this project synergize well with ongoing research efforts funded by DOE-BER, and will reveal critical attributes of fungal cellulosomes that can be engineered and exploited for lignocellulose breakdown and bio-based fuel & chemical production.
Project Details
Project type
FICUS Research
Start Date
2022-10-01
End Date
N/A
Status
Active
Released Data Link
Team
Principal Investigator
Team Members