Spatially-resolved -omics to target reaction partitioning mechanisms in a unique lignocellulose-degrading fungus
EMSL Project ID
49816
Abstract
Brown rot fungi are among a select group of filamentous fungi able to deconstruct lignocellulose, and their biochemical pathways have potential biofuels applications and carbon cycling implications. These unique fungi combine oxidative and enzymatic mechanisms to remove wood carbohydrates selectively, with minimal lignin removal. This 'two-step' mechanism has not been resolved, but has long been theorized to involve a temporal staggering of the two incompatible steps.We recently showed that carbohydrate-selective brown rot-type fungi partition, via differential expression, a brief (<48-hr) oxidative pretreatment ahead of most carbohydrate-active enzymes (CAZys). Our current sectioning approach to elucidate this staggered two-step mechanism, however, achieves only 1-mm resolution in the best case. This lack of resolution leaves two key questions related to gene discovery that beg for higher-resolution approaches to answer them. 1) Can we reduce the number of candidate genes critical to this unique pretreatment step by differentiating the expression at mycelial front hyphal tips from the 'noise' at sub-apical branching tips? 2) Can we further narrow these gene targets by overlaying maps of metabolites (e.g. glycoproteomics) to integrate functional information?
With our goal of addressing these questions with EMSL collaboration and via microscopy-enabled -omics approaches, our specific aims are as follows:
Aim 1: Image and map, via fluorescence in situ hybridization (FISH) and super-resolution microscopy, expression of 6-8 genes we identified at coarser scale as differentially expressed genes (DEGs) for the fungus Postia placenta.
Aim 2: Use laser capture microdissection (LCM) to isolate, physically, hyphal tips of P. placenta at the hyphal front from tips and hyphae in sub-apical regions, for RNAseq and whole-transcriptome comparisons.
Aim 3: Apply microscopy-enabled mass spectrometry (MS) with increasing resolution to overlay metabolite information, including glycoproteomics to address a likely oxidative stress adaptation.
This work is a tight fit in EMSL's Biosystem Dynamics & Design Science Theme, with our focus on metabolic compartmentalization (extracellular) to enable net energy gains from lignocellulose conversion, including the post-transcriptional and -translational pathways enabling the mechanism. Wood-degrading fungi are biotechnology-relevant and recycle Earth's largest pool of aboveground carbon, hitting all three EMSL focus areas (biology, energy, environment). Defining the metabolic mechanisms and the underlying genes involved with brown rot pretreatment and saccharification mechanisms similarly dovetails with DOE, particularly in BER.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2017-10-01
End Date
2019-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Zhang J., D. Hu, G. Orr, and J.S. Schilling. 2019. "Fluorescence In Situ mRNA Hybridization for Gene Expression Detection in a Wood Decay Fungus." International Biodeterioration & Biodegradation 143. PNNL-SA-140382. doi:10.1016/j.ibiod.2019.104731
Zhang J., H.D. Mitchell, L. Markillie, M.J. Gaffrey, G. Orr, and J.S. Schilling. 2019. "Reference genes for accurate normalization of gene expression in wood-decomposing fungi." Fungal Genetics and Biology : FG & B 123. PNNL-SA-142134. doi:10.1016/j.fgb.2018.11.005