Heterologous expression of fungal glycoside hydrolase family 115 alpha-glucuronidases

Authors: Bowman, Michael; Nichols, Nancy; Hector, Ronald

Submitted to: Protein Expression and Purification
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/31/2026
Publication Date: 3/31/2026
Citation: Bowman, M.J., Nichols, N.N., Hector, R.E. 2026. Heterologous expression of fungal glycoside hydrolase family 115 alpha-glucuronidases. Protein Expression and Purification. https://doi.org/10.1016/j.pep.2026.106927.
DOI: https://doi.org/10.1016/j.pep.2026.106927

Interpretive Summary: Turning plant material like corn stalks, grain husks, and other farm leftovers into simple sugars is a critical step in making bio-based fuels and chemicals. Multiple proteins called enzymes are required to break down tough plant fibers into sugars. This enzyme market is worth about $2.5 billion a year worldwide. However, the enzymes currently available can’t fully break down all parts of plant fibers, which means valuable sugars get left behind. This study addressed this challenge by finding and producing four new enzymes that do a better job of breaking down these stubborn fibers. The new enzyme production system can be used by biotech companies and enzyme suppliers to improve their products. The improved enzymes will help get more sugar out of crop residues like corn stover, straw, or agricultural waste fibers. Improved break-down of resistant fiber would benefit ethanol producers by increasing ethanol production and corn oil recovery, while also increasing the nutrient value of co-products sold as animal feed, particularly for pigs and chickens.

Technical Abstract: Deconstruction of plant xylan is challenging due to the structural complexity arising from side groups on the polymer. One of the side groups, glucuronic acid, can be further modified with a methyl group at the 4-hydroxy position, as well as modification by the addition of sugar, acetate, and/or ferulate. This heterogeneity necessitates multiple enzymes capable of recognizing and hydrolyzing each side group type for complete hydrolysis of xylan to xylose. In this study, four fungal alpha-glucuronidase genes were identified and cloned into Coniochaeta ligniaria, an ascomycete isolated from soil. An E. coli-C. ligniaria shuttle vector was constructed with transcription promoter and termination sequences, as well as a secretion signal sequence for secretion of enzymes and a his6-tag sequence for antibody recognition and for convenience in affinity purification. This cloning and expression strategy allowed secretion of heterologous fungal proteins in a microbe well-suited for growth in a biomass environment. alpha-Glucuronidase enzymes were successfully expressed and secreted by Coniochaeta ligniaria grown under four media conditions: defined mineral medium with glucose or acetate as sole carbon source, richer medium containing peptone and yeast extract, and corn stover hydrolysate. The four recombinant glycoside hydrolases were incubated with beech xylan, and alpha-glucuronidase activity for all four enzymes was detected as release of 4-methylglucuronic acid monomers from xylan. Hydrolysis of 4-methylglucuronic acid from xylan polymer is consistent with designation of the enzymes in glycoside hydrolase family 115.