Metagenomic discovery of feruloyl esterases from rumen microflora

Authors: Wong, Dominic; Chan, Victor; LIAO, HANS - Cargill, Incorporated

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/26/2019
Publication Date: 9/12/2019
Citation: Wong, D., Chan, V.J., Liao, H. 2019. Metagenomic discovery of feruloyl esterases from rumen microflora. Applied Microbiology and Biotechnology. 103:8449-8457. https://doi.org/10.1007/s00253-019-10102-y.
DOI: https://doi.org/10.1007/s00253-019-10102-y

Interpretive Summary: Ferulic acid crosslinking is a major cause of recalcitrance of biomass materials. The removal of ferulic acid crosslinks will increase the digestibility of lignocellulose as feedstock for biofuels and bioproducts. This report describes the metagenomic approach to explore the diversity and complexity of genes and enzymes in the unculturable microbial world. Seven novel ferulic acid esterase genes were isolated from the microbial metagenome of a cow’s rumen, cloned, and expressed. The purified enzymes were characterized for their actions on natural plant polysaccharides. The synergistic effect with xylanases which attack the main chain of hemicellulose was found to significantly enhance the release of ferulic acid. This will lead to high-efficiency breakdown rendering the cell wall polysaccharides more accessible for complete degradation.

Technical Abstract: Feruloyl esterases (FAEs) are a key group of enzymes that hydrolyze ferulic acids ester-linked to plant polysaccharides. The cow's rumen is a highly evolved ecosystem of complex microbial microflora capable of converting fibrous substances to energy. From direct cloning of the rumen microbial metagenome, we identified seven active phagemids conferring feruloyl esterase activity. The genomic inserts ranged from 1,633 to 4,143 bp, and the ORFs from 681 to 1,359 bp. BLAST search of the gene sequences reveals similarities to a/b fold hydrolases and esterase/lipase in the GenBank database. The genes were expressed in E. coli, and the enzyme proteins were purified to homogeneous and characterized using model hydroxycinnamic acid esters. The seven FAEs were found to cover type B, C and D in feruloyl esterase classification. That all catalyzed the release of ferulic acid was firmly established using the natural substrates corn fiber and wheat insoluble arabinoxylan. Three of the enzymes were demonstrated to cleave diferulates, and hence the capability to break down crosslinks in the substrates. The wide variation in the sequence, activity, and substrate specificity observed in the FAEs discovered in this study is a confirming evidence that combined actions of a full range of FAE enzymes contribute to the high-efficiency fiber digestion in the rumen microbial ecosystem.