Lignin modifying enzymes from bacteria: discovery and engineering
Monday, April 28, 2014
Exhibit/Poster Hall, lower level (Hilton Clearwater Beach)
Rahul Singh1, Cameron R. Strachan2, Kateryna Levdokymenko3, Karen Budwill4, Steven J. Hallam3 and Lindsay D. Eltis3, (1)Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada, (2)MetaMixis, Vancouver, BC, Canada, (3)Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada, (4)Environment and Carbon Management Division, Alberta Innovates-Technology Futures, Edmonton, AB, Canada
Lignin, the most abundant aromatic biopolymer on earth, represents a potential source of high value aryl compounds. Efficient conversion of lignin into bio-fuels and value-added products is also fundamental to sustainable bio-refining. Although efforts to establish lignin-depolymerizing biocatalyst have focused on fungal enzymes, recent advances in genomics and other high-throughput approaches are revealing the lignolytic potential of bacteria. We identified Rhodococcus jostii RHA1 as a lignin-degrading actinomycete and characterized DypB – a dye-decolorizing peroxidase – as the first bacterial lignolytic enzyme capable of oxidizing Mn2+. We engineered the heme binding pocket of DypB and produced a variant, DypBN246A, with improved Mn2+-oxidizing activity (80- and 15-fold, respectively, higher kcat and kcat/Km values). DypBN246A catalyzed the manganese-dependent transformation of hard wood kraft lignin and produced 2,6-dimethoxybenzoquinone and syringaldehyde as major degradation products.  We also devised a high-throughput screening method to identify lignin-modifying enzymes from metagenomic libraries based on a co-culture screening method that utilizes a biosensor responsive to lignin-derived aromatic compounds. Using this approach we identified 24 metagenomic fosmid clones producing compounds such as vanillin, 1,4-dihydroxy-2,6-dimethoxybenzene, and syringaldehyde. Mutagenesis and sequence analyses of these clones revealed the presence of numerous genes encoding enzymes with lignolytic potential, such as multi-copper oxidases and aryl alcohol oxidases. The ongoing work aims to characterize bacterial lignin-modifying enzymes, develope tunable scaffolds from metagenomic clones and ultimately engineer improved biorefining strains capable of efficient lignin transformation.