Database Mining for Novel Bacterial β-Etherases, Glutathione-Dependent Lignin-Degrading Enzymes

Abstract

Lignin is the most abundant aromatic polymer in nature and a promising renewable source for the provision of aromatic platform chemicals and biofuels. β-Etherases are enzymes with a promising potential for application in lignin depolymerization due to their selectivity in the cleavage of β-O-4 aryl ether bonds. However, only a very limited number of these enzymes have been described and characterized so far. Using peptide pattern recognition (PPR) as well as phylogenetic analyses, 96 putatively novel β-etherases have been identified, some even originating from bacteria outside the order Sphingomonadales. A set of 13 diverse enzymes was selected for biochemical characterization, and β-etherase activity was confirmed for all of them. Some enzymes displayed up to 3-fold higher activity than previously known β-etherases. Moreover, conserved sequence motifs specific for either LigE- or LigF-type enzymes were deduced from multiple-sequence alignments and the PPR-derived peptides. In combination with structural information available for the β-etherases LigE and LigF, insight into the potential structural and/or functional role of conserved residues within these sequence motifs is provided. Phylogenetic analyses further suggest the presence of additional bacterial enzymes with potential β-etherase activity outside the classical LigE- and LigF-type enzymes as well as the recently described heterodimeric β-etherases. IMPORTANCE The use of biomass as a renewable source and replacement for crude oil for the provision of chemicals and fuels is of major importance for current and future societies. Lignin, the most abundant aromatic polymer in nature, holds promise as a renewable starting material for the generation of required aromatic structures. However, a controlled and selective lignin depolymerization to yield desired aromatic structures is a very challenging task. In this regard, bacterial β-etherases are especially interesting, as they are able to cleave the most abundant bond type in lignin with high selectivity. With this study, we significantly expanded the toolbox of available β-etherases for application in lignin depolymerization and discovered more active as well as diverse enzymes than previously known. Moreover, the identification of further β-etherases by sequence database mining in the future will be facilitated considerably through our deduced etherase-specific sequence motifs.

How Do Shipworms Eat Wood? Screening Shipworm Gill Symbiont Genomes for Lignin-Modifying Enzymes

“…From these four databases, terms specifically associated with the eight proteins of interest were found and organized under a single Function Set for downstream use. Since β-etherases are part of the glutathione S-transferase (GST) family, a large protein family associated with a wide breath of functions, we could only find general functional IDs relating to GSTs (Allocati et al, 2009;Voß et al, 2020). We were also unable to find functional terms relating to lignin peroxidases or manganese peroxidases.…”

“…Since β-etherases are part of the glutathione S-transferase (GST) family, a large protein family associated with a wide breath of functions, we could only find general functional IDs relating to GSTs ( Allocati et al, 2009 ; Voß et al, 2020 ). We were also unable to find functional terms relating to lignin peroxidases or manganese peroxidases.…”

“…A sequence with the highest percent identity match (64.32% identity; GenBank Accession: PZR21549.1 ) was one belonging to a member of the Enterobacteriaceae family, and this sequence as well as one from a member of the Alteromonadaceae family (61.62% identity; GenBank Accession: MAO07630.1 ) and one from a member of the Moraxellaceae family (60.48% identity; GenBank Accession: RZA00329.1 ) were used for subsequent analyses. LigE (GenBank Accession: WP_044331491.1 ) and LigF (GenBank Accession: OGT78215.1 ) β-etherase sequences from a Sphingomonas species and a Gammaproteobacterium were, respectively, acquired from Voß et al (2020) . A LigP (GenBank Accession: BAK67935.1 ) sequence and two additional β-etherases from species of Acinetobacter (GenBank Accession: ATY43794.1 and VAX44176.1 ) were obtained from the NCBI protein database.…”

“…Only the catalase-peroxidases had average percent identities greater than 50% (Supplementary Table 4). We blasted our genomes against five β-etherase reference sequences derived from Voß et al (2020) and NCBI GenBank. The LigF β-etherase sequence (OGT78215.1) produced blastp hits against at least one gene in each genome, with the exception of T. waterburyi BS02 (Supplementary Table 6).…”

“…In some bacteria, these proteins can be secreted from the cell to interact with dioxygen in the presence of wood to produce a reactive species and break lignin bonds ( Rashid et al, 2015 ). Various glutathione-dependent β-etherases (denoted as LigE, LigF, and LigP) have also been shown to modify lignin and its partially degraded intermediates by cleaving the abundant β- O -4 aryl ether bonds present throughout lignin’s complex structure ( Allocati et al, 2009 ; Scully et al, 2013 ; Picart et al, 2015 ; de Gonzalo et al, 2016 ; Lee et al, 2019 ; Voß et al, 2020 ). Classes of dioxygenases (intradiol, extradiol, and LigAB) have also been documented to attack a variety of aromatic substances, including lignin and its derivatives ( Sugimoto et al, 1999 ; Sonoki et al, 2009 ; Barry and Taylor, 2013 ; Bianchetti et al, 2013 ; Scully et al, 2013 ; de Gonzalo et al, 2016 ; Burroughs et al, 2019 ; Lee et al, 2019 ).…”