Discovery of novel lignin oxidizing enzymes from Sphingobacterium sp.T2

Bacterial lignin-degrading strain Sphingobacterium sp. T2 is reported as most active strain among 12 isolates and proteomic analysis of extracellular fractions gave hits for two superoxide dismutase enzymes. Two novel lignin-oxidising enzymes from Sphingobacterium sp. T2 were identified. Experimental and bioinformatic evidence revealed that these enzymes are extracellular superoxide dismutases, namely SOD1 and SOD2. Both enzyme genes have been cloned and overexpressed in Escherichia coli. The metal specificity of both enzymes was investigated using ICP-OES, UV-vis spectra and superoxide dismutation activity of different metal-containing enzyme preparations, the results indicate that both enzymes (SOD1 and SOD2) are manganese-containing SOD. MnSOD1 and MnSOD2 from Sphingobacterium sp. T2 (SpMnSODs) exhibit time-dependent lignin-oxidising ability, they can depolymerise wheat straw organosolv lignin, modify Kraft lignin, and lignocellulose materials (pine and miscanthus). Analysis of reaction components of SpMnSOD enzymes with organosolv lignin by HPLC, LC/MS and GC/MS show that SOD1 and SOD2 generate 10 new products. These metabolites were identified by comparison of their retention times and mass fragmentation pattern produced by LC/MS and GC/MS with their authentic analogue. The mechanism of reaction and reactive species generated by SpMnSODs were elucidated from their identified reaction products. Results indicate that SpMnSOD enzymes produce highly reactive species (hydroxyl radical) and catalyse several types of reactions which include: Ca-Cß cleavage, arylC-Ca Cleavage, -OCH3 group replacement with -OH group, hydroxylation of aromatic rings, Ipso-substitution and decarboxylation. The X-ray crystal structure of MnSOD1 was determined to 1.35 Å resolution, showing that MnSOD1 has a typical homodimer with a Mn(II) ion in each active site ligated by three His, one Asp, and a water/hydroxide forming trigonal bipyramidal geometry. The reaction of SpMnSOD enzymes with lignin model compounds confirms that these enzymes can catalyse Ca-Cß cleavage, arylC-Ca Cleavage, ipso-substitution, decarboxylation, demethoxylation, hydroxylation of aromatic rings and fragmentation of propenoic acid side chains which in turn are evidence of generation of highly oxidising species (hydroxyl radical)

Enhanced biocatalytic degradation of lignin using combinations of lignin-degrading enzymes and accessory enzymes

Methods for screening combinations of lignin-degrading enzymes and accessory enzymes for product release from polymeric lignin have been developed, using two colorimetric assays that can be applied in microtiter plate format. A set of 3 bacterial DyP-type peroxidase enzymes from Pseudomonas fluorescens, Comamonas testosteroni and Agrobacterium sp., two bacterial multi-copper oxidase enzymes CueO from Ochrobactrum sp. and CopA from Pseudomonas putida, and Sphingobacterium sp. T2 manganese superoxide dismutase have been tested in combination with one LigE β-etherase enzyme from Agrobacterium sp., two dihydrolipoamide dehydrogenase enzymes from Sphingobacterium sp. T2, Burkholderia cenocepacia peroxiredoxin, and Desulfitobacterium hafniense arylsulfotransferase. Combinations of Agrobacterium LigE with DyP-type peroxidases gave 4–10 enhancement in low molecular weight product release from technical lignins, and enhancements in product release were observed for all lignins tested, using different accessory enzymes. Analysis of products formed by reverse phase HPLC verified increases in concentrations of specific low molecular weight products

Delignification and enhanced gas release from soil containing Lignocellulose by treatment with bacterial lignin degraders

Aims The aim of the study was to isolate bacterial lignin-degrading bacteria from municipal solid waste soil, and to investigate whether they could be used to delignify lignocellulose-containing soil, and enhance methane release. Methods and Results A set of 20 bacterial lignin degraders, including 11 new isolates from municipal solid waste soil, were tested for delignification and phenol release in soil containing 1% pine lignocellulose. A group of 7 strains were then tested for enhancement of gas release from soil containing 1% lignocellulose in small-scale column tests. Using an aerobic pre-treatment, aerobic strains such as Pseudomonas putida showed enhanced gas release from the treated sample, but four bacterial isolates showed 5-10 fold enhancement in gas release in an in situ experiment under microanaerobic conditions: Agrobacterium sp., Lysinibacillus sphaericus, Comamonas testosteroni, and Enterobacter sp.. Conclusions The results show that facultative anaerobic bacterial lignin degraders found in landfill soil can be used for in situ delignification and enhanced gas release in soil containing lignocellulose. Significance & impact of the study The study demonstrates the feasibility of using an in situ bacterial treatment to enhance gas release and resource recovery from landfill soil containing lignocellulosic waste

Characterisation of thiamine diphosphate-dependent 4-hydroxybenzoylformate decarboxylase enzymes from Rhodococcus jostii RHA1 and Pseudomonas fluorescens Pf-5 involved in degradation of aryl-C2 lignin degradation fragments

A thiamine diphosphate-dependent enzyme annotated as a benzoylformate decarboxylase is encoded in gene cluster ro02984-ro02986 in Rhodococcus jostii RHA1 previously shown to generate vanillin and 4-hydroxybenzaldehyde from lignin oxidation, and a closely related gene cluster is also found in the genome of Pseudomonas fluorescens Pf-5. Two hypotheses for possible pathways involving a thiamine diphosphate-dependent cleavage, either C-C cleavage of a ketol or diketone aryl C3 substrate, or decarboxylation of an aryl C2 substrate, were investigated by expression and purification of the recombinant enzymes, and expression of dehydrogenase and oxidase enzymes also found in the gene clusters. The ThDP-dependent enzymes showed no activity for cleavage of aryl C3 ketol or diketone substrates, but showed activity for decarboxylation of benzoylformate and 4-hydroxybenzoylformate. A flavin-dependent oxidase encoded by gene ro02984 was found to oxidise either mandelic acid or phenylglyoxal. The crystal structure of the P. fluorescens decarboxylase enzyme was determined at 1.69 Å resolution, showing similarity to known benzoylformate decarboxylase enzymes. The P. fluorescens decarboxylase enzyme showed enhanced carboligase activity between vanillin and acetaldehyde, rationalised by the presence of alanine vs serine at residue 73 in the enzyme active site, which was investigated further by site-directed mutagenesis of this residue. A hypothesis for a pathway for degradation of aryl-C2 fragments arising from oxidative cleavage of phenylcoumaran and diarylpropane structures in lignin is proposed

Sphingobacterium sp. T2 manganese superoxide dismutase catalyses the oxidative demethylation of polymeric lignin via generation of hydroxyl radical

Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes which exhibit unprecedented activity for lignin oxidation but via an unknown mechanism. Enzymatic treatment of lignin model compounds gave products whose structures were indicative of aryl–Cα oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol oxidation. 18O labeling studies on the SpMnSOD-catalyzed oxidation of lignin model compound guiaiacylglycerol-β-guaiacyl ether indicated that the an oxygen atom inserted by the enzyme is derived from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative 31P NMR spectroscopy demonstrated 20–40% increases in phenolic and aliphatic OH content, consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the release of 4.1 molar equivalents of hydroxyl radical by SpMnSOD1. Four amino acid replacements in SpMnSOD1 were investigated, and A31H or Y27H site-directed mutant enzymes were found to show no lignin demethylation activity according to 31P NMR analysis. Structure determination of the A31H and Y27H mutant enzymes reveals the repositioning of an N-terminal protein loop, leading to widening of a solvent channel at the dimer interface, which would provide increased solvent access to the Mn center for hydroxyl radical generation

Bacterial enzymes for lignin depolymerisation : new biocatalysts for generation of renewable chemicals from biomass

The conversion of polymeric lignin from plant biomass into renewable chemicals is an important unsolved problem in the biorefinery concept. This article summarises recent developments in the discovery of bacterial enzymes for lignin degradation, our current understanding of their molecular mechanism of action, and their use to convert lignin or lignocellulose into aromatic chemicals. The review also discusses the recent developments in screening of metagenomic libraries for new biocatalysts, and the use of protein engineering to enhance lignin degradation activity

Investigation of the chemocatalytic and biocatalytic valorization of a range of different lignin preparations: The importance of β-O-4 content

A set of seven different lignin preparations was generated from a range of organosolv (acidic, alkaline, ammonia-treated, and dioxane-based), ionic liquid, autohydrolysis, and Kraft pretreatments of lignocelluloses. Each lignin was characterized by 2D HSQC NMR spectroscopy, showing significant variability in the β-O-4 content of the different lignin samples. Each lignin was then valorised using three biocatalytic methods (microbial biotransformation with Rhodococcus jostii RHA045, treatment with Pseudomonas fluorescens Dyp1B or Sphingobacterium sp. T2 manganese superoxide dismutase) and two chemocatalytic methods (catalytic hydrogenation using Pt/alumina catalyst, DDQ benzylic oxidation/Zn reduction). Highest product yields for DDQ/Zn valorization were observed from poplar ammonia percolation-organosolv lignin, which had the highest β-O-4 content of the investigated lignins and also gave the highest yield of syringaldehyde (243 mg L -1 ) when using R. jostii RHA045 and the most enzymatic products using P. fluorescens Dyp1B. The highest product yield from the Pt/alumina hydrogenation was observed using oak dioxasolv lignin, which also had a high β-O-4 content. In general, highest product yields for both chemocatalytic and biocatalytic valorization methods were obtained from preparations that showed highest β-O-4 content, while variable yields were obtained with preparations containing intermediate β-O-4 content, and little or no product was obtained with preparations containing low β-O-4 content

Overexpression of endogenous multi‐copper oxidases mcoA and mcoC in Rhodococcus jostii RHA1 enhances lignin bioconversion to 2,4‐pyridine‐dicarboxylic acid

To improve the titre of lignin-derived pyridine-dicarboxylic acid (PDCA) products in engineered Rhodococcus jostii RHA1 strains, plasmid-based overexpression of seven endogenous and exogenous lignin-degrading genes was tested. Overexpression of endogenous multi-copper oxidases mcoA, mcoB, and mcoC was found to enhance 2,4-PDCA production by 2.5-, 1.4-, and 3.5-fold, respectively, while overexpression of dye-decolorizing peroxidase dypB was found to enhance titre by 1.4-fold, and overexpression of Streptomyces viridosporus laccase enhanced titre by 1.3-fold. The genomic context of the R. jostii mcoA gene suggests involvement in 4-hydroxybenzoate utilization, which was consistent with enhanced whole cell biotransformation of 4-hydroxybenzoate by R. jostii pTipQC2-mcoA. These data support the role of multi-copper oxidases in bacterial lignin degradation, and provide an opportunity to enhance titres of lignin-derived bioproducts

Identification of manganese superoxide dismutase from Sphingobacterium sp. T2 as a novel bacterial enzyme for lignin oxidation

The valorisation of aromatic heteropolymer lignin is an important unsolved problem in the development of a biomass-based biorefinery, for which novel high-activity biocatalysts are needed. Sequencing of the genomic DNA of lignin-degrading bacterial strain Sphingobacterium sp. T2 revealed no matches to known lignin-degrading genes. Proteomic matches for two manganese superoxide dismutase proteins were found in partially purified extracellular fractions. Recombinant MnSOD1 and MnSOD2 were both found to show high activity for oxidation of Organosolv and Kraft lignin, and lignin model compounds, generating multiple oxidation products. Structure determination revealed that the products result from aryl-Cα and Cα-Cβ bond oxidative cleavage and O-demethylation. The crystal structure of MnSOD1 was determined to 1.35 Å resolution, revealing a typical MnSOD homodimer harbouring a 5-coordinate trigonal bipyramidal Mn(II) centre ligated by three His, one Asp and a water/hydroxide in each active site. We propose that the lignin oxidation reactivity of these enzymes is due to the production of hydroxyl radical, a highly reactive oxidant. This is the first demonstration that MnSOD is a microbial lignin-oxidising enzyme

Elucidation of microbial lignin degradation pathways using synthetic isotope-labelled lignin

Pathways by which the biopolymer lignin is broken down by soil microbes could be used to engineer new biocatalytic routes from lignin to renewable chemicals, but are currently not fully understood. In order to probe these pathways, we have prepared synthetic lignins containing 13C at the sidechain β-carbon. Feeding of [β-13C]-labelled DHP lignin to Rhodococcus jostii RHA1 has led to the incorporation of 13C label into metabolites oxalic acid, 4-hydroxyphenylacetic acid, and 4-hydroxy-3-methoxyphenylacetic acid, confirming that they are derived from lignin breakdown. We have identified a glycolate oxidase enzyme in Rhodococcus jostii RHA1 which is able to oxidise glycolaldehyde via glycolic acid to oxalic acid, thereby identifying a pathway for the formation of oxalic acid. R. jostii glycolate oxidase also catalyses the conversion of 4-hydroxyphenylacetic acid to 4-hydroxybenzoylformic acid, identifying another possible pathway to 4-hydroxybenzoylformic acid. Formation of labelled oxalic acid was also observed from [β-13C]-polyferulic acid, which provides experimental evidence in favour of a radical mechanism for α,β-bond cleavage of β-aryl ether units