Lignin is a heterogeneous aromatic polymer found as 10-35% of lignocellulose, found in plant cell walls. The bio-conversion of plant lignocellulose to glucose is an important part of second generation biofuel production, but the resistance of lignin to breakdown is a major obstacle in this process, hence there is considerable interest in the microbial breakdown of lignin. White-rot fungi are known to break down lignin with the aid of extracellular peroxidase and laccase enzymes. There are also reports of bacteria that can degrade lignin, and recent work indicates that bacterial lignin breakdown may be more significant than previously thought. The review will discuss the enzymes for lignin breakdown in fungi and bacteria, and the catabolic pathways for breakdown of the β-aryl ether, biphenyl and other components of lignin in bacteria and fungi. The review will also discuss small molecule phenolic breakdown products from lignin that have been identified from lignin-degrading microbes, and includes a bioinformatic analysis of the occurrence of known lignin-degradation pathways in Gram-positive and Gram-negative bacteria.

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Rhodococcus jostii RHA1, a polychlorinated biphenyl-degrading soil bacterium whose genome has been sequenced, shows lignin degrading activity in two recently developed spectrophotometric assays. Bioinformatic analysis reveals two unannotated peroxidase genes present in the genome of R. jostii RHA1 with sequence similarity to open reading frames in other lignin-degrading microbes. They are members of the Dyp peroxidase family and were annotated as DypA and DypB, on the basis of bioinformatic analysis. Assay of gene deletion mutants using a colorimetric lignin degradation assay reveals that a ΔdypB mutant shows greatly reduced lignin degradation activity, consistent with a role in lignin breakdown. Recombinant DypB protein shows activity in the colorimetric assay and shows Michaelis-Menten kinetic behavior using Kraft lignin as a substrate. DypB is activated by Mn(2+) by 5-23-fold using a range of assay substrates, and breakdown of wheat straw lignocellulose by recombinant DypB is observed over 24-48 h in the presence of 1 mM MnCl(2). Incubation of recombinant DypB with a β-aryl ether lignin model compound shows time-dependent turnover, giving vanillin as a product, indicating that C(α)-C(β) bond cleavage has taken place. This reaction is inhibited by addition of diaphorase, consistent with a radical mechanism for C-C bond cleavage. Stopped-flow kinetic analysis of the DypB-catalyzed reaction shows reaction between the intermediate compound I (397 nm) and either Mn(II) (k(obs) = 2.35 s(-1)) or the β-aryl ether (k(obs) = 3.10 s(-1)), in the latter case also showing a transient at 417 nm, consistent with a compound II intermediate. These results indicate that DypB has a significant role in lignin degradation in R. jostii RHA1, is able to oxidize both polymeric lignin and a lignin model compound, and appears to have both Mn(II) and lignin oxidation sites. This is the first detailed characterization of a recombinant bacterial lignin peroxidase.

The aromatic polymer lignin represents a possible renewable source of aromatic chemicals, if biocatalytic routes for lignin breakdown can be developed. The availability of a genome sequence for Rhodococcus jostii RHA1, a bacterium that breaks down lignin, has allowed the application of a targeted pathway engineering strategy to lignin breakdown to produce vanillin, a valuable food/flavor chemical. A gene deletion strain of R. jostii RHA1 in which the vanillin dehydrogenase gene had been deleted, when grown on minimal medium containing 2.5% wheat straw lignocellulose and 0.05% glucose, was found to accumulate vanillin with yields of up to 96 mg/L after 144 h, together with smaller amounts of ferulic acid and 4-hydroxybenzaldehyde.

Aims: To develop a method to detect bacteria from environmental samples that are able to metabolize lignin. Methods and Results: A previously developed UV-vis assay method for lignin degradation activity has been developed for use as a spray assay on agar plates. Nine mesophilic strains were isolated using this method from woodland soil incubated in enrichment cultures containing wheat straw lignocellulose: four Microbacterium isolates, two Micrococcus isolates, Rhodococcus erythropolis (all Actinobacteria) and two Ochrobactrum isolates (Alphaproteobacteria). Three thermotolerant isolates were isolated from the same screening method applied at 45°C to samples of composted wheat straw from solid-state fermentation: Thermobifida fusca and two isolates related to uncharacterized species of Rhizobiales and Sphingobacterium (Bacteroidetes), the latter strain showing tenfold higher lignin degradation activity than other isolates. The isolated strains were able to depolymerize samples of size-fractionated high molecular weight and low molecular weight Kraft lignin, and produced low molecular weight metabolites oxalic acid and protocatechuic acid from incubations containing wheat straw lignocellulose. Conclusions: A new method for the isolation of bacteria able to metabolize lignin has been developed, which has been used to identify 12 bacterial isolates from environmental sources. The majority of isolates cluster into the Actinobacteria and the Alphaproteobacteria. Significance and Impact of the Study: Lignin-degrading bacterial strains could be used to convert lignin-containing feedstocks into renewable chemicals and to identify new bacterial lignin-degrading enzymes.

Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called random drift mutagenesis (RNDM). Mutant populations are screened and all functional mutants are collected and put forward into the next round of mutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a beta-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant beta-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (k(cat)/K(M)) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (k(cat)) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (K(i)) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site.

Flow cytometry (FCM) is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It uses the principles of light scattering, light excitation and the emission from fluorescent molecules to generate specific multiparameter data from particles and cells. The cells are hydrodynamically focussed in a sheath solution before being intercepted by a focused light source provided by a laser. FCM has been used primarily in medical applications but is being used increasingly for the examination of individual cells from environmental samples. It has found uses in the isolation of both culturable and hitherto non-culturable bacteria present infrequently in environmental samples using appropriate growth conditions. FCM lends itself to high-throughput applications in directed evolution for the analysis of single cells or cell populations carrying mutant genes. It is also suitable for encapsulation studies where individual bacteria are compartmentalised with substrate in water-in-oil-in-water emulsions or with individual genes in transcriptional/translational mixtures for the production of mutant enzymes. The sensitivity of the technique has allowed the examination of gene optimisation by a procedure known as random or neutral drift where screening and selection is based on the retention of some predetermined level of activity through multiple rounds of mutagenesis.

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