Discovery of lignin-transforming bacteria and enzymes in thermophilic environments using stable isotope probing

Characterizing microorganisms and enzymes involved in lignin biodegradation in thermal ecosystems can identify thermostable biocatalysts. We integrated stable isotope probing (SIP), genome-resolved metagenomics, and enzyme characterization to investigate the degradation of high-molecular weight, 13C-ring-labeled synthetic lignin by microbial communities from moderately thermophilic hot spring sediment (52 °C) and a woody “hog fuel” pile (53 and 62 °C zones). 13C-Lignin degradation was monitored using IR-GCMS of 13CO2, and isotopic enrichment of DNA was measured with UHLPC-MS/MS. Assembly of 42 metagenomic libraries (72 Gb) yielded 344 contig bins, from which 125 draft genomes were produced. Fourteen genomes were significantly enriched with 13C from lignin, including genomes of Actinomycetes (Thermoleophilaceae, Solirubrobacteraceae, Rubrobacter sp.), Firmicutes (Kyrpidia sp., Alicyclobacillus sp.) and Gammaproteobacteria (Steroidobacteraceae). We employed multiple approaches to screen genomes for genes encoding putative ligninases and pathways for aromatic compound degradation. Our analysis identified several novel laccase-like multi-copper oxidase (LMCO) genes in 13C-enriched genomes. One of these LMCOs was heterologously expressed and shown to oxidize lignin model compounds and minimally transformed lignin. This study elucidated bacterial lignin depolymerization and mineralization in thermal ecosystems, establishing new possibilities for the efficient valorization of lignin at elevated temperature.

Roughly 1:1 molar ratio of vanillin and malonic acid (1:1.2 if using 13 C-labeled vanillin), amounting to 2.5 g vanillin and 2.0 g malonic acid, were mixed in the presence of catalytic amounts of piperidine and analine (6 drops) in pyridine in a 2 neck round bottom flask (~25 mLs) and "refluxed" at 55°C for 16 h.
After 16 h, disappearance of vanillin was verified by thin-layer chromatography (TLC) in comparison to a standard to ensure reaction completion using 1:1 ethyl acetate:petroleum ether as the solvent. Upon completion, pyridine was removed using a rotary evaporator (RotoVap). Roughly 100 mLs of ethyl acetate was added to the mixture, which was then washed 3x with 1 M HCL. TLC was again used to ensure all product was located in the organic layer. Product was re-extracted with ethyl acetate if necessary. The solution was dried with MgSO4 and the organic layer was recovered and the solvent was removed by RotoVap.

Esterification of reaction
Ferulic acid was dissolved in ~10-15 mL of MeOH. Roughly 50 ul of concentrated HCl was added, and the reaction was allowed to reflux for 4 hours to overnight. Progress was again monitored using TLC. Once the reaction reached completion, MeOH was removed using the RotoVap and the remaining product was dissolved in ethyl acetate, washed with H2O, then wash again with NaHCO3. The remaining product was dried using MgSO4 then the ethyl acetate was removed by RotoVap.
The compound was dry loaded onto a silica column (dissolve in MeOH and add silica to round bottom flask and rotovap out MeOH) for additional purification. The column was run using 1:1 ethyl acetate:petroleum ether as a solvent. The column was washed using a 3:2 ethyl acetate:petroleum ether, and pure ethyl acetate was used to elute the compound. The compound was verified using HSQC NMR and mass spectrometry.

Reduction of methyl ester
Reduction of the methyl ester to coniferyl alcohol is light sensitive and should be performed in the dark. Failure to do so results in the dimerization of coniferyl alcohol. A 1:1 molar ratio of LiAlH4:compound was used for the reduction reaction. The reaction used a two neck round bottom flask with a large stir bar. The reaction was performed under positive N2 pressure. Roughly 60 mL of freshly distilled tetrahydrofuran (THF) was slowly added to the LiAlH4 while stirring in an ice bath. Next, the methyl ester was dissolved in ~14 mL of freshly distilled THF and added dropwise to the reaction. The reaction was kept on ice until all methyl ester was added. The add funnel was rinsed with ~10 mL of THF and added to the reaction. Following addition of all methyl ester, the flask was removed from the ice bath and stirring continued for ~2 hours. The reaction was monitored by TLC until the reaction was complete. Upon completion, the reaction was quenched by adding 12 mL ethyl acetate dropwise. Ethyl acetate must be added slowly at first, as the LiAlH4 remains reactive. Next, 70 mL of 2 M HCl was added. Coniferyl alcohol was extracted 3x using 50 mL ethyl acetate, and washed once with 100 mL H2O. TLC was used to confirm reaction completion and purity. Extraction with ethyl acetate was repeated if required. The ethyl acetate mixture was dried with MgSO4, and solvent was removed by RotoVap.
A silica column was used to clean-up the coniferyl alcohol. It was first dissolved in dichloromethane (DCM) prior to loading, and eluted using 3:2 ethyl acetate:petroleum ether. Fractions were once again analyzed with TLC to ensure completeness and purity of the coniferyl alcohol product.

DHP synthesis
Coniferyl alcohol [1.7 g (9.45 mmol)] and 80 mg of peroxidase (HRP type II, Sigma-Aldrich, St. Louis, U.S.A.) were dissolved in 400 ml of degassed sodium phosphate buffer (100 mM) at pH 7.5. This solution and 9.45 mmol of H2O2 in 400 ml of degassed buffer were added simultaneously and separately to 200 ml of well-stirred degassed buffer containing 20 mg of peroxidase and 30 mg (9.45 mmol) of vanillyl alcohol, over a 16 h period with a syringe pump (~ 0.4 ml/min). Coniferyl alcohol can be dissolved in a small volume (10-20 ml) of organic solvent (Acetone or DMSO) prior to addition to 400 ml buffer. All solutions were kept under N2 and the reaction vessel was kept in dark (covered with the aluminum foil). After final addition, the mixture was stirred an additional 10 h. The insoluble polymer was separated by centrifugation at ~3000 x g for 30 minutes at 4°C and washed with deionized water (100 mLs). The washed polymer was freeze dried and stored at -20 °C.

DNA extractions and fractionation
DNA concentration was measured using the Qubit high-sensitivity DNA assay (Thermo Fisher Scientific, Waltham, U.S.A.). DNA was stored at -80 o C for up to five days prior to cesium chloride density gradient centrifugation and fractionation, according to published protocols [13,14]. Briefly, 1 ml gradient buffer (0.1 M Tris, 0.1 M KCl and 1 mM EDTA) containing 5µg DNA was mixed with 4.9 ml CsCl (1.878 g ml -1 ) to a final density of 1.725 g ml −1 in 5.1 ml centrifuge tubes (Beckman Coulter, Brea, U.S.A). Density gradients were established over 42 hr at 44,100 rpm and 20 o C in an Optima L-90K ultracentrifuge (Beckman Coulter) with a Vti 65.2 vertical rotor (Beckman Coulter) in a vacuum, and without braking. Following centrifugation, a 23-gauge needle attached with tubing to a 60-ml syringe in an RE syringe pump (Razel Scientific, Saint Albans, U.S.A.) was used to pump water into the top of the tube at a rate of 0.5 ml min -1 . Twelve 0.42-ml fractions were collected dropwise in sterile 1.7-ml microtubes. The refractivity index (RI) of 20 μl of each fraction was immediately measured by an r 2 mini handheld refractometer (Reichert Technologies, Depew, U.S.A.), and density (D) in calculated using the equation: (1) D = (RI × 10.927) − 13.593 DNA in each fraction was immediately precipitated using 1 μl glycogen (Sigma-Aldrich) and 1.0 ml PEG6000 (Sigma-Aldrich), which was mixed, precipitated for 2 hr at room temperature, and centrifuged at 13,000 g at room temperature for 30 min. Supernatant was removed and the pellet was washed with 0.5 ml 70% ethanol. Following re-centrifugation as above, the ethanol was aspirated, the pellet dried for ~15 min and suspended in sterile 30 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The level of 13 C enrichment in each purified DNA fraction was quantified using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Details are provided in [15] and in supplementary methods. This method allowed us to target sequencing of DNA fractions with higher confidence that originated from cells "enriched" by incorporation of 13 C-DHP.