Bacterial catabolism of acetovanillone, a lignin-derived compound

Significance Upgrading lignin, an underutilized component of biomass, is essential for sustainable biorefining. Biocatalysis has considerable potential for upgrading lignin, but our lack of knowledge of relevant enzymes and pathways has limited its application. Herein, we describe a microbial pathway that catabolizes acetovanillone, a major component of several industrial lignin streams. This pathway is unusual in that it involves phosphorylation and carboxylation before conversion to the intermediate, vanillate, which is degraded via the β-ketoadipate pathway. Importantly, the hydroxyphenylethanone catabolic pathway enables bacterial growth on softwood lignin pretreated by oxidative catalytic fractionation. Overall, these insights greatly facilitate the engineering of bacteria to biocatalytically upgrade lignin.

Substrate depletion was monitored using an Agilent Technologies (Santa Clara, U.S.A.) 6890N gas chromatograph equipped with a 30-m Agilent 190915-433 capillary column and a 5973 mass-selective detector (GC/MS). 300 μl of culture was spiked with 3chlorobenzoic acid as an internal standard, acidified with 1% acetic acid and then extracted 1:1 (v/v) with ethyl acetate and dried under nitrogen. Samples were then derivatized with equal volumes of pyridine and trimethylsilyl (TMS).
Transcriptomic analyses -GD02 was grown in triplicate on M9+minerals with 1 mM organic substrate (AV, HAP, AP, or citrate) with shaking at 200 rpm at 37 °C to mid-log phase. Extracted RNA was run on a 1% (w/v) agarose gel and its concentration was determined by Qubit (Thermo-Fisher). Ribodepletion, library preparation (Nextera) and sequencing (NextSeq, 2x150) were performed by the Sequencing and Bioinformatics Consortium at The University of British Columbia. Transcripts were quantified using Salmon 0.8.1. Differential expression was analyzed using DeSeq2 1.18.1 in R 3.4.4.
DNA manipulation -DNA was propagated, purified and manipulated using standard protocols 1 . E. coli and RHA1 were transformed with DNA by electroporation using a MicroPulser with GenePulser cuvettes (Bio-Rad). The expression vector pTip-hpeCBA and pTip-hpeC were constructed by amplifying hpeCBA and hpeC as single fragments from GD02 genomic DNA using hpeCBA-For and hpeCBA-Rev for hpeCBA and hpeC-For and hpeC-Rev for hpeC (Table S4). The resulting amplicons were inserted into NdeI/HindIII-linearized pTipQC2 using T4 ligase. The pET-hpeD vector was constructed by amplifying hpeD using hpeD-For and hpeD-Rev (Table S4). The resulting amplicon was cloned into pET15b that had been linearized using NdeI/HindIII using Gibson Assembly. The nucleotide sequences of the constructs were verified (GENEWIZ). Water for buffers was purified using a Barnstead Nanopure Diamond TM system to a resistivity of 18 MΩ.
HpeH, HpeI and HpeD production and purification -HpeH, HpeI and HpeD were produced heterologously as N-terminal polyHis-tagged (Ht-) proteins using E. coli BL-21 λ (DE3) containing pET-hpeH, pET-hpeI, or pET-hpeD. Freshly transformed cells were grown with shaking at 200 rpm at 37 °C in LB supplemented with 50 mg L -1 of ampicillin, until the culture reached an OD600 of ~0.6. Expression was induced with 0.5 mM isopropyl β-D-thiogalactopyranoside, and the cells were incubated at 30 °C for an additional 16 h. Cells from 1 L of culture were pelleted by centrifugation at 4 °C and resuspended in 20 mL of lysis buffer containing 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole (omitted for HpeH and HpeI), pH 8.0, 2 tablets of proteinase inhibitor (cOmplete™, Mini) and DNAseI (2 μg mL -1 ). Cells were lysed at 4 °C using an EmulsiFlex-C5 homogenizer (Avestin). Cellular debris was removed by centrifugation and the soluble portion was filtered (0.45 μm). Proteins were purified from the cell extract using immobilized metal affinity chromatography (Ni-NTA, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. The fractions containing HpeH, HpeI, or HpeD, as judged by SDS-PAGE, were pooled and dialyzed overnight against 50 mM sodium phosphate, 150 mM NaCl, pH 8.0. Protein preparations were concentrated, flash frozen in liquid N2, and stored at -80 °C until further use.
HpeCBA production and purification -RHA1 freshly transformed with pTip-hpeCBA was grown in LB with shaking at 200 rpm at 30 ˚C. This culture was used to inoculate 1 L of LB supplemented with 34 μg mL -1 chloramphenicol and 20 µg mL -1 of biotin and grown until it reached an OD600 of ~0.8. Expression of hpeCBA was induced with 5 µg mL -1 thiostrepton, and the cells were incubated for an additional 24 h. Cells were harvested by centrifugation. Cells collected from 1 L of culture were suspended in 20 mL of buffer A (20 mM MOPS, I = 0.1 M, pH 7.5) containing 2 tablets of proteinase inhibitor (cOmplete™, Mini). The cell suspension was subjected to five rounds of beadbeating at 6 m/s using a FastPrep®-24 (MP Biomedicals) with 5 min on ice between rounds. Cellular debris was removed by centrifugation (40,000 RCF for 40 min) and ammonium sulfate was added to the supernatant to a final concentration of 1.3 M followed by another round of centrifugation. The supernatant was removed. The pellet of precipitated proteins containing HpeCBA was suspended in buffer A supplemented with 2 mM DTT and 10% glycerol, and dialyzed overnight against the same buffer to remove residual ammonium sulfate. The protein preparation was loaded onto a MonoQ 10/100 GL column (GE Healthcare) run using an ÄKTA Purifier. Proteins were eluted with a linear gradient of buffer B (buffer A + 2 mM DTT + 10% glycerol + 1 M NaCl). HpeCBA-containing fractions, as judge by SDS-PAGE, were pooled and concentrated to ~5 mg mL -1 using an Amicon Ultra-15 centrifugal filtration unit (Millipore) equipped with a 30 kDa cut-off membrane, flash frozen in liquid N2, and stored at -80 °C.
HpeC production and purification -HpeC was produced in RHA1 as an N-terminal polyHis-tagged (Ht-) protein using RHA1 cells containing pTip-hpeC. Expression was conducted as described for HpeCBA. Cells collected from 1 L of culture were suspended in 20 mL of of 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8, containing 2 tablets of proteinase inhibitor (cOmplete™, Mini) and DNAseI (2 μg mL -1 ). The cell suspension was subjected to five rounds of bead-beating at 6 m/s using a FastPrep®-24 (MP Biomedicals) with 5 min on ice between rounds. Cellular debris was removed by centrifugation (40,000 × g for 40 min) and the soluble portion was filtered (0.45 μm). Ht-HpeC was purified from the cell extract using immobilized metal affinity chromatography (Ni-NTA, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. The fractions containing Ht-HpeC, as judged by SDS-PAGE, were pooled and dialyzed overnight against 50 mM sodium phosphate, 150 mM NaCl, pH 8.0. Protein preparation was concentrated to ~1 mg mL -1 , flash frozen in liquid N2, and stored at -80 °C until further use.
Protein analytical methods -The molecular weight and purity of the protein were analyzed using SDS-PAGE. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin as a standard. Mass spectrometry was performed by MSL/ChiBi Proteomics Core Facility (UBC).
Streptavidin mobility shift assay -Biotinylation was determined using a previously described mobility shift assay with minor modifications 12 . Briefly, 4 µL of 10 µM HpeCBA was mixed with 2 µL of 5× PAGE loading dye and heated at 95 °C for 5 min. Samples were cooled to room temperature and 4 µL of streptavidin (5, 10, or 20 µM) were added and incubated for 5 min. Samples were then loaded onto a 15% Mini-PROTEAN SDS-PAGE gel and separated at 120 V. Running buffer was prechilled and the gel box was kept on ice during separation. Gels were stained overnight using SYPRO Ruby and imaged on a Typhoon laser scanner using a 488 nm excitation laser and a 670BP30 (655 to 685 nm) filter.
Steady-state kinetic assays -HpeHI assays were performed with 200 µM AV or HAP in 20 mM HEPPS, pH 8.0, 2 mM MgCl2, 1 mM MnCl2, 2 mM DTT, 1 mM ATP, and 20 µg each HpeH and HpeI at 30°C. Reactions were initiated by the addition of enzyme. Extinction coefficients for substrates and products were determined in the same buffer without ATP, as follows, ε340 (AV) = 12.40 mM -1 cm -1 ; ε323 (HAP) = 11.94 mM -1 cm -1 ; ε340 (PAV) = 0.29 mM -1 cm -1 ; and ε323 (PAP) = 0.17 mM -1 cm -1 (SI Appendix Fig. S8). Turnover assays were performed in the same buffer with 50 µM substrate and 1 µM each HpeH and HpeI. The reaction progress was measured by change in absorbance at 323 nm for HAP and 340 nm for AV on a Cary 60 UV-Vis spectrophotometer equipped with a thermostatted cuvette holder. Rates were calculated using the Δε between the substrate and product. Assays were performed in triplicate.
HpeCBA activity was coupled to NADH oxidation (ε340 = 6.3 mM -1 cm -1 ). The standard reaction was performed by incubating 266 nM HpeCBA, 0.2 mM NADH, 0.5 mM phosphoenolpyruvate, 0.5 mM ATP, 6 units of LDH and 4 units of PK (Sigma-Aldrich) in 20 mM MOPS, pH 7.5 (I = 0.1 M), 40 mM NaHCO3, 4 mM MgCl2, 80 mM KCl at 25 °C and was initiated by the addition of PAV or PAP. Progress curves were recorded using a Cary5000 UV-Vis spectrophotometer (Agilent Technologies). Steady-state kinetic parameters were evaluated using concentrations of substrates from 20 to 2000 µM. Data were fit to steady-state kinetic equations using LEONORA 13 . The pH optimum of the reaction was determined using 20 mM MOPS, 80 mM KCl (I = 0.1 M). HpeCBA reactions performed in the presence of HpeD were corrected for the background absorbance of end product (i.e., the phenolate anion of hydroxyphenyl-β-ketopropionate). The background absorbance was recorded by performing the reaction in the absence of NADH. These experiments were performed at pH 7.0 and 7.5 as the background absorbance was much lower at pH 7.0.
4-Hydroxyacetophenone (250 mg, 1.84 mmol) was dissolved in dry acetonitrile (9 mL) in a round bottom flask under an argon atmosphere. The solution was cooled to -10 °C and dry CCl4 (0.88 mL, 9.2 mmol), N,N-diisopropylethylamine (0.67 mL, 3.86 mmol), and N,N-dimethylpyridin-4-amine (23 mg, 0.18 mmol) were added sequentially. Dibenzyl phosphite (0.58 mL, 2.67 mmol) was next added to the solution dropwise and the reaction was stirred at -10 °C for 1 h until TLC indicated the consumption of the phenol. After quenching with 3 mL of 0.5 M KH2PO4, the mixture was extracted with ethyl acetate (15 mL × 3). The organic layers were combined and washed with water (15 mL) and brine (15 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1 to 1:1) to yield 1 as a clear, colorless oil (634 mg, 87%). 1 (480 mg, 1.13 mmol) was dissolved in 10 mL of ethyl acetate, 48 mg of Pd/C was added, and the mixture was put under an atmosphere of H2. The reaction was stirred at room temperature for 1 hour and monitored by TLC. Upon completion, the reaction mixture was filtered over Celite and concentrated in vacuo. The mixture was then purified by reverse phase chromatography (Waters C18 Sep-Pak, 20 cc Vac Cartridge, 5 g Sorbent per Cartridge, 37-55 µm) using water and acetonitrile as eluent to yield 2 as a light brown solid (178 mg, 0.82 mmol, 73%). This compound was found to contain a small amount of an impurity with very similar retention time to the product. Low resolution ESI-MS suggests that it is the compound resulting from reduction of the ketone to alcohol.  Compound 3 (50 mg, 0.12 mmol) was subjected to the same deprotection procedure detailed above to yield 4 as the product in sufficient purity (18 mg, 0.076 mmol, 65% yield, ~95% pure). Similar to the case for compound 1, an impurity resulting from reduction of the ketone functional group was observed, but the amount of this impurity was more noticeable in this case, about 5% as estimated by H-NMR. The product was therefore further purified by HPLC to yield pure compound for analytical purposes. Characterization of OCF extracts -The wood sample was ground into a powder with a knife, milled so it could pass through a 40-mesh screen, and subsequently Soxhlet extracted with acetone to make extractive free wood powder. 10 g of dried wood powder was dispersed in 500 mL of 7.5% NaOH solution, placed in stirred Parr reactor and pressurized with O2 to 1 MPa after purging the reactor. The samples were stirred at 400 RPM for 1 h at 160 °C. The reaction slurry was acidified with HCl to pH 2 and then further extracted. Compounds were extracted from the OCF products using ethyl acetate, in 25:10 ratio (OCF:solvent, v/v) and dehydrated with Na2SO4 prior to analysis. Note, no solid residuals were detected after the OCF treatment.
Aromatic compounds in extracts were identified using an Agilent Technologies (Santa Clara, U.S.A.) 6890N gas chromatograph equipped with a 30-m Agilent 190915-433 capillary column and an Agilent 5973 mass-selective detector. Samples were dried and derivatized using N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane in a 50/50 mixture with pyridine. Runs were held at 90 °C for 3 min, and then ramped to 290 °C at 12 °C min -1 with a 10 min final hold. Authentic standards of vanillin, acetovanillone, vanillate, lactate, gylocate and fumarate were run in parallel. Aliphatic acids were quantified by GC-MS using standard curves. For monoaromatics quantification in OCF slurry and extracts, HPLC analysis was performed using a Waters 2695 HPLC (Waters, Milford, MA) equipped with a 250 × 4.6 mm Luna ® 5 µm C18 column (Phenomenex, Torrance, CA) and a UV detector, as described above. OCF slurry samples were diluted 1:100 in 10% acetic acid and centrifuged for 5 min at maximum velocity. Filtered (0.2 μm) samples were injected. For OCF extracts, ethyl acetate was evaporated, the extracts suspended in methanol, filtered (0.2 μm) and injected. Vanillin, acetovanillone and vanillate were quantified by HPLC using standard curves.
GD02 growth on OCF extracts -For growth of GD02 on OCF extracts, a single colony was inoculated in 5 mL LB broth and grown overnight. Cells were pelleted at 1000 RCF, washed twice with M9, then used to inoculate M9+minerals at OD600 ~0.05. For toxicity experiments, LB broth was amended with different amounts of OCF extracts and cells inoculated at OD600 ~0.05. LB without OCF extract served as a control. For substrate preparation, solvent was evaporated under N2 and suspended in DMSO to prepare a stock solution. Growth studies in 96-well plate were performed using a Tecan Spark-Multimode Microplate Reader with shaking at 250 rpm and OD600 recorded every 30 min. For growth experiments in flasks, cells were incubated with 2 mM (monoaromatic compounds) OCF extracts at 200 rpm, and the growth was followed by measuring OD600. To evaluate the monoaromatic compounds depletion in culture supernatant, 100-µL samples were withdrawn, acidified to 10% acetic acid, and processed and analyzed by HPLC as described above. To evaluate the small acids depletion, 300-µL samples were withdrawn, acidified with 10% acetic acid, extracted with equal volume of ethyl acetate, and processed and analyzed by GC-MS as described above  (Table 1, Table S1) are concentrated in one region of the chromosome, but the hpe, acp, vdh and pcaGH genes are not in this region. Gene cluster abbreviations: Pca, protocatechuate; MDF, methionine-dependent formaldehyde oxidation, βKA, beta-ketoadipate; Van, vanillate; Cat, catechol; Phe, phenol; Gco, guaiacol. pRGD2 Vdh Figure S2. Evidence for co-transcription and horizontal transfer of the hpe gene cluster. The graph shows transcriptional read coverage in the region of the hpe gene cluster during growth of GD02 on AV. Coverage of junctions between hpe genes indicates co-transcription of the entire region, but relatively low coverage of the 5-prime end of hpeI suggests factors may affect transcriptional regulation of individual genes. Dark shading at the junctions of hpeICBADEF indicates four-nucleotide overlaps of the putative open reading frames at each of these junctions. These overlaps provide additional evidence for co-transcription of these genes. The putative transcriptional regulators encoded upstream of the hpe gene cluster are IclR family transcriptional regulators that may modulate transcription of hpe genes. Transposase and recombinase genes flanking the hpe gene cluster suggest horizontal transfer of the cluster. The GC content of the GD02 chromosome and the hpe gene cluster are 68% and 67%, respectively, which is probably too small a difference to be evidence for horizonal transfer.        Reactions performed in the absence of HpeD (blue trace) showed faster rates of A340 decrease than in its presence (black trace). However, a control reaction containing no NADH (grey trace) showed a strong increase in A340 due to the absorbance of HAPC, the dephosphorylated product of the HpeD-catalyzed reaction, whose production was confirmed by HPLC (Fig. 4). Correcting the black trace for this absorbance yielded the red trace. Maximal rates calculated from the blue and red traces were similar (see text). Reactions were performed in triplicate to calculate the specific activity. Single representative traces are shown. Similar results were obtained at pH 7.5. However, HAPC absorbs more strongly at pH 7.5, so reactions were performed at pH 7.0 to mitigate this absorbance HAPC.