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. 2026 Mar 24;74(12):10406–10415. doi: 10.1021/acs.jafc.5c16625

Platform Potential of Sphingobium lignivorans SYK‑6 for Lignin Valorization via Biological Funneling

Naofumi Kamimura †, Mina Endo †, Banri Nakamura †, Takuya Akiyama ‡, Ryo Kato †, Mitsuru Kawazoe †, Masaya Fujita †, Takuma Araki §, Yuzo Suzuki §, Tsuyoshi Michinobu ∥, Yuichiro Otsuka §, Masaya Nakamura §, Eiji Masai †,*
PMCID: PMC13047686  PMID: 41873666

Abstract

Microbial lignin valorization is promising, yet the heterogeneity of aromatic compounds generated from lignin depolymerization remains a significant challenge. Here, we engineered Sphingobium lignivorans SYK-6 to funnel aromatic mixtures in black liquor from oxygen-soda-anthraquinone pulping into the polymer building block 2-pyrone-4,6-dicarboxylic acid (PDC). In softwood (Japanese cedar) black liquor, rich in guaiacyl-type aromatics, a PDC hydrolase gene-deficient strain (ΔligI) achieved a 135% (mol/mol) PDC yield based on identified compounds, suggesting conversion of unidentified aromatics, potentially including oligomers. Hardwood (birch) black liquor contains abundant syringyl-type aromatics metabolized via branched pathways in SYK-6. By identifying a previously unidentified catabolism gene (desY) and disrupting ligI, ligM, and desY, we redirected metabolic flux toward PDC, obtaining a 91% (mol/mol) PDC yield from syringic acid and 62% from birch black liquor. These results demonstrate the strong potential of engineered SYK-6 for efficient PDC production from diverse lignin streams through biological funneling.

Keywords: lignin; Sphingobium lignivorans; black liquor; 2-pyrone-4,6-dicarboxylic acid; biological funneling

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Introduction

Lignin, the second most abundant bioresource on Earth after cellulose, is expected to be crucial for replacing petroleum-based resources. Conversion of lignin into value-added chemicals such as precursors for high-performance polymers is essential for establishing a lignocellulosic bioeconomy. , Lignin is a heterogeneous aromatic plant cell wall polymer synthesized via radical coupling reactions of three distinct monolignols, p-coumaryl (H unit), coniferyl (G unit), and sinapyl (S unit) alcohols. The composition of monolignols varies among plant species. Softwood lignin is composed primarily of guaiacyl (G)-type units, while hardwood lignin has a mixture of G- and syringyl (S)-types. Due to the compositional heterogeneity of lignin, selective depolymerization to produce plastic monomers or direct utilization as a material is challenging. Biological funneling, an approach that employs microbial biocatalysts to convert heterogeneous aromatic mixtures into a single target compound, has emerged as a promising strategy to address this challenge. ,, Previous studies have demonstrated biological funneling of depolymerized lignin-derived aromatics into value-added bioproducts, including 2-pyrone-4,6-dicarboxylic acid (PDC), − cis,cis-muconic acid, , polyhydroxyalkanoates, − vanillic acid, and β-ketoadipic acid.

PDC has a pseudoaromatic pyrone ring and two carboxy groups. The introduction of PDC into the polymer backbone is expected to enhance the mechanical strength and thermal stability of the biobased polymers. So far, various PDC-based polyesters have demonstrated excellent mechanical strength and biodegradability, , and PDC-based polyurethanes have shown superior adhesion properties. , Due to its unique molecular structure, the production of PDC from biomass via chemical conversion presents significant challenges, and microbial fermentation is currently the only feasible production method. PDC is a promising alternative to petroleum-based chemicals as a precursor for biodegradable polymers with excellent performance.

A bacterial strain, Sphingobium lignivorans SYK-6 (formerly Sphingobium sp. strain SYK-6) isolated from a waste treatment pond at a kraft pulp mill, catabolizes a remarkably broad range of lignin-derived aromatic compounds, including dimers with β-aryl ether, biphenyl, phenylcoumaran, and diarylpropane structures. , SYK-6 is the best-characterized bacterium regarding its catabolic system of lignin-derived aromatic compounds, including substrate uptake, catabolic pathways, enzyme properties, gene function, and transcriptional regulation. In SYK-6, G-type compounds, predominant components derived from softwood lignin, are funneled into vanillic acid, which is further catabolized via the protocatechuic acid (PCA) 4,5-cleavage pathway (Figure ). PDC is produced as an intermediate of the PCA 4,5-cleavage pathway. H-type compounds are funneled into p-hydroxybenzoic acid and further catabolized via PCA. On the other hand, S-type compounds derived from hardwood lignin are funneled into syringic acid and converted to 3-O-methylgallic acid (3MGA). 3MGA is further catabolized via branched pathways involving multiple enzymes to PDC and 4-oxalomesaconic acid (OMA), intermediate metabolites in the PCA 4,5-cleavage pathway.

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Catabolic pathways of lignin-derived aromatic compounds in S. lignivorans SYK-6. G-, H-, and S-type aromatic compounds are funneled into vanillic acid, p-hydroxybenzoic acid, and syringic acid, respectively. While the catabolic pathways for G- and H-type aromatic compounds pass through PDC, the pathway for S-type aromatic compounds branches at 3MGA, and part of the branched pathways do not pass through PDC. The CHMOD hydrolase gene, previously unknown, was identified in this study. Enzymes: PobA, p-hydroxybenzoic acid hydroxylase; LigM, vanillic acid/3MGA O-demethylase; DesA, syringic acid/vanillic acid O-demethylase; LigAB, PCA 4,5-dioxygenase; LigC, CHMS dehydrogenase; LigI, PDC hydrolase; LigU, OMA δ-isomerase; LigJ, KCH hydratase; LigK, 4-carboxy-4-hydroxy-2-oxoadipic acid aldolase; DesZ, 3MGA 3,4-dioxygenase; DesB, gallic acid dioxygenase. Abbreviations: PCA, protocatechuic acid; 3MGA, 3-O-methylgallic acid, CHMS, 4-carboxy-2-hydroxymuconate-6-semialdehyde; PDC, 2-pyrone-4,6-dicarboxylic acid; CHMOD, 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoic acid; OMA, 4-oxalomesaconic acid; KCH, 2-keto-4-carboxy-3-hexenedioic acid.

Pseudomonas putida has frequently been used as a platform for the production of valuable chemicals from aromatic mixtures prepared by chemical treatment of lignocellulosic biomass. , The engineered P. putida strains basically target aromatic monomers for biological funneling. Recent studies have reported that lignin depolymerization often yields a wide variety of aromatic dimers and oligomers, rather than only expected aromatic monomers, regardless of the method. This fact suggests that the potential degradation range of the host strain for dimers and oligomers is crucial to achieving high product yields. Since SYK-6 can catabolize lignin-derived dimers, unlike commonly used platform organisms, it is potentially superior to the previously tested hosts with respect to the range of lignin degradation products that can be funneled into PDC.

Industrial black liquors, major lignin-rich side streams from the pulping industry, represent a readily available and abundant feedstock for valorization. However, their direct microbial conversion is often hindered by their complex chemical composition, including not only diverse aromatic compounds but also residual chemicals used in the pulping process, which can act as microbial inhibitors. Therefore, we focused on black liquors prepared by oxygen-soda-anthraquinone pulping. This sulfur-free pulping method is expected to integrate more easily with bioprocesses than conventional kraft pulping. Furthermore, it inherently avoids the generation of strong odors, negating the requirement for extensive mitigation infrastructure. In our previous study, although the potential of engineered SYK-6 to produce PDC has been demonstrated with structurally defined discrete β-aryl ether and diarylpropane dimers, its effectiveness in funneling aromatic compounds from industrial black liquor into PDC has been rarely investigated. Thus, key knowledge gaps remain regarding its conversion efficiency with lignocellulosic feedstocks containing numerous unidentified compounds. In this study, to address these limitations, we investigated the potential of SYK-6 as a PDC production platform for lignin decomposition products in industrial black liquors from softwood Japanese cedar and hardwood birch, prepared by oxygen-soda-anthraquinone pulping.

Materials and Methods

Bacterial Strains, Plasmids, Culture Conditions, Primers, and Chemicals

The strains, plasmids, and PCR primers used in this study are listed (Table S1). Sphingobium lignivorans SYK-6 and its mutant derivatives were cultured at 30 °C with shaking (160 rpm) in Lysogeny Broth (LB), Wx medium, or LB supplemented with the Wx solution I [1.7 g/L KH2PO4, 9.8 g/L Na2HPO4·12H2O, and 1.0 g/L (NH4)2SO4] (designated as LB-Wx). Whenever necessary, the media for SYK-6, its mutants, and its transformants were supplemented with 50 mg/L kanamycin, 12.5 mg/L nalidixic acid, or 1 mM m-toluic acid (an inducer of expression from P m promoter in pJB861). P. putida PpY1100 and PpY1100 harboring pVapoligVABC (designated PpY-PDC) were cultured at 30 °C with shaking (160 rpm) in LB or LB-Wx. The media for PpY-PDC was supplemented with 50 mg/L kanamycin. Escherichia coli NEB 10-beta used for the cloning experiments was cultured at 37 °C with shaking (160 rpm) in LB. The media for E. coli transformants were supplemented with 100 mg/L ampicillin or 25 mg/L kanamycin.

Vanillin, vanillic acid, syringaldehyde, syringic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, acetovanillone, and acetosyringone were purchased from Tokyo Chemical Industry Co., Ltd. 5-carboxyvanillic acid, PDC, and 5,5′-dehydrodivanillin were prepared as described previously. 5-Carboxyvanillin, 5-formylvanillin, and 5-formylvanillic acid were chemically synthesized from o-vanillic acid, vanillin, and vanillic acid, respectively by the Duff reaction as described previously. , Black liquors of softwood Japanese cedar (Cryptomeria japonica) and hardwood birch (Betula pendula) were prepared by oxygen-soda-anthraquinone pulping method as described previously. Softwood chips (12% w/v) were treated with 12% (w/v) NaOH and 0.02% (w/v) anthraquinone at 170 °C for 60 min under pressured oxygen (0.9 MPa). Hardwood chips (12% w/v) were similarly treated at 155 °C for 90 min under pressured oxygen (0.65 MPa). The reaction yielded a soluble fraction (black liquor) and solid pulp. Neutralized black liquor was prepared by adjusting the pH to 6.5–7.0 by adding 6–8% (v/v) phosphoric acid.

Analytical Methods

High-performance liquid chromatography (HPLC) analysis was performed using an ACQUITY iClass UPLC system (Waters). Samples were filtered through a 0.20 μm PTFE membrane filter (Captiva Econofilter, Agilent). Analyses were conducted using a CORTECS T3 C18 column (1.6 μm, 2.1 × 100 mm; Waters) with a flow rate of 0.5 mL/min and a column temperature of 30 °C. Electrospray ionization-mass spectrometry (ESI–MS) was performed using an ACQUITY TQ Detector (Waters) coupled to the HPLC system. Mass spectra were acquired in both positive and negative ion modes with the following settings: capillary voltage, 3.0 kV; cone voltage, 10–40 V; source temperature, 120 °C; desolvation temperature, 350 °C; desolvation gas flow rate, 650 L/h; and cone gas flow rate, 50 L/h. For the analysis of neutralized cedar black liquor and the conversion of 5-carboxyvanillin, 5-formylvanillin, 5-formylvanillic acid, and 5,5′-dehydrodivanillin, the mobile phase consisted of a mixture of solution A (acetonitrile with 0.1% formic acid) and B (water with 0.1% formic acid) using the following gradient conditions: 0–1.0 min, 10% A; 1.0–3.0 min, 10–20% A (linear gradient); 3.0–4.0 min, 20–40% A (linear gradient); 4.0–5.0 min, 100% A; 5.0–6.0 min, 10% A. For the analysis of unidentified compounds in neutralized cedar black liquor, the following gradient conditions were used: 0–1.5 min, 8% A; 1.5–2.5 min, 8–30% A (linear gradient); 2.5–4.5 min, 30–45% A (linear gradient); 4.5–6.4 min, 45–60% A (linear gradient); 6.4–6.5 min, 60–100% A (linear gradient); 6.5–8.0 min, 100% A, 8.0–8.1 min, 100–8% A (linear gradient); 8.1–9.0 min, 8% A. For the analysis of neutralized birch black liquor, the mobile phase consisted of a mixture of solution C (acetonitrile with 0.5% phosphoric acid) and solution D (water with 0.5% phosphoric acid) using the following gradient conditions: 0–3.0 min, 1–20% C (linear gradient); 3.0–5.0 min, 20–7% C (linear gradient); 5.0–6.1 min, 7–30% C (linear gradient); 6.1–7.1 min, 30–50% C (linear gradient); 7.1–8.0 min, 1% C. Vanillin, vanillic acid, syringaldehyde, syringic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, acetovanillone, acetosyringone, 5-carboxyvanillin were detected at 279, 260, 308, 275, 284, 255, 276, 300, and 318 nm, respectively. 5-Formylvanillin, 5-formylvanillic acid, and 5,5′-dehydrodivanillin were detected by ESI–MS in selected ion monitoring mode at m/z 179 (negative ion), m/z 195 (negative ion), and m/z 303 (positive ion), respectively.

Construction of SYK-6 Mutants and PpY-PDC

To construct SYK-6 mutants, 0.5–1.0 kb fragments upstream and downstream of SLG_12720, SLG_25010, and ligM were amplified by PCR using the primer pairs and SYK-6 genomic DNA as a template. The resulting fragments were cloned into pAK405 or pAK405GFP for constructing markerless gene deletion plasmids using the NEBuilder HiFi DNA assembly cloning kit (New England Biolabs). Each resulting plasmid was introduced into cells of ΔligI or ΔligIM by triparental mating, and the resulting mutants were selected as described previously. Gene deletion was confirmed through colony PCR. To create complementary plasmids for SLG_25010, a DNA fragment containing SLG_25010 was amplified by PCR using the primers and the SYK-6 genomic DNA as a template. This fragment was then introduced into the downstream of the Pm promoter in pJB861 to generate pJB25010. The nucleotide sequences of the PCR-amplified fragments were confirmed by DNA sequencing by Eurofins Genomics (Japan).

For the construction of PpY-PDC, pVapoligVABC was introduced into P. putida PpY1100 by electroporation. pVapoligVABC contains the vanillin dehydrogenase (ligV), PCA 4,5-dioxygenase (ligAB), and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC) genes of SYK-6, as well as the vanillic acid O-demethylase (vanAB) and p-hydroxybenzoic acid hydroxylase (pobA) genes of P. putida KT2440 in pKT230 vector. Since P. putida PpY1100 lacks the ability to catabolize PCA, the introduction of pVapoligVABC enables PpY-PDC to produce PDC from vanillin and vanillic acid.

PDC Production from Aromatic Compounds, Model Black Liquors, and Neutralized Black Liquors

To prepare cell suspensions, SYK-6 derivatives (ΔligI, ΔligU, ΔligIM, and ΔligIMY) and PpY-PDC were grown in LB for 24 h, collected by centrifugation (14,000g for 1 min), and washed twice with Wx solution I. To examine PDC production from 5-carboxyvanillin, 5-formylvanillin, 5-formylvanillic acid, and 5,5′-dehydrodivanillin, cells of ΔligI were resuspended in LB supplemented with 100 μM of each substrate to an optical density at 600 nm (OD600) of 0.2 and incubated for 12 h. To investigate PDC production from model black liquors or neutralized black liquors (cedar or birch) by ΔligI, ΔligIMY, and PpY-PDC, cells were suspended in LB-Wx (pH 7.0) supplemented with 5% (v/v) of the respective neutralized black liquor to an OD600 of 0.2 and incubated for 24 or 48 h. To assess PDC production from syringic acid, model black liquors, or neutralized cedar black liquor by ΔligU, cells were resuspended in LB-Wx (pH 6.6 or pH 7.0) supplemented with 5 mM syringic acid or 5% (v/v) of neutralized birch black liquor to an OD600 of 0.2 and cultured for 24 h. Samples were collected periodically to monitor culture pH and OD600. For PDC production from syringic acid by ΔligIM and ΔligIMY, cells were resuspended in LB-Wx (pH 7.0) supplemented with 5 mM syringic acid to an OD600 of 0.2 and cultured for 24 h. Samples were collected periodically to determine culture pH and OD600. For each experiment, substrate conversion and PDC production were monitored by HPLC–MS analysis of the culture supernatants. PDC yield was calculated based on the total molar amount of identified aromatic compounds in the black liquor. For birch black liquor, it was based on the total molar amount of G- and S-type compounds.

Growth Analysis

SYK-6, its mutants, and complemented strains were grown in LB for 24 h, collected by centrifugation (14,000g for 1 min), and washed twice with Wx solution I. The cell suspensions were inoculated into Wx medium containing 5 mM syringic acid to an OD600 of 0.1. Cultures were incubated in an Epoch 2 microplate reader (Agilent BioTek), and OD600 was monitored.

Sequence Analysis

The genomic sequence of SYK-6 (Assembly ID: ASM28351v1) and protein sequences were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov). Homologous genes were searched using NCBI Protein BLAST (BLAST-P). Pairwise alignment was carried out using EMBOSS Needle.

RNA-Seq Analysis

SYK-6 cells were cultured in Wx-SEMP until the OD600 reached 0.5, followed by the addition of 5 mM syringic acid or 5 mM acetosyringone and incubation for an additional 6 h. Total RNA was subsequently isolated from the cells using the Illustra RNAspin Mini RNA Isolation Kit (Cytiva). RNA-Seq analysis of SYK-6 was performed on the Illumina platform (Novogene). Read quality was filtered using Trimmomatic version 0.3949 and mapped to the reference genome of SYK-6 (GCA_000283515.1) by bowtie2 version 2.5.0.50. Relative transcript abundance was calculated as transcripts per million (TPM).

Safety Considerations

The oxygen-soda-anthraquinone pulping process involves high temperatures and pressures under an oxygen atmosphere, presenting potential explosion hazards. This reaction must be conducted in a properly rated high-pressure reactor with appropriate shielding. Sodium hydroxide, phosphoric acid, and formic acid are corrosive and cause severe skin and eye burns. Acetonitrile is toxic and flammable. Appropriate personal protective equipment and fume hoods were used for all procedures involving hazardous chemicals. Standard biosafety level 1 (BSL-1) practices were followed for bacterial cultivation.

Results and Discussion

Composition of Aromatic Compounds in Black Liquors Prepared from Cedar and Birch

Aromatic monomers were reported to be obtained in 17% (w/w) yield from cedar lignin by oxygen-soda-anthraquinone pulping. Ten aromatic compounds were identified in the neutralized softwood cedar black liquor used in this study (Figure ). The G-type monomer vanillin was predominant (20.4 mM), accounting for 63% (mol/mol) of the identified compounds. In addition, the G-type monomers 5-carboxyvanillin, vanillic acid, acetovanillone, 5-carboxyvanillic acid, 5-formylvanillin, and 5-formylvanillic acid; the H-type monomers p-hydroxybenzaldehyde and p-hydroxybenzoic acid; and a biphenyl-type dimer 5,5′-dehydrodivanillin were identified as minor components. In the hardwood birch black liquor, seven S-type and G-type monomers were identified (Figure ). Among them, syringaldehyde was the major component (19.2 mM), accounting for 57% (mol/mol) of the identified compounds. The total concentrations of the identified aromatic compounds (32.3 mM in cedar black liquor and 33.9 mM in birch black liquor) were similar between the two black liquors. The H/G/S-type monomer composition in the neutralized cedar and birch black liquors reflected the monolignol ratios in their original lignins. , No biphenyl-type compounds were detected in the birch black liquor, nor were any 5-position substituted G-type monomers detected. These results are reasonable given that the 5-5 linkages are largely absent in hardwood lignin. Additionally, no hydroxycinnamic acids, such as ferulic acid and p-coumaric acid, were detected in either black liquor.

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Compositions of aromatic compounds in neutralized black liquors. Compounds detected in cedar black liquor and birch black liquor are enclosed in green and blue boxes, respectively. Concentrations (mM) and molar fractions (%) are presented relative to the total identified aromatic compounds. The classification of each compound into guaiacyl, p-hydroxyphenyl, or syringyl units is indicated by the letters G, H, and S, respectively.

PDC Production from the Cedar Black Liquor

Conversion capability of SYK-6 for 5-carboxyvanillin, 5-formylvanillin, 5-formylvanillic acid, and 5,5′-dehydrodivanillin found in the neutralized cedar black liquor has yet to be investigated. We first examined whether SYK-6 could convert these compounds to PDC. Cells of a PDC hydrolase gene (ligI) mutant (ΔligI) were grown in the presence of 5-carboxyvanillin, 5-formylvanillin, 5-formylvanillic acid, or 5,5′-dehydrodivanillin. After cultivation for 12 h, all of these compounds were completely converted, and PDC production was observed (Figure S1). In SYK-6, 5,5′-dehydrodivanillic acid and 5-carboxyvanillic acid are catabolized via PDC (Figure S2). Therefore, we propose that 5,5′-dehydrodivanillin is oxidized at the formyl groups at the 1- and 1′-positions of each aromatic ring to form 5,5′-dehydrodivanillic acid, and 5-formylvanillin is oxidized at both formyl groups at the 1- or 5-positions to form 5-carboxyvanillic acid via 5-formylvanillic acid and/or 5-carboxyvanillin. These oxidation reactions are likely catalyzed by benzaldehyde dehydrogenases. SYK-6 possesses multiple benzaldehyde dehydrogenases, including the vanillin and syringaldehyde dehydrogenases LigV and DesV, which exhibit broad substrate specificity toward benzaldehydes. In addition, FerD catalyzes the oxidation of 5-formylferulic acid, which bears a 5-formyl group like 5-formylvanillin and 5-formylvanillic acid. Among these aldehyde dehydrogenases, an enzyme(s) is thought to be responsible for the oxidation of the formyl groups in the above substrates.

Next, the PDC production capacity of SYK-6 using cedar black liquor was investigated (Figure A–C). Because G- and H-type aromatic compounds are catabolized via one unbranched pathway in SYK-6, ΔligI was employed for PDC production (Figure S3A–C). ΔligI was cultivated in LB with 5% (v/v) neutralized cedar black liquor. All ten identified aromatic compounds (1600 μM in total) were consumed within 24 h of cultivation, and 2160 μM PDC (0.396 g/L) was produced at 48 h. This PDC yield corresponds to approximately 7% (w/w) relative to the original wood and 20% (w/w) of lignin. In addition, the PDC yield based on the identified compounds was 135% (mol/mol). The yield exceeding 100% is likely due to the conversion of unidentified compounds to PDC by ΔligI. Based on mass balance calculations using both the amount of the original wood feedstock and the lignin fraction estimated from the Klason lignin content (35.4%), of the total 0.396 g/L PDC produced, approximately 0.099 g/L could be attributed to unidentified components. To investigate the unidentified compounds contributing to the PDC yield observed in ΔligI, LC–MS analysis was performed on neutralized cedar black liquor before and after cultivation. Most of the unidentified peaks detected in both positive- and negative-ion modes were markedly reduced or disappeared after 48 h of cultivation (Figure S4). The estimated parent ion m/z values of these peaks were all above 275, suggesting that these components include oligomer-sized aromatic compounds.

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PDC production from cedar black liquor by engineered S. lignivorans SYK-6 and P. putida strains. Cells of ΔligI and PpY1100 harboring pVapoligVABC (PpY-PDC) were cultivated in LB-Wx medium containing 5% (v/v) neutralized cedar black liquor. Panels (A–F) show the profiles for ΔligI and PpY-PDC, respectively. Panels (A,D) show the conversion of major aromatic compounds and the production of PDC. Panels (B,E) represent the conversion of minor aromatic compounds. Panels (C,F) compare the molar percentage (mol %) of identified aromatic compounds in the cedar black liquor at the start of incubation and after 48 h, alongside the PDC yield (mol %). Each value represents the mean ± standard deviation (error bars) from three independent experiments. Abbreviations: PDC, 2-pyrone-4,6-dicarboxylic acid; VN, vanillin; VA, vanillic acid; CVN, 5-carboxyvanillin; AV, acetovanillone; CVA, 5-carboxyvanillic acid; DDV, 5,5′-dehydrodivanillin; HN, p-hydroxybenzaldehyde; FVN, 5-formylvanillin; HA, p-hydroxybenzoic acid; FVA, 5-formylvanillic acid.

To determine whether the high PDC production yield observed above originated from a unique characteristic of SYK-6, similar experiments were conducted using P. putida PpY1100 as a PDC-producing host strain. P. putida PpY1100 harboring pVapoligVABC (designated PpY-PDC) produced 1250 μM PDC from the neutralized black liquor, achieving a yield of 75% (mol/mol) (Figure D–F). The accumulation of acetovanillone, 5-carboxyvanillic acid, and 5,5′-dehydrodivanillin was observed, and their total residual concentration was 480 μM after 48 h. These results suggest that P. putida PpY1100 is unable to convert these compounds. Among the three compounds, 5-carboxyvanillic acid was particularly highly accumulated (380 μM at 48 h).

Taken together, these results demonstrate that ΔligI is a highly effective platform strain for PDC production from cedar black liquor. SYK-6 appears to possess a remarkably broad substrate range for lignin-derived aromatic compounds, potentially including aromatic oligomers. Supporting this hypothesis, the β-etherase system in SYK-6, for example, comprised of LigD, LigF, and LigG, which is responsible for cleaving the key β-O-4 linkage in lignin, exhibits activity toward both synthetic lignin polymer and oligomers containing β-O-4 bonds. , However, a critical question concerns whether these relatively large aromatic oligomers can be transported into the cell. Membrane permeation simulations of lignin-derived monomers and dimers suggest that uncharged aromatic compounds can be transported by passive processes. Furthermore, bacteria capable of assimilating uncharged tetrameric lignin model compounds have been found. , In addition, SYK-6 specifically transports the charged dimer 5,5′-dehydrodivanillic acid across the outer membrane via the TonB-dependent transporter DdvT. This strain possesses 73 TonB-dependent transporter-like genes, in addition to ddvT. Some of these transporters may be involved in transporting aromatic oligomers.

pH-Dependent PDC Production from Birch Black Liquor Using ligU Mutant

To achieve high-yield PDC production from birch black liquor, it is necessary to establish a production route from the predominant component, S-type compounds (72% mol/mol in neutralized birch black liquor, Figure ), in addition to the G-type compounds. The S-type compound catabolic pathway of SYK-6 branches at 3MGA, including a route that does not produce PDC as an intermediate (Figure ). However, all of the branched pathways converge at OMA. In addition, OMA is also an intermediate of the G-type compound catabolism. Furthermore, PDC hydrolase LigI also catalyzes the conversion of OMA to PDC via a reverse reaction. We initially devised a strategy to accumulate OMA and then convert it to PDC by the reverse reaction of LigI. By disrupting the OMA tautomerase gene ligU in SYK-6, a dead-end point in the catabolic pathways for both S- and G-type compounds was created at OMA (Figure S3B). The reaction catalyzed by LigI favors the conversion of PDC to OMA at higher pH, while the reverse reaction from OMA to PDC is promoted under acidic conditions (below pH 7.0). ,, PDC production from syringic acid, an S-type model compound, was examined using the ligU mutant (ligU:kan) and buffered LB (LB-Wx) with pH 6.6 and 7.0 (Figure A,B). The ligU mutant produced PDC with maximum molar yields of 75% and 62% from 5 mM syringic acid when using LB-Wx with pH 6.6 and 7.0, respectively. The culture medium pH was maintained for the first 15 h of incubation. Subsequently, as the pH increased, the concentration of PDC decreased accordingly. These results suggest that PDC production yields from S-type compounds can be enhanced by controlling the culture medium pH when the ligU mutant is used as the host.

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PDC production from syringic acid and birch black liquor by the ligU mutant of SYK-6. Cells of the ligU mutant were cultivated in LB-Wx medium containing 5 mM syringic acid (A,B) or 5% (v/v) neutralized birch black liquor (C,D). The medium was adjusted to an initial pH of 6.6 (A,C) or 7.0 (B,D). Conversion of aromatic compounds, PDC production, cell growth (OD600), and culture medium pH were monitored. Each value represents the mean ± standard deviation (error bars) from three independent experiments. Abbreviations: PDC, 2-pyrone-4,6-dicarboxylic acid; SA, syringic acid; SN, syringaldehyde; VN, vanillin; VA, vanillic acid; AS, acetosyringone; CVN, 5-carboxyvanillin; AV, acetovanillone.

Next, PDC production from neutralized birch black liquor (5% [v/v]) was conducted using the ligU mutant at initial pH values of 6.6 and 7.0 (Figure C,D). Syringaldehyde and vanillin were rapidly converted into syringic acid and vanillic acid, and PDC was subsequently produced along with the transformation of these compounds. The substrate conversion rate at pH 7.0 was significantly faster than at pH 6.6, and all aromatic compounds identified in the black liquor were completely transformed within 15 h at pH 7.0 and 24 h at pH 6.6. In both conditions, the PDC concentration reached its maximum when the substrates were completely consumed; however, the PDC yield was higher at pH 6.6 (54% mol/mol) than at pH 7.0 (40% mol/mol). These results indicate that controlling the medium pH improves PDC production yields using the ligU mutant as the host, even when neutralized birch black liquor was used as the feedstock. The yield could be further improved by more precise control of the medium pH during cultivation or by adding a step at a lower pH of LigI-catalyzed conversion of OMA to PDC after the cultivation is completed.

Engineering S-Type Compound Catabolism in SYK-6 for Enhanced PDC Production

It was hypothesized that the simultaneous disruption of ligI and vanillic acid/3MGA O-demethylase gene ligM, in addition to the unidentified 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoic acid (CHMOD) hydrolase gene involved in the branched 3MGA catabolic pathway, would enable high-yield production of PDC from S-type compounds (Figure S3C). Therefore, we first constructed a ligI ligM double mutant (ΔligIM) (Figure S5A). As observed in our previous study, ΔligIM exhibited delayed growth on syringic acid compared to the wild-type strain (Figure A). Furthermore, the only functioning O-demethylase, DesA, is highly specific for syringic acid, exhibiting almost negligible activity toward 3MGA. These facts suggest that the demethylation route of the 3MGA catabolic pathway is blocked in ΔligIM. However, this strain did not produce PDC from SA (Figure B). Next, we focused on the CHMOD hydrolase gene in SYK-6. The CHMOD hydrolase gene (desC, Saro_2864) was found in Novosphingobium aromaticivorans DSM 12444. In SYK-6, a putative α/β hydrolase gene SLG_12720 located in the vicinity of ligM showed the highest similarity to desC (45% identity at the amino acid sequence level). On the other hand, SLG_25010, located upstream of desA, forms a syringic acid-inducible SLG_25010-desA operon and encodes a predicted hydrolase. Since SLG_12720 and SLG_25010 were considered candidate genes for CHMOD hydrolase, deletion mutants of these genes in ΔligIM (ΔligIM 12720 and ΔligIM 25010) were constructed (Figure S5A,B). Under cultivation on syringic acid, ΔligIM 12720 showed growth comparable to ΔligIM, whereas ΔligIM 25010 lost the ability to grow (Figure A). ΔligIM 25010 expressing SLG_25010 grew on syringic acid, showing that SLG_25010 is involved in syringic acid catabolism, thus this gene was designated desY (Figure S3C). The PDC production from syringic acid was evaluated using ΔligIM 25010 (hereafter, ΔligIMY). ΔligIMY produced PDC with a 91% yield (mol/mol) at 14 h of cultivation, dramatically improving PDC productivity over ΔligIM (Figure B,C). These results indicate that the disruption of desY in ΔligIM blocked CHMOD hydrolysis, directing the metabolic flow toward the nonenzymatic conversion of CHMOD to PDC (Figure S3C).

5.

5

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Characterization of ΔligIM, ΔligIM 12720, and ΔligIM 25010. (A) Growth of SYK-6, ΔligI, ΔligIM, ΔligIM 12720, and ΔligIM 25010 (ΔligIMY) in Wx medium containing 5 mM syringic acid. (B,C) Syringic acid conversion and PDC production by ΔligIM (B) and ΔligIM 25010 (ΔligIMY) (C) during the cultivation in LB-Wx medium supplemented with 5 mM syringic acid. Each value represents the mean ± standard deviation (error bars) from three independent experiments. Abbreviations: PDC, 2-pyrone-4,6-dicarboxylic acid; SA, syringic acid.

DesY possesses the MenH motif (COG0596), which is frequently found in α/β hydrolases, but shares only 22.3% amino acid sequence identity with DesC from N. aromaticivorans DSM 12444. Thus, DesY is a novel CHMOD hydrolase distinct from DesC. In SYK-6, 3MGA is primarily catabolized via O-demethylation catalyzed by LigM, yielding gallic acid. However, alternative pathways also function, where 3MGA undergoes aromatic ring cleavage by DesZ and LigAB and is catabolized via PDC or CHMOD. The fact that desY and desA constitute a syringic acid-inducible operon implies that the CHMOD pathway plays a significant complementary role in the syringic acid catabolism in SYK-6.

PDC Production from Birch Black Liquor Using ΔligIMY

A synthetic black liquor model was prepared to mimic the composition of birch black liquor, containing syringaldehyde, syringic acid, acetosyringone, vanillin, vanillic acid, and acetovanillone at a total concentration of 1 mM. ΔligIMY was cultivated in LB-Wx medium supplemented with 1 mM model black liquor. All compounds were completely consumed within 8 h, resulting in a PDC yield of 81% (mol/mol) (Figure A). Subsequently, PDC production from neutralized birch black liquor (5% [v/v]) was evaluated using ΔligIMY. All identified aromatic compounds were completely consumed within 15 h, resulting in a PDC yield of 62% (mol/mol) (Figure B).

6.

6

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PDC production from a model of black liquor and birch black liquor by the ΔligIMY. Cells of ΔligIMY were cultivated in LB-Wx medium containing 1 mM model mixture mimicking birch black liquor (A) or 5% (v/v) neutralized birch black liquor (B). Cell growth (OD600) is shown in the upper panels, while the conversion of aromatic compounds and PDC production are indicated in the lower panels. Each value represents the mean ± standard deviation (error bars) from three independent experiments. Abbreviations: PDC, 2-pyrone-4,6-dicarboxylic acid; SA, syringic acid; SN, syringaldehyde; VN, vanillin; VA, vanillic acid; AS, acetosyringone; CVN, 5-carboxyvanillin; AV, acetovanillone.

When using syringic acid, birch black liquor, or its model mixture, the PDC yield failed to reach a molar yield of 100%. The most plausible explanation for this problem is the presence of enzymes involved in the conversion of CHMOD other than DesY in ΔligIMY. Although SLG_12720 was found not to be involved in syringic acid catabolism (Figure A), the possibility that its product possesses CHMOD hydrolytic activity cannot be excluded. Indeed, RNA-seq analysis revealed that the transcript level of SLG_12720 was significantly higher in the presence of acetosyringone (192 transcripts per million, TPM) than in the presence of syringic acid (54 TPM). This fact is consistent with the lower PDC yields observed when using birch black liquor (62% mol/mol) and its model mix (81% mol/mol) as substrates compared to syringic acid alone (91% mol/mol). These results suggest that SLG_12720 may be involved in CHMOD degradation, although its catalytic activity toward CHMOD has not yet been confirmed.

Interestingly, vanillic acid was converted more efficiently than expected in the ΔligIMY (Figure ). The deletion of ligM was made to suppress the O-demethylation of 3MGA in the S-type compounds catabolic pathway. However, LigM is also the major O-demethylase for vanillic acid in SYK-6, exhibiting significantly higher activity toward vanillic acid than DesA. Therefore, disruption of ligM was thought to significantly impede the conversion of vanillic acid. Contrary to this prediction, vanillic acid accumulated temporarily but was entirely consumed during the production of PDC from both the model compound mixture and the birch black liquor (Figure ). These results indicate that the O-demethylase activity of DesA in ΔligIMY toward vanillic acid is sufficient to convert the guaiacyl-type compounds present in birch black liquor into PDC at the concentrations used in this study.

We focused on the exceptionally broad metabolic capability of the nonmodel bacterium SYK-6 toward lignin-derived aromatic compounds and investigated its potential as a platform for biological funneling. Based on identified monomers, a PDC yield exceeding 100% (135% mol/mol) was achieved using 5% (v/v) cedar black liquor as the feedstock, corresponding to a PDC titer of 0.396 g/L. Notably, all identified aromatic compounds in the black liquor were completely converted, representing a clear advancement beyond previous demonstrations based on individual model compounds. This result indicates that SYK-6 can metabolize a broader spectrum of aromatic compounds than previously anticipated, likely including unidentified oligomers. In addition, we identified the CHMOD hydrolase gene (desY), which is involved in the catabolism of S-type compounds in SYK-6. This finding enabled metabolic pathway engineering to redirect metabolic flux from S-type compounds toward PDC production. Collectively, our findings demonstrate that SYK-6 not only serves as a valuable model for studying the catabolic system of lignin-derived aromatic compounds but also holds significant potential as a platform for lignin valorization in biorefinery applications.

Supplementary Material

jf5c16625_si_001.pdf (5.1MB, pdf)

Acknowledgments

We thank Drs. Kengo Magara and Satoshi Kubo at the Forestry and Forest Products Research Institute for providing black liquors.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.5c16625.

  • Bacterial strains, plasmids, and primers; model compound conversion assays; proposed catabolic pathways, pathway engineering strategies, and gene disruption and complementation analyses (PDF)

⊥.

N.K. and M.E. contributed equally to this work.

This research was partially supported by the Project of the NARO Bio-oriented Technology Research Advancement Institution (Research Program on Development of Innovative Technology) Grant Number 01014B; JST Grant Number JPMJPF2104; JST CREST Grant Number JPMJCR23L4; JSPS KAKENHI Grant Numbers 24K01891 and 25H01196, and the JSPS Program for Forming Japan’s Peak Research Universities (J-PEAKS), Grant Number JPJS00420240017.

The authors declare no competing financial interest.

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