Catabolic System of 5-Formylferulic Acid, a Downstream Metabolite of a ß-5-Type Lignin-Derived Dimer, in Sphingobium lignivorans SYK-6.

Sphingobium lignivorans SYK-6 can assimilate various lignin-derived aromatic compounds, including a ß-5-type (phenylcoumaran-type) dimer, dehydrodiconiferyl alcohol (DCA). SYK-6 converts DCA to a stilbene-type intermediate via multiple reaction steps and then to vanillin and 5-formylferulic acid (FFA). Here, we first elucidated the catabolic pathway of FFA, which is the only unknown pathway in DCA catabolism. Then, we identified and characterized the enzyme-encoding genes responsible for this pathway. Analysis of the metabolites revealed that FFA was converted to 5-carboxyferulic acid (CFA) through oxidation of the formyl group, followed by conversion to ferulic acid by decarboxylation. A comprehensive analysis of the aldehyde dehydrogenase genes in SYK-6 indicated that NAD+-dependent FerD (SLG_12800) is crucial for the conversion of FFA to CFA. LigW and LigW2, which are 5-carboxyvanillic acid decarboxylases involved in the catabolism of a 5,5-type dimer, were found to be involved in the conversion of CFA to ferulic acid, and LigW2 played a significant role. The ligW2 gene forms an operon with ferD, and their transcription was induced during growth in DCA.

Successful selective production of vanillic acid from depolymerized sulfite lignin and its application to poly(ethylene vanillate) synthesis.

Towards lignin upgrading, vanillic acid (VA), a lignin-derived guaiacyl compound, was produced from sulfite lignin for successfully synthesizing poly(ethylene vanillate), an aromatic polymer. The engineered Sphingobium sp. SYK-6-based strain in which the genes responsible for VA/3-O-methyl gallic acid O-demethylase and syringic acid O-demethylase were disrupted was able to produce vanillic acid (VA) from the mixture consisting of acetovanillone, vanillin, VA, and other low-molecular-weight aromatics obtained by Cu(OH)2-catalyzed alkaline depolymerization of sulfite lignin and membrane fractionation. From the bio-based VA, methyl-4-(2-hydroxyethoxy)-3-methoxybenzoate was synthesized via methylesterification, hydroxyethylation, and distillation, and then it was subjected to polymerization catalyzed by titanium tetraisopropoxide. The molecular weight of the obtained poly(ethylene vanillate) was evaluated to be Mw = 13,000 (Mw/Mn = 1.99) and its melting point was 261 °C. The present work proved that poly(ethylene vanillate) is able to be synthesized using VA produced from lignin for the first time.

Multiplexed fitness profiling by RB-TnSeq elucidates pathways for lignin-related aromatic catabolism in Sphingobium sp. SYK-6.

Bioconversion of lignin-related aromatic compounds relies on robust catabolic pathways in microbes. Sphingobium sp. SYK-6 (SYK-6) is a well-characterized aromatic catabolic organism that has served as a model for microbial lignin conversion, and its utility as a biocatalyst could potentially be further improved by genome-wide metabolic analyses. To this end, we generate a randomly barcoded transposon insertion mutant (RB-TnSeq) library to study gene function in SYK-6. The library is enriched under dozens of enrichment conditions to quantify gene fitness. Several known aromatic catabolic pathways are confirmed, and RB-TnSeq affords additional detail on the genome-wide effects of each enrichment condition. Selected genes are further examined in SYK-6 or Pseudomonas putida KT2440, leading to the identification of new gene functions. The findings from this study further elucidate the metabolism of SYK-6, while also providing targets for future metabolic engineering in this organism or other hosts for the biological valorization of lignin.

Stereoinversion via Alcohol Dehydrogenases Enables Complete Catabolism of ß-1-Type Lignin-Derived Aromatic Isomers.

Sphingobium sp. strain SYK-6 is an efficient aromatic catabolic bacterium that can consume all four stereoisomers of 1,2-diguaiacylpropane-1,3-diol (DGPD), which is a ring-opened ß-1-type dimer. Recently, LdpA-mediated catabolism of erythro-DGPD was reported in SYK-6, but the catabolic pathway for threo-DGPD was as yet unknown. Here, we elucidated the catabolism of threo-DGPD, which proceeds through conversion to erythro-DGPD. When threo-DGPD was incubated with SYK-6, the Cα hydroxy groups of threo-DGPD (DGPD I and II) were initially oxidized to produce the Cα carbonyl form (DGPD-keto I and II). This initial oxidation step is catalyzed by Cα-dehydrogenases, which belong to the short-chain dehydrogenase/reductase (SDR) family and are involved in the catabolism of ß-O-4-type dimers. Analysis of seven candidate genes revealed that NAD+-dependent LigD and LigL are mainly involved in the conversion of DGPD I and II, respectively. Next, we found that DGPD-keto I and II were reduced to erythro-DGPD (DGPD III and IV) in the presence of NADPH. Genes involved in this reduction were sought from Cα-dehydrogenase and ldpA-neighboring SDR genes. The gene products of SLG_12690 (ldpC) and SLG_12640 (ldpB) catalyzed the NADPH-dependent conversion of DGPD-keto I to DGPD III and DGPD-keto II to DGPD IV, respectively. Mutational analysis further indicated that ldpC and ldpB are predominantly involved in the reduction of DGPD-keto. Together, these results demonstrate that SYK-6 harbors a comprehensive catabolic enzyme system to utilize all four ß-1-type stereoisomers through successive oxidation and reduction reactions of the Cα hydroxy group of threo-DGPD with a net stereoinversion using multiple dehydrogenases. IMPORTANCE In many catalytic depolymerization processes of lignin polymers, aryl-ether bonds are selectively cleaved, leaving carbon-carbon bonds between aromatic units intact, including dimers and oligomers with ß-1 linkages. Therefore, elucidating the catabolic system of ß-1-type lignin-derived compounds will aid in the establishment of biological funneling of heterologous lignin-derived aromatic compounds to value-added products. Here, we found that threo-DGPD was converted by successive stereoselective oxidation and reduction at the Cα position by multiple alcohol dehydrogenases to erythro-DGPD, which is further catabolized. This system is very similar to that developed to obtain enantiopure alcohols from racemic alcohols by artificially combining two enantiocomplementary alcohol dehydrogenases. The results presented here demonstrate that SYK-6 has evolved to catabolize all four stereoisomers of DGPD by incorporating this stereoinversion system into its native ß-1-type dimer catabolic system.

Click Synthesis of Triazole Polymers Based on Lignin-Derived Metabolic Intermediate and Their Strong Adhesive Properties to Cu Plate.

2-Pyrone-4,6-dicarboxylic acid (PDC) is a chemically stable metabolic intermediate of lignin that can be produced on a large scale by transforming bacteria. Novel biomass-based polymers based on PDC were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and fully characterized by nuclear magnetic resonance, infrared spectroscopies, thermal analysis, and tensile lap shear strength measurements. The onset decomposition temperatures of these PDC-based polymers were all above 200 °C. In addition, the PDC-based polymers exhibited strong adhesive properties to various metal plates, with the highest adhesion to a copper plate of 5.73 MPa. Interestingly, this result was in contrast to our previous findings that PDC-based polymers weakly adhere to copper. Furthermore, when bifunctional alkyne and azide monomers were polymerized in situ under hot-press conditions for 1 h, the resulting PDC-based polymer displayed a similar adhesion to a copper plate of 4.18 MPa. The high affinity of the triazole ring to copper ions improved the adhesive ability and selectivity of the PDC-based polymers to copper while still maintaining the strong adhesive ability to other metals, which is conducive to enhancing the versatility of PDC-based polymers as adhesives.

High-level production of 2-pyrone-4,6-dicarboxylic acid from vanillic acid as a lignin-related aromatic compound by metabolically engineered fermentation to realize industrial valorization processes of lignin.

2-Pyrone-4,6-dicarboxylic acid (PDC) is a valuable building block molecule produced from lignin-derived aromatic compounds by biological funneling. This study aimed to design a fermentation process for producing PDC from vanillic acid, which could be applied at an industrial production. Metabolomic analysis revealed that a high primary metabolic activity within cells was required to improve the production efficiency. Moreover, a medium with ammonium salts and no alkali metals was advantageous because it suppressed the formation of PDC-metal complexes. Resulting optimized process yielded the highest PDC titer and productivity ever reported: 99.9 g/L and 1.69 g/L/h, respectively. Per batch, 190 g of PDC was produced per liter of initial culture media, and the final liquid volume was 1.9 L. This study demonstrates the design of fermentation processes for the advanced industrial utilization of lignin by biological funneling.

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation.

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived ß-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

The Catabolic System of Acetovanillone and Acetosyringone in Sphingobium sp. Strain SYK-6 Useful for Upgrading Aromatic Compounds Obtained through Chemical Lignin Depolymerization.

Acetovanillone is a major aromatic monomer produced in oxidative/base-catalyzed lignin depolymerization. However, the production of chemical products from acetovanillone has not been explored due to the lack of information on the microbial acetovanillone catabolic system. Here, the acvABCDEF genes were identified as specifically induced genes during the growth of Sphingobium sp. strain SYK-6 cells with acetovanillone and these genes were essential for SYK-6 growth on acetovanillone and acetosyringone (a syringyl-type acetophenone derivative). AcvAB and AcvF produced in Escherichia coli phosphorylated acetovanillone/acetosyringone and dephosphorylated the phosphorylated acetovanillone/acetosyringone, respectively. AcvCDE produced in Sphingobium japonicum UT26S carboxylated the reaction products generated from acetovanillone/acetosyringone by AcvAB and AcvF into vanilloyl acetic acid/3-(4-hydroxy-3,5-dimethoxyphenyl)-3-oxopropanoic acid. To demonstrate the feasibility of producing cis,cis-muconic acid from acetovanillone, a metabolic modification on a mutant of Pseudomonas sp. strain NGC7 that accumulates cis,cis-muconic acid from catechol was performed. The resulting strain expressing vceA and vceB required for converting vanilloyl acetic acid to vanillic acid and aroY encoding protocatechuic acid decarboxylase in addition to acvABCDEF successfully converted 1.2 mM acetovanillone to approximately equimolar cis,cis-muconic acid. Our results are expected to help improve the yield and purity of value-added chemical production from lignin through biological funneling. IMPORTANCE In the alkaline oxidation of lignin, aromatic aldehydes (vanillin, syringaldehyde, and p-hydroxybenzaldehyde), aromatic acids (vanillic acid, syringic acid, and p-hydroxybenzoic acid), and acetophenone-related compounds (acetovanillone, acetosyringone, and 4'-hydroxyacetophenone) are produced as major aromatic monomers. Also, base-catalyzed depolymerization of guaiacyl lignin resulted in vanillin, vanillic acid, guaiacol, and acetovanillone as primary aromatic monomers. To date, microbial catabolic systems of vanillin, vanillic acid, and guaiacol have been well characterized, and the production of value-added chemicals from them has also been explored. However, due to the lack of information on the microbial acetovanillone and acetosyringone catabolic system, chemical production from acetovanillone and acetosyringone has not been achieved. This study elucidated the acetovanillone/acetosyringone catabolic system and demonstrates the potential of using these genes for the production of value-added chemicals from these compounds.

Integrated process development for grass biomass utilization through enzymatic saccharification and upgrading hydroxycinnamic acids via microbial funneling.

In grass biomass, hydroxycinnamic acids (HCAs) play crucial roles in the crosslinking of lignin and polysaccharides and can be easily extracted by mild alkaline pretreatment, albeit heterogeneously. Here, HCAs were extracted from bamboo and rice straw as model grass biomass with different HCAs composition, and microbial funneling was then conducted to produce 2-pyrone-4,6-dicarboxylic acid (PDC) and (4S)-3-carboxymuconolactone (4S-3CML), promising building blocks for bio-based polymers, respectively. Pseudomonas putida PpY1100 engineered for efficient microbial funneling completely converted HCAs to PDC and 4S-3CML with high titers of 3.9-9.3 g/L and molar yields of 92-99%, respectively. The enzymatic saccharification efficiencies of lignocellulose after HCAs extraction were 29.5% in bamboo and 73.8% in rice straw, which are 8.9 and 6.8 times higher than in alkaline-untreated media, respectively. These results provide a green-like process for total valorization of grass biomass through enzymatic saccharification integrated with upgrading heterogeneous HCAs to a valuable single chemical via microbial funneling.

Pseudomonas sp. NGC7 as a microbial chassis for glucose-free muconate production from a variety of lignin-derived aromatics and its application to the production from sugar cane bagasse alkaline extract.

cis,cis-Muconate (ccMA) is a promising platform for use in synthesizing various polymers. A glucose-free ccMA production using Pseudomonas sp. NGC7 from hardwood lignin-derived aromatic compounds was previously reported. In that system, syringyl nucleus compounds were essential for growth. Here, it is shown that NGC7 is available for glucose-free ccMA production even from a mixture of lignin-derived aromatics that does not contain syringyl nucleus compounds. By introducing a gene set for the protocatechuate (PCA)-shunt consisting of PCA 3,4-dioxygenase and PCA decarboxylase into an NGC7-derived strain deficient in PCA 3,4-dioxygenase and ccMA cycloisomerase, it was succeeded in constructing a ccMA-producing strain that grows on a lignin-derived aromatics mixture containing no syringyl nucleus compounds. Finally, it is demonstrated that the engineered strain produced ccMA from sugar cane bagasse alkaline extract in 18.7 mol%. NGC7 is thus shown to be a promising microbial chassis for biochemicals production from lignin-derived aromatics.

Exploration and structure-based engineering of alkenal double bond reductases catalyzing the CαCß double bond reduction of coniferaldehyde.

Lignin, a complex aromatic polymer, represents a significant obstacle in lignocellulosic biomass utilization. The polymerization of lignin occurs by radical couplings, which mainly form ether and C-C bonds between monolignol units. The chemical stability of these bonds between monolignol units causes the recalcitrant nature of lignin. Since the Cα-Cß double bond in the monolignols is a crucial chemical feature for the radical coupling, reduction of the double bond would decrease the degree of lignin polymerization, avoiding the recalcitrance of lignin. To develop a method of lignin engineering, we have focused on alkenal double bond reductases (DBR), which can reduce the Cα-Cß double bond of a monolignol precursor. Here, a novel bacterial DBR from Parvibaculum lavamentivorans DS-1 (PlDBR) was found. This enzyme can reduce the side-chain double bond of coniferaldehyde (CALD) and has a 41% amino-acid sequence identity with CALD DBR from Arabidopsis thaliana (AtDBR). The crystal structure of the PlDBR showed that it has a larger substrate-binding pocket than AtDBR, conferring broader substrate specificity on the former. Structural and mutation analyses of PlDBR and AtDBR suggested that Tyr51 and Try252 are critical residues for the catalytic activity of PlDBR. In addition, Tyr81 of AtDBR appears to cause substrate inhibition. Replacing Tyr81 of AtDBR with a smaller amino-acid residue, as in the AtDBR variants Tyr81Leu and Tyr81Ala, resulted in a substantially higher CALD-reducing activity compared to the wild type. These variants would be promising candidates for lignin manipulation to decrease the recalcitrance of lignocellulosic biomass.

Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen.

Lignin is a phenolic polymer deposited in the plant cell wall, and is mainly polymerized from three canonical monomers (monolignols), i.e. p-coumaryl, coniferyl and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway that provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of p-coumaroyl-CoA to p-coumaroyl shikimate, one of the key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase (SK). This expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high SK activity produced a lignin content comparable to that in wild-type plants, and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated SK activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

Functional roles of multiple Ton complex genes in a Sphingobium degrader of lignin-derived aromatic compounds.

TonB-dependent transporters (TBDTs) mediate outer membrane transport of nutrients using the energy derived from proton motive force transmitted from the TonB-ExbB-ExbD complex localized in the inner membrane. Recently, we discovered ddvT encoding a TBDT responsible for the uptake of a 5,5-type lignin-derived dimer in Sphingobium sp. strain SYK-6. Furthermore, overexpression of ddvT in an SYK-6-derivative strain enhanced its uptake capacity, improving the rate of platform chemical production. Thus, understanding the uptake system of lignin-derived aromatics is fundamental for microbial conversion-based lignin valorization. Here we examined whether multiple tonB-, exbB-, and exbD-like genes in SYK-6 contribute to the outer membrane transport of lignin-derived aromatics. The disruption of tonB2-6 and exbB3 did not reduce the capacity of SYK-6 to convert or grow on lignin-derived aromatics. In contrast, the introduction of the tonB1-exbB1-exbD1-exbD2 operon genes into SYK-6, which could not be disrupted, promoted the conversion of ß-O-4-, ß-5-, ß-1-, ß-ß-, and 5,5-type dimers and monomers, such as ferulate, vanillate, syringate, and protocatechuate. These results suggest that TonB-dependent uptake involving the tonB1 operon genes is responsible for the outer membrane transport of the above aromatics. Additionally, exbB2/tolQ and exbD3/tolR were suggested to constitute the Tol-Pal system that maintains the outer membrane integrity.

Lignin valorization through efficient microbial production of ß-ketoadipate from industrial black liquor.

Vanillin and vanillate are the major lignin-derived aromatic compounds produced through the alkaline oxidation of softwood lignin. Because the production of higher-value added chemicals from these compounds is essential for lignin valorization, the microbial production of ß-ketoadipate, a promising raw material for the synthesis of novel nylons, from lignin was considered. Pseudomonas putida KT2440 was engineered to convert vanillin and vanillate to ß-ketoadipate. By examining the culture conditions with an initial culture volume of 1 L, the engineered strain completely converted 25 g of vanillin and 25 g of vanillate and produced approximately 23 g of ß-ketoadipate from each of them with a yield of 93% or higher. Furthermore, this strain showed the ability to efficiently produce ß-ketoadipate from softwood lignin extracts in black liquor, a byproduct of pulp production. These results suggest that the production of ß-ketoadipate from industrial black liquor is highly feasible for substantial lignin valorization.

Caffeoylquinic acids: chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity.

Caffeoylquinic acids (CQAs) are specialized plant metabolites we encounter in our daily life. Humans consume CQAs in mg-to-gram quantities through dietary consumption of plant products. CQAs are considered beneficial for human health, mainly due to their anti-inflammatory and antioxidant properties. Recently, new biosynthetic pathways via a peroxidase-type p-coumaric acid 3-hydroxylase enzyme were discovered. More recently, a new GDSL lipase-like enzyme able to transform monoCQAs into diCQA was identified in Ipomoea batatas. CQAs were recently linked to memory improvement; they seem to be strong indirect antioxidants via Nrf2 activation. However, there is a prevalent confusion in the designation and nomenclature of different CQA isomers. Such inconsistencies are critical and complicate bioactivity assessment since different isomers differ in bioactivity and potency. A detailed explanation regarding the origin of such confusion is provided, and a recommendation to unify nomenclature is suggested. Furthermore, for studies on CQA bioactivity, plant-based laboratory animal diets contain CQAs, which makes it difficult to include proper control groups for comparison. Therefore, a synthetic diet free of CQAs is advised to avoid interferences since some CQAs may produce bioactivity even at nanomolar levels. Biotransformation of CQAs by gut microbiota, the discovery of new enzymatic biosynthetic and metabolic pathways, dietary assessment, and assessment of biological properties with potential for drug development are areas of active, ongoing research. This review is focused on the chemistry, biosynthesis, occurrence, analytical challenges, and bioactivity recently reported for mono-, di-, tri-, and tetraCQAs.

Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics.

Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H+ symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. The mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using 14C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.

Roles of two glutathione S-transferases in the final step of the ß-aryl ether cleavage pathway in Sphingobium sp. strain SYK-6.

Sphingobium sp. strain SYK-6 is an alphaproteobacterial degrader of lignin-derived aromatic compounds, which can degrade all the stereoisomers of ß-aryl ether-type compounds. SYK-6 cells convert four stereoisomers of guaiacylglycerol-ß-guaiacyl ether (GGE) into two enantiomers of α-(2-methoxyphenoxy)-ß-hydroxypropiovanillone (MPHPV) through GGE α-carbon atom oxidation by stereoselective Cα-dehydrogenases encoded by ligD, ligL, and ligN. The ether linkages of the resulting MPHPV enantiomers are cleaved by stereoselective glutathione (GSH) S-transferases (GSTs) encoded by ligF, ligE, and ligP, generating (ßR/ßS)-α-glutathionyl-ß-hydroxypropiovanillone (GS-HPV) and guaiacol. To date, it has been shown that the gene products of ligG and SLG_04120 (ligQ), both encoding GST, catalyze GSH removal from (ßR/ßS)-GS-HPV, forming achiral ß-hydroxypropiovanillone. In this study, we verified the enzyme properties of LigG and LigQ and elucidated their roles in ß-aryl ether catabolism. Purified LigG showed an approximately 300-fold higher specific activity for (ßR)-GS-HPV than that for (ßS)-GS-HPV, whereas purified LigQ showed an approximately six-fold higher specific activity for (ßS)-GS-HPV than that for (ßR)-GS-HPV. Analyses of mutants of ligG, ligQ, and both genes revealed that SYK-6 converted (ßR)-GS-HPV using both LigG and LigQ, whereas only LigQ was involved in converting (ßS)-GS-HPV. Furthermore, the disruption of both ligG and ligQ was observed to lead to the loss of the capability of SYK-6 to convert MPHPV. This suggests that GSH removal from GS-HPV catalyzed by LigG and LigQ, is essential for cellular GSH recycling during ß-aryl ether catabolism.

Author Correction: Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Syringate O-Demethylase Gene of Sphingobium sp. Strain SYK-6 Is Regulated by DesX, while Other Vanillate and Syringate Catabolism Genes Are Regulated by DesR.

Syringate and vanillate are the major metabolites of lignin biodegradation. In Sphingobium sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of desA, ligM, and desB are induced by syringate and vanillate, while those of ligM and desB are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in desA regulation. Here, we clarified the regulatory system for desA transcription by analyzing the IclR-type transcriptional regulator desX, located downstream of desA Quantitative reverse transcription (RT)-PCR analyses of a desX mutant indicated that the transcription of desA was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of ligM and desB The ferulate catabolism genes (ferBA), under the control of a MarR-type transcriptional regulator, FerC, are located upstream of desA RT-PCR analyses suggested that the ferB-ferA-SLG_25010-desA gene cluster consists of the ferBA operon and the SLG_25010-desA operon. Promoter assays revealed that a syringate- and vanillate-inducible promoter is located upstream of SLG_25010. Purified DesX bound to this promoter region, which overlaps an 18-bp inverted-repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that the compounds are effector molecules of DesX.IMPORTANCE Syringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and to elucidate their transcriptional regulation. In this study, we found that the transcription of desA, encoding syringate O-demethylase in SYK-6, is regulated by an IclR-type transcriptional regulator, DesX. The findings of this study, combined with our previous results on desR (encoding a MarR transcriptional regulator that controls the transcription of ligM and desB), provide an overall picture of the transcriptional-regulatory systems for syringate and vanillate catabolism in SYK-6.

Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds.

Iron, an essential element for all organisms, acts as a cofactor of enzymes in bacterial degradation of recalcitrant aromatic compounds. The bacterial family, Sphingomonadaceae comprises various degraders of recalcitrant aromatic compounds; however, little is known about their iron acquisition system. Here, we investigated the iron acquisition system in a model bacterium capable of degrading lignin-derived aromatics, Sphingobium sp. strain SYK-6. Analyses of SYK-6 mutants revealed that FiuA (SLG_34550), a TonB-dependent receptor (TBDR), was the major outer membrane iron transporter. Three other TBDRs encoded by SLG_04340, SLG_04380, and SLG_10860 also participated in iron uptake, and tonB2 (SLG_34540), one of the six tonB comprising the Ton complex which enables TBDR-mediated transport was critical for iron uptake. The ferrous iron transporter FeoB (SLG_36840) played an important role in iron uptake across the inner membrane. The promoter activities of most of the iron uptake genes were induced under iron-limited conditions, and their regulation is controlled by SLG_29410 encoding the ferric uptake regulator, Fur. Although feoB, among all the iron uptake genes identified is highly conserved in Sphingomonad strains, the outer membrane transporters seem to be diversified. Elucidation of the iron acquisition system promises better understanding of the bacterial degradation mechanisms of aromatic compounds.