Pathway discovery and engineering for cleavage of a β-1 lignin-derived biaryl compound

Presley, Gerald N.; Werner, Allison Z.; Katahira, Rui; Garcia, David; Haugen, Stefan J.; Ramirez, Kelsey J.; Giannone, Richard J.; Beckham, Gregg T.

doi:10.1016/j.ymben.2021.02.003

Cited by 28 publications

(42 citation statements)

References 68 publications

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“…Our results showed β-5 linkage was cleavage by choline dehydrogenase (betA) and aldehyde dehydrogenase (aldA/aldB-I) which was partially consistent with literature [56]. In addition, no homologous proteins for lsdE and lsdA were found in P. putida KT2440 showed there might be a different β-1 linkage breakdown mechanism compared to gram-negative bacteria Novosphingobium aromaticivorans DSM12444 [41].…”

Section: Discussionsupporting

confidence: 89%

“…Based on the enzyme activity analysis, GC-MS, NMR, proteomics results, the proposed lignin degradation reaction pathways are shown in Fig. 5 [21,27,37,[40][41][42][43][44][45][46].The enzymes identi ed that might be involved in the lignin catabolism pathway were summarized in Table S4. Overall, the proposed lignin degradation pathways include cleavage of β-aryl ether (β-O-4), resinol (β-β), phenylcoumaran (β-5), and spirodienone (β-1) linkages.…”

Section: Gc-ms Analysis Of Lignin Breakdown Productsmentioning

confidence: 99%

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Understanding of Bacterial Lignin Extracellular Degradation Mechanisms by Pseudomonas putida KT2440 via Secretomic Analysis

Peng

Kitata

et al. 2022

Preprint

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Background: Bacterial lignin degradation is believed to be primarily achieved by a secreted enzyme system. Effects of such extracellular enzyme systems on lignin structural changes and degradation pathways are still not clearly understood, which remains as a bottleneck in the bacterial lignin bioconversion process. Results: This study investigated lignin degradation using an isolated secretome secreted by Pseudomonas putida KT2440 that grew on glucose as the only carbon source. Enzyme assays revealed that the secretome harbored oxidase and peroxidase/Mn2+-peroxidase capacity and reached the highest activity at 120 h of the fermentation time. The degradation rate of alkali lignin was found to be only 8.1% by oxidases, but increased to 14.5% with the activation of peroxidase/Mn2+-peroxidase. Gas chromatography-mass spectrometry (GC-MS) and two-dimensional 1H-13C heteronuclear single-quantum coherence (HSQC) NMR analysis revealed that the oxidases exhibited strong C-C bond (β-β, β-5, and β-1) cleavage. The activation of peroxidases enhanced lignin degradation by stimulating C-O bond (β-O-4) cleavage, resulting in increased yields of aromatic monomers and dimers. Further mass spectrometry-based quantitative proteomics measurements comprehensively identified different groups of enzymes particularly oxidoreductases in P. putida secretome, including reductases, peroxidases, monooxygenases, dioxygenases, oxidases, and dehydrogenases, potentially contributed to the lignin degradation process. Conclusions: Overall, we discovered that bacterial extracellular degradation of alkali lignin to vanillin, vanillic acid, and other lignin derived-aromatics involved a series of oxidative cleavage, catalyzed by active DyP-type peroxidase, multicopper oxidase, and other accessory enzymes. These resutls will guide further metabolic engineering design to improve the efficiency of lignin bioconversion.

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Section: Discussionsupporting

confidence: 89%

Section: Gc-ms Analysis Of Lignin Breakdown Productsmentioning

confidence: 99%

Understanding of Bacterial Lignin Extracellular Degradation Mechanisms by Pseudomonas putida KT2440 via Secretomic Analysis

Peng

Kitata

et al. 2022

Preprint

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“…Cleavage of ring-opened β-1 C–C linkages or stilbene linkages can also yield two molar equivalents of p -vanillin. 31 − 34 β-5 linkages can conceptually yield one molar equivalent of p -vanillin and o -vanillin (2-hydroxy-3-methoxybenzaldehyde) ( Scheme 1 B, C). 35 , 36 The Amycolatopsis sp.…”

mentioning

confidence: 99%

“…As shown in Scheme B, β-O-4 bond cleavage, by far the most well-studied reaction in lignin depolymerization chemistry, − ,, can yield the p -vanillin aldehyde (4-hydroxy-3-methoxybenzaldehyde, commonly referred to simply as “vanillin”). Cleavage of ring-opened β-1 C–C linkages or stilbene linkages can also yield two molar equivalents of p -vanillin. − β-5 linkages can conceptually yield one molar equivalent of p -vanillin and o -vanillin (2-hydroxy-3-methoxybenzaldehyde) (Scheme B, C). , The Amycolatopsis sp. ATCC 39116 GcoAB cytochrome P450 system (WP_020419855, WP_020419854) was therefore engineered for efficient turnover of aromatic aldehydes, specifically targeting p- and o -vanillin: substrates with which GcoAB has little to no native activity.…”

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confidence: 99%

Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes

Ellis

Hinchen

Bleem

et al. 2021

JACS Au

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Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O -methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O- methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o -and p -vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o - and p -isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p -vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo . This work expands the known aromatic O- demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.

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“…In bacteria, LSDs have been assigned catabolic and protective roles. For example, the catabolism of β-1-diaryl propane by Sphingomonas paucimobilis TMY1009 (TMY1009 hereafter), which, like SYK-6, grows on a variety of lignin-derived aromatic compounds, involves a stilbenoid intermediate in the form of lignostilbene ( 25 , 26 ). TMY1009 harbors four isoforms of LSD, which cleave lignostilbene to two equivalents of vanillin ( 19 , 27 ).…”

mentioning

confidence: 99%

Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6

Kuatsjah

Chan

Katahira

et al. 2021

Journal of Biological Chemistry

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Lignostilbene-α,β-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from β-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼10 6 M −1 s −1 ) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.

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Pathway discovery and engineering for cleavage of a β-1 lignin-derived biaryl compound

Cited by 28 publications

References 68 publications

Understanding of Bacterial Lignin Extracellular Degradation Mechanisms by Pseudomonas putida KT2440 via Secretomic Analysis

Understanding of Bacterial Lignin Extracellular Degradation Mechanisms by Pseudomonas putida KT2440 via Secretomic Analysis

Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes

Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6

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