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

Abstract

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.

Keywords: Sphingobium sp. strain SYK-6; acetophenone; biotin-dependent carboxylase; cis; cis-muconic acid; lignin.

FIG 1

(A) Catabolic pathway of AV and AS in Sphingobium sp. strain SYK-6. The pathways for both guaiacyl (R = H)- and syringyl (R = OCH₃)-type compounds are shown. VAA, an intermediate metabolite of GGE, has been suggested to be spontaneously decarboxylated to AV (45, 46). Enzymes: AcvAB, AVP/ASP synthetase; AcvF, AVP/ASP phosphatase; AcvCDE, biotin-dependent carboxylase; LigD, LigL, and LigN, Cα-dehydrogenases; LigF, LigE, and LigP, β-etherases; LigG and LigQ, glutathione S-transferases; HpvZ, HPV/HPS oxidase; ALDHs, aldehyde dehydrogenases; SLG_20400, vanilloyl acetaldehyde dehydrogenase. AV, acetovanillone; AS, acetosyringone; AVP, 4-acetyl-2-methoxyphenylphosphate; ASP, 4-acetyl-2,6-dimethoxyphenylphosphate; VAA, vanilloyl acetic acid; SAA, 3-(4-hydroxy-3,5-dimethoxyphenyl)-3-oxopropanoic acid; GGE, guaiacylglycerol-β-guaiacyl ether; HPV, β-hydroxypropiovanillone; HPS, β-hydroxypropiosyringone. (B) Gene organization of acvABCDEF. Arrows indicate the genes from SLG_06570 to SLG_06480. (C) RT-PCR analysis of acvABCDEF. Total RNA used for cDNA synthesis was isolated from SYK-6 cells grown in Wx-SEMP containing 5 mM AV. The regions to be amplified are indicated by black bars below the genetic map. Lanes: M, molecular size markers; g, control PCR with the SYK-6 genomic DNA; + and −, RT-PCR with and without reverse transcriptase, respectively.

FIG 2

HPLC-MS analysis of AV metabolites. Cells of SYK-6 grown with AV (OD₆₀₀ = 0.2) were incubated with 1 mM AV in Wx medium. Portions of the reaction mixtures were collected at the start (A) and after 33 h (B) of incubation and then analyzed by HPLC-MS. The ESI-MS spectra of compounds I and II (negative mode) are shown in panels C and D, respectively. (E and F) Chemical structures of compound I (vanillic acid) and compound II (VAA), respectively.

FIG 3

Conversions of AV and AS by resting cells of S. japonicum UT26S carrying acvABCDEF. Resting cells of UT26S harboring pJB861 (OD₆₀₀ = 10.0; A and B) and resting cells of UT26S harboring pJBacv (OD₆₀₀ = 10.0; C to F) were incubated with AV (200 μM; A to D) or AS (200 μM; E and F). Portions of the reaction mixtures were collected at the start (A, C, and E), after 1 h (B and D), and after 4 h (F) of incubation and analyzed by HPLC-MS. The ESI-MS spectrum of compound III (negative mode) is shown in panel G. (H) Chemical structure of compound III (SAA).

FIG 4

Conversion of AV by crude AcvA and AcvB. AV (200 μM) was incubated with a mixture of cell extracts of E. coli BL21(DE3) harboring pE16acvA and E. coli BL21(DE3) harboring pET-16b (500 μg protein/mL each; A), a mixture of cell extracts of E. coli BL21(DE3) harboring pE16acvB and E. coli BL21(DE3) harboring pET-16b (500 μg protein/mL each; B), and a mixture of cell extracts of E. coli BL21(DE3) harboring pE16acvA and E. coli BL21(DE3) harboring pE16acvB (500 μg protein/mL each; C). Reactions were performed in the presence of 2 mM ATP, 2 mM MgCl₂, and 200 μM MnCl₂. Portions of the reaction mixtures were collected after 30 min of incubation and analyzed by HPLC. The ESI-MS spectrum of compound IV (negative mode) is shown in panel D. (E) Chemical structure of compound IV (AVP).

FIG 5

Conversions of AVP and ASP by AcvF. AVP (100 μM; A and B) and ASP (100 μM; C and D) were incubated with purified AcvF (5 μg protein/mL). Portions of the reaction mixtures were collected at the start (A and C) and 60 min (B and D) of incubation and analyzed by HPLC.

FIG 6

A mixture of AcvA-AcvB, AcvF, and AcvC-AcvD-AcvE catalyzed carboxylation of AV and AS. AV (100 μM; A to C) and AS (100 μM; D to F) were incubated with a cell extract of S. japonicum UT26S harboring pQF (1 mg protein/mL; A, B, D, and E) or a cell extract of UT26S harboring pQFacvCDE (1 mg protein/mL; C and F) in the presence of AcvA-AcvB and AcvF. Specifically, the reactions were performed in the presence of a cell extract of E. coli BL21(DE3) harboring pE16acvA (1 mg protein/mL), a cell extract of E. coli BL21(DE3) harboring pE16acvB (1 mg protein/mL), purified AcvF (10 μg protein/mL), 2 mM ATP, 2 mM MgCl₂, 200 μM MnCl₂, and 10 mM NaHCO₃. Portions of the reaction mixtures were collected at the start (A and D) and after 60 min (B, C, E, and F) of incubation and analyzed by HPLC.

FIG 7

Production of ccMA from AV through the engineered metabolic pathway constructed in Pseudomonas sp. NGC7. (A) Engineered route for ccMA production from AV. Enzymes: AcvAB, AVP/ASP synthetase; AcvF, AVP/ASP phosphatase; AcvCDE, biotin-dependent carboxylase; VceA, VAA/SAA-converting enzyme; VceB, vanilloyl-CoA/syringoyl-CoA thioesterase; VanAB, vanillate O-demethylase (the vanillic acid-converting enzyme gene has not yet been identified); AroY, protocatechuic acid decarboxylase; PcaHG, protocatechuic acid 3,4-dioxygenase; CatA, catechol 1,2-dioxygenase; CatB, ccMA cycloisomerase. Abbreviations: AV, acetovanillone; AVP, 4-acetyl-2-methoxyphenylphosphate; VAA, vanilloyl acetic acid; ccMA, cis,cis-muconic acid. (B) Schematic representations of the NGC703 recombinant strain, which contains pSEVAacv and pTS093vce-aroY. (C) Conversion of 1.2 mM AV by NGC703(pSEVAacv + pTS093vce-aroY) cells during growth in MMx-3 medium containing 15 g/L glucose. The concentrations of AV (red), VAA (purple), vanillic acid (green), protocatechuic acid (mustard), ccMA (blue), and cis,trans-muconic acid (gray) were periodically measured by HPLC. The concentration of glucose (triangles) was measured by a glucose electrode. Cell growth (squares) was monitored by measuring the OD₆₀₀. All experiments were performed in triplicate, and values represent the averages ± standard deviations.