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.

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.