Redundancy in aromatic O-demethylation and ring opening reactions in Novosphingobium aromaticivorans and their impact in the metabolism of plant derived phenolics.

Lignin is a plant heteropolymer composed of phenolic subunits. Because of its heterogeneity and recalcitrance, the development of efficient methods for its valorization still remains an open challenge. One approach to utilize lignin is its chemical deconstruction into mixtures of monomeric phenolic compounds followed by biological funneling into a single product. Novosphingobium aromaticivorans DSM12444 has been previously engineered to produce 2-pyrone-4,6-dicarboxylic acid (PDC) from depolymerized lignin by simultaneously metabolizing multiple aromatics through convergent routes involving the intermediates 3-methoxygallic acid (3-MGA) and protocatechuic acid (PCA). We investigated enzymes predicted to be responsible for O-demethylation and oxidative aromatic ring opening, two critical reactions involved in the metabolism of phenolics compounds by N. aromaticivorans The results showed the involvement of DesA in O-demethylation of syringic and vanillic acids, LigM in O-demethylation of vanillic acid and 3-MGA, and a new O-demethylase, DmtS, in the conversion of 3-MGA into gallic acid (GA). In addition, we found that LigAB was the main aromatic ring opening dioxygenase involved in 3-MGA, PCA, and GA metabolism, and that a previously uncharacterized dioxygenase, LigAB2, had high activity with GA. Our results indicate a metabolic route not previously identified in N. aromaticivorans that involves O-demethylation of 3-MGA to GA. We predict this pathway channels ∼15% of the carbon flow from syringic acid, with the rest following ring opening of 3-MGA. The new knowledge obtained in this study allowed for the creation of an improved engineered strain for the funneling of aromatic compounds that exhibits stoichiometric conversion of syringic acid into PDC.IMPORTANCE For lignocellulosic biorefineries to effectively contribute to reduction of fossil fuel use, they need to become efficient at producing chemicals from all major components of plant biomass. Making products from lignin will require engineering microorganisms to funnel multiple phenolic compounds to the chemicals of interest, and N. aromaticivorans is a promising chassis for this technology. The ability of N. aromaticivorans to efficiently and simultaneously degrade many phenolic compounds may be linked to having functionally redundant aromatic degradation pathways and enzymes with broad substrate specificity. A detailed knowledge of aromatic degradation pathways is thus essential to identify genetic engineering targets to maximize product yields. Furthermore, knowledge of enzyme substrate specificity is critical to redirect flow of carbon to desired pathways. This study described an uncharacterized pathway in N. aromaticivorans and the enzymes that participate in this pathway, allowing the engineering of an improved strain for production of PDC from lignin.

A heterodimeric glutathione S-transferase that stereospecifically breaks lignin's ß(R)-aryl ether bond reveals the diversity of bacterial ß-etherases.

Lignin is a heterogeneous polymer of aromatic subunits that is a major component of lignocellulosic plant biomass. Understanding how microorganisms deconstruct lignin is important for understanding the global carbon cycle and could aid in developing systems for processing plant biomass into valuable commodities. Sphingomonad bacteria use stereospecific glutathione S-transferases (GSTs) called ß-etherases to cleave the ß-aryl ether (ß-O-4) bond, the most common bond between aromatic subunits in lignin. Previously characterized bacterial ß-etherases are homodimers that fall into two distinct GST subclasses: LigE homologues, which cleave the ß(R) stereoisomer of the bond, and LigF homologues, which cleave the ß(S) stereoisomer. Here, we report on a heterodimeric ß-etherase (BaeAB) from the sphingomonad Novosphingobium aromaticivorans that stereospecifically cleaves the ß(R)-aryl ether bond of the di-aromatic compound ß-(2-methoxyphenoxy)-γ-hydroxypropiovanillone (MPHPV). BaeAB's subunits are phylogenetically distinct from each other and from other ß-etherases, although they are evolutionarily related to LigF, despite the fact that BaeAB and LigF cleave different ß-aryl ether bond stereoisomers. We identify amino acid residues in BaeAB's BaeA subunit important for substrate binding and catalysis, including an asparagine that is proposed to activate the GSH cofactor. We also show that BaeAB homologues from other sphingomonads can cleave ß(R)-MPHPV and that they may be as common in bacteria as LigE homologues. Our results suggest that the ability to cleave the ß-aryl ether bond arose independently at least twice in GSTs and that BaeAB homologues may be important for cleaving the ß(R)-aryl ether bonds of lignin-derived oligomers in nature.