Production of carotenoids from aromatics and pretreated lignocellulosic biomass by Novosphingobium aromaticivorans.

IMPORTANCE: There is economic and environmental interest in generating commodity chemicals from renewable resources, such as lignocellulosic biomass, that can substitute for chemicals derived from fossil fuels. The bacterium Novosphingobium aromaticivorans is a promising microbial platform for producing commodity chemicals from lignocellulosic biomass because it can produce these from compounds in pretreated lignocellulosic biomass, which many industrial microbial catalysts cannot metabolize. Here, we show that N. aromaticivorans can be engineered to produce several valuable carotenoids. We also show that engineered N. aromaticivorans strains can produce these lipophilic chemicals concurrently with the extracellular commodity chemical 2-pyrone-4,6-dicarboxylic acid when grown in a complex liquor obtained from alkaline pretreated lignocellulosic biomass. Concurrent microbial production of valuable intra- and extracellular products can increase the economic value generated from the conversion of lignocellulosic biomass-derived compounds into commodity chemicals and facilitate the separation of water- and membrane-soluble products.

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.