Engineering Novosphingobium aromaticivorans to produce cis,cis-muconic acid from biomass aromatics

Abstract

The platform chemical cis,cis-muconic acid (ccMA) provides facile access to a number of monomers used in the synthesis of commercial plastics. It is also a metabolic intermediate in the β-ketoadipic acid pathway of many bacteria and, therefore, a current target for microbial production from abundant renewable resources via metabolic engineering. This study investigates Novosphingobium aromaticivorans DSM12444 as a chassis for the production of ccMA from biomass aromatics. The N. aromaticivorans genome predicts that it encodes a previously uncharacterized protocatechuic acid (PCA) decarboxylase and a catechol 1,2-dioxygenase, which would be necessary for the conversion of aromatic metabolic intermediates to ccMA. This study confirmed the activity of these two enzymes in vitro and compared their activity to ones that have been previously characterized and used in ccMA production. From these results, we generated one strain that is completely derived from native genes and a second that contains genes previously used in microbial engineering synthesis of this compound. Both of these strains exhibited stoichiometric production of ccMA from PCA and produced greater than 100% yield of ccMA from the aromatic monomers that were identified in liquor derived from alkaline pretreated biomass. Our results show that a strain completely derived from native genes and one containing homologs from other hosts are both capable of stoichiometric production of ccMA from biomass aromatics. Overall, this work combines previously unknown aspects of aromatic metabolism in N. aromaticivorans and the genetic tractability of this organism to generate strains that produce ccMA from deconstructed biomass.IMPORTANCEThe production of commodity chemicals from renewable resources is an important goal toward increasing the environmental and economic sustainability of industrial processes. The aromatics in plant biomass are an underutilized and abundant renewable resource for the production of valuable chemicals. However, due to the chemical composition of plant biomass, many deconstruction methods generate a heterogeneous mixture of aromatics, thus making it difficult to extract valuable chemicals using current methods. Therefore, recent efforts have focused on harnessing the pathways of microorganisms to convert a diverse set of aromatics into a single product. Novosphingobium aromaticivorans DSM12444 has the native ability to metabolize a wide range of aromatics and, thus, is a potential chassis for conversion of these abundant compounds to commodity chemicals. This study reports on new features of N. aromaticivorans that can be used to produce the commodity chemical cis,cis-muconic acid from renewable and abundant biomass aromatics.

Keywords: Novosphingobium; aromatic compounds; carbon metabolism; decarboxylases; extradiol; intradiol; lignin; metabolic engineering; muconic acid; sphingomonads.

Fig 1

Metabolic pathways for aromatic catabolism from H and G phenolic monomers and other aromatics (benzoic acid, phenol, and guaiacol, right). Two central intermediates, PCA and catechol of their metabolism, are shown.

Fig 2

Cell density (a) and extracellular concentrations of vanillic acid (b) and PCA (c) in cultures of the parent strain 12444 (purple), the 12444_ΔligAB1/2 (yellow) strain, and the ΔligAB1/2 ΔnadBCD (12444_PCA, green) strain. Cells were grown in batch cultures in minimal media containing 2 mM vanillic acid and 10 mM glucose. No other aromatics were detected in the media. All experiments were performed in triplicate. Error bars represent one standard deviation above and below the mean.

Fig 3

The dependence of NadCD on a prFMN source for PCA decarboxylase activity. Experiments were performed with 100 nM NadCD with 1 mM PCA in either the presence or absence of a prFMN lysate. The bars represent the concentration of either PCA (red) or catechol (blue) after 18 h of incubation at room temperature. A control of lysate with 1 mM PCA was also performed to ensure no PCA to catechol conversion with lysate only. NadCD only produced catechol (blue) from PCA (red) when in the presence of a prFMN source. Each bar is the average of three trials with error bars representing one standard deviation.

Fig 4

Time-dependent conversion of PCA (red) to catechol (blue) by either 100 nM EcAroY (a) or 100 nM NadCD (b) with 1 mM PCA in prFMN lysate in 50 mM HEPES, 150 mM NaCl pH 7.5. Each data point represents the average of three trials with error bars representing one standard deviation.

Fig 5

Cell density (a) and extracellular metabolite concentration of vanillic acid (b) and PCA (c) of LigAB1_EcDec (red) and LigAB1_NaDec (blue). Cultures were grown with 2 mM vanillic acid and 10 mM glucose, and metabolite concentrations were analyzed by liquid chromatography mass spectroscopy. The data points are the average of three trials with error bars representing one standard deviation.

Fig 6

Representative time courses of the formation of ccMA with either EcCatA (red) or NaCatA (blue). Both reactions were performed in 50 mM HEPES 150 mM NaCl pH 7.5 with either 0.5 µM EcCatA (a) or NaCatA (b) and initiated with 100 μM catechol. The formation of ccMA was monitored by UV/vis absorption spectroscopy at λ_260-nm and the data points were best fit to a linear equation ([ccMA] = kt + [ccMA]₀). The resulting fit is shown as the black line. The inset shows the average rate after three trials with error represented by one standard deviation above and below the mean.

Fig 7

Cell density (a) of EcDec_ccMA (red) and NaDec_ccMA (blue) and extracellular metabolite concentration of PCA (b), catechol (c), and ccMA (d). Cultures were grown in a shake flask with 2 mM PCA and 10 mM glucose. Metabolite concentrations were analyzed by liquid chromatography mass spectroscopy. Each data point represents the average of three trials with error bars representing one standard deviation.

Fig 8

Conversion of Qsub APL into ccMA with either EcDec_ccMA or NaDec_ccMA N. aromaticivorans strains. The first bar represents the total concentration of the major free and glycosylated aromatics identified from Qsub APL with PCA (red), vanillic acid (blue), and 4-hydroxybenzoic acid (gray). Next, the bars represent the concentration of ccMA produced (green) from either the EcDec_ccMA or NaDec_ccMA strains after 48h incubation with Qsub APL. Each bar is the average of three trials with error bars representing one standard deviation.