Rapid, Parallel Identification of Catabolism Pathways of Lignin-Derived Aromatic Compounds in Novosphingobium aromaticivorans

Abstract

Transposon mutagenesis is a powerful technique in microbial genetics for the identification of genes in uncharacterized pathways. Recently, the throughput of transposon mutagenesis techniques has been dramatically increased through the combination of DNA barcoding and high-throughput sequencing. Here we show that, when applied to catabolic pathways, barcoded transposon libraries can be used to distinguish redundant pathways, decompose complex pathways into substituent modules, discriminate between enzyme homologs, and rapidly identify previously-hypothetical enzymes in an unbiased genome-scale search. We use this technique to identify two genes, which we name and, are involved in the degradation of the lignin-derived aromatic compound sinapic acid in the non-model bacterium We show that DesC is a methyl-esterase acting on an intermediate formed during sinapic acid catabolism, providing the last enzyme in a proposed catabolic pathway. This approach will be particularly useful in the identification of complete pathways suitable for heterologous expression in metabolic engineering. Identification of the genes involved in specific biochemical transformations is a key step in predicting microbial function from nucleic acid sequences and in engineering microbes to endow them with new functions. We have shown that new techniques for transposon mutagenesis can dramatically simplify this process and allow the rapid identification of genes in uncharacterized pathways. These techniques provide the necessary scale to fully elucidate complex biological networks such as those used to degrade mixtures of lignin-derived aromatic compounds.

Funneling aromatic products of chemically depolymerized lignin into 2-pyrone-4-6-dicarboxylic acid withNovosphingobium aromaticivorans

“…In N. aromaticivorans, LigAB has been proposed to catalyze ring opening to produce a mixture of stereoisomers of 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD); a cis-trans isomerase, DesD, isomerizes one of the stereoisomers, and the methylesterase DesC completes demethylation of CHMOD to OMA. 2 Two other routes of 3-MGA degradation are proposed in Sphingobium sp. SYK-6, one requiring ring opening by the 3,4dioxygenase DesZ and cyclization to PDC and another one requiring O-demethylation to gallate by LigM followed by ring opening by the dioxygenase DesB.…”

“…1 While LigM is present in N. aromaticivorans, homologues of DesZ and DesB are not encoded in its genome. 2 In addition, the LigAB of Sphingobium sp. SYK-6 has been shown to produce a combination of CHMOD and PDC when 3-MGA is the substrate, 28 and there are reports of slow abiotic transformation of CHMOD to PDC.…”

“…34 The presence of a catechol degradation pathway in N. aromaticivorans that uses 2,3-cleavage of the aromatic ring has been suggested as a possible alternative pathway for protocatechuic acid degradation. 2 Such alternative non-specific reaction of a catechol dioxygenase could explain the observed lower efficiencies in the transformation of some G and H aromatics to PDC. This hypothesis is supported by the increased cell density observed in cultures of strain 12444ΔligIΔdesCD grown in media containing glucose plus protocatechuic acid compared to cultures only fed glucose ( Table 2).…”

“…5A), indicating that either desC, desD or both genes are essential for growth on syringic acid, in agreement with observations reported previously. 2 To test the 12444ΔdesCD strain for a defect in S aromatic metabolism when growth was occurring, we inoculated the strain into media containing both 3 mM glucose and 3 mM syringic acid (Fig. 5C).…”

Horizontal transfer of a pathway for coumarate catabolism unexpectedly inhibits purine nucleotide biosynthesis

“…The extraction was then placed at −20°C for 15 min, cells/debris pelleted at 21,000 × g for 5 min at 4°C, and supernatants transferred to cold autosampler vials. Five microliters of each extract was then analyzed by high‐resolution LC‐MS/MS using a Vanquish UHPLC plumbed directly in‐line with a Q Exactive Plus mass spectrometer (Thermo Scientific) outfitted with an in‐house pulled nanospray emitter, as previously described (Cecil et al , ). Samples ( n = 4 per strain; biological replicates) were analyzed across 3 separate LC‐MS injects, with technical replicates ( n = 3) and blank runs spread across the entire sampling campaign time of 20 h. Precursor abundances were derived for specific nucleotide phosphates (AxP, GxP, IxP) via Skyline (MacLean et al , ) at 5 ppm accuracy and normalized to IPTG, which is taken up by cells but not metabolized.…”