Discovery and Functional Analysis of a Salicylic Acid Hydroxylase from Aspergillus niger

Abstract

Salicylic acid plays an important role in the plant immune response, and its degradation is therefore important for plant-pathogenic fungi. However, many nonpathogenic microorganisms can also degrade salicylic acid. In the filamentous fungus Aspergillus niger, two salicylic acid metabolic pathways have been suggested. The first pathway converts salicylic acid to catechol by a salicylate hydroxylase (ShyA). In the second pathway, salicylic acid is 3-hydroxylated to 2,3-dihydroxybenzoic acid, followed by decarboxylation to catechol by 2,3-dihydroxybenzoate decarboxylase (DhbA). A. niger cleaves the aromatic ring of catechol catalyzed by catechol 1,2-dioxygenase (CrcA) to form cis,cis-muconic acid. However, the identification and role of the genes and characterization of the enzymes involved in these pathways are lacking. In this study, we used transcriptome data of A. niger grown on salicylic acid to identify genes (shyA and crcA) involved in salicylic acid metabolism. Heterologous production in Escherichia coli followed by biochemical characterization confirmed the function of ShyA and CrcA. The combination of ShyA and CrcA demonstrated that cis,cis-muconic acid can be produced from salicylic acid. In addition, the in vivo roles of shyA, dhbA, and crcA were studied by creating A. niger deletion mutants which revealed the role of these genes in the fungal metabolism of salicylic acid.IMPORTANCE Nonrenewable petroleum sources are being depleted, and therefore, alternative sources are needed. Plant biomass is one of the most abundant renewable sources on Earth and is efficiently degraded by fungi. In order to utilize plant biomass efficiently, knowledge about the fungal metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable compounds such as cis,cis-muconic acid. cis,cis-Muconic acid is an important platform chemical that is used to synthesize nylon, polyethylene terephthalate (PET), polyurethane, resins, and lubricants. Currently, cis,cis-muconic acid is mainly produced through chemical synthesis from petroleum-based chemicals. Here, we show that two enzymes from fungi can be used to produce cis,cis-muconic acid from salicylic acid and contributes in creating alternative methods for the production of platform chemicals.

Keywords: catechol-dioxygenase; chemical building block; intradiol ring fission; platform chemical; salicylic acid metabolism.

FIG 1

Expression of A. niger shyA in two independent E. coli strains, ShyA.1 and ShyA.2. (a and b) LB plates (a) and liquid cultures (b) contained 1 mM salicylic acid and were incubated for 2 days. An E. coli strain containing the empty vector without an insert was used as a control. After incubation, the plates were stained with FeCl₃, coloring salicylic acid purple and catechol black. (c) Salicylic acid concentrations in culture medium were determined by HPLC. Error bars represent the standard deviations of three biological replicates. Asterisks indicate significant differences in salicylic acid concentration compared with the empty vector control (Student’s t test, P value ≤ 0.01).

FIG 2

Conversion of salicylic acid by cell extracts of the E. coli strains producing ShyA, CrcA, and HqdA from A. niger. Black, light gray, and dark gray bars represent salicylic acid, catechol, and cis,cis-muconic acid, respectively. The concentration of cis,cis-muconic acid was slightly overestimated due to the impurity of the standard. Error bars represent the standard deviations of three biological replicates. Asterisks indicate significant differences in salicylic acid concentration compared with the empty-vector control (Student’s t test, P value ≤ 0.01).

FIG 3

Effects of pH and temperature on ShyA activity with 1 mM salicylic acid. (a). ShyA activity at 30°C after 15 min under different pH conditions; (b) ShyA activity at pH 6.0 after 15 min of incubation at different temperatures. Error bars represent the standard deviations between three replicates. (c) Rate of reaction of ShyA with salicylic acid. The assay was performed at pH 6.0 and 30°C. Error bars represent the standard deviations between three experiments.

FIG 4

Growth profile of the A. niger ΔshyA, ΔdhbA, ΔcrcA, ΔhqdA, Δ43, Δ2597, and Δ5330 deletion mutants and the reference strain on aromatic compounds. Phenotypes were examined after 7 days at 30°C. Fructose and a no-carbon-source condition served as growth controls.

FIG 5

Growth profile of the deletion strains and the reference on MM agar plates containing salicylic acid, 2,3-dihydroxybenzoic acid, and catechol after 7 days of growth and stained with FeCl₃. As a control, noninoculated plates were stained with FeCl₃.

FIG 6

The salicylic acid metabolic pathway and oxoadipate pathway in A. niger. Confirmed pathways are shown with black arrows, and the suggested m-hydroxylation pathway is shown with a gray dashed arrow. Boxed in gray are the gene expression fold change values on salicylic acid compared to the no-carbon-source control for the corresponding gene. NRRL3_10508, putative muconate isomerase; NRRL3_10507, putative muconolactone isomerase; NRRL3_4788, putative 3-oxoadipate enol-lactone hydrolase; NRRL3_1886, putative 3-oxoadipate CoA transferase; NRRL3_1526, putative 3-oxoacyl CoA thiolase.