The pathway intermediate 2-keto-3-deoxy-L-galactonate mediates the induction of genes involved in D-galacturonic acid utilization in Aspergillus niger

Abstract

In Aspergillus niger, the enzymes encoded by gaaA, gaaB, and gaaC catabolize d-galacturonic acid (GA) consecutively into l-galactonate, 2-keto-3-deoxy-l-galactonate, pyruvate, and l-glyceraldehyde, while GaaD converts l-glyceraldehyde to glycerol. Deletion of gaaB or gaaC results in severely impaired growth on GA and accumulation of l-galactonate and 2-keto-3-deoxy-l-galactonate, respectively. Expression levels of GA-responsive genes are specifically elevated in the ∆gaaC mutant on GA as compared to the reference strain and other GA catabolic pathway deletion mutants. This indicates that 2-keto-3-deoxy-l-galactonate is the inducer of genes required for GA utilization.

Keywords: d-galacturonic acid catabolism; gene regulation; pectinase.

Figure 1

(A) The evolutionarily conserved GA catabolic pathway in filamentous fungi as proposed by Martens‐Uzunova and Schaap 5. GA is converted in pyruvate and glycerol by consecutive action of GaaA, GaaB, GaaC, and GaaD enzymes. Growth profile of the reference strain (MA249.1) and GA catabolic pathway deletion mutants ∆gaaA, ∆gaaB, ∆gaaC, and ∆gaaD (B) on solid MM without any carbon source, or with 50 mm monomeric or 1% polymeric carbon sources after 7 days at 30 °C, and (C) in microtiter plate in liquid medium with 50 mm GA at 30 °C. Error bars represent standard deviation of six biological replicates.

Figure 2

Metabolic and gene expression analyses of Aspergillus niger GA catabolic pathway deletion mutants ∆gaaA, ∆gaaB, ∆gaaC, and ∆gaaD (A) Extracellular GA, l‐galactonate, and 2‐keto‐3‐deoxy‐l‐galactonate concentration in cultures of the reference strain (FP‐1132.1) and GA catabolic pathway deletion mutants. GA concentration is given in mm and l‐galactonate and 2‐keto‐3‐deoxy‐l‐galactonate amounts are presented as ion chromatogram peak areas relative to ∆gaaB 55 h and ∆gaaC 55 h samples, respectively. (B) Northern blot analysis of selected GA‐responsive genes in the reference strain (MA249.1) and GA catabolic pathway deletion mutants. Actin (NRRL3_03617) was used as a control. (C) RNA‐seq analysis of pectinase genes in the reference strain (FP‐1132.1) and ∆gaaC in GA (FPKM). Expression in ∆gaaR in GA (FPKM) 19 and in the reference strain (MA234.1) and ∆gaaX in d‐fructose (TPM) 20 was shown for comparison. Pectinase genes that belong to the GaaR/GaaX panregulon 20 are indicated with an asterisk. Strains were pregrown in CM with 2% d‐fructose. For metabolic analysis, mycelia were transferred to and grown in MM containing 50 mm GA. For northern blot analysis, mycelia were transferred to and grown in MM containing 50 mm d‐fructose (F) or GA for 2 h. For RNA‐seq analysis, mycelia were transferred to and grown in MM containing 25 mm GA for 2 h.