Abstract
Tannins and hydroxylated aromatic acids, such as gallic acid (3,4,5-trihydroxybenzoic acid), are plant secondary metabolites which protect plants against herbivores and plant-associated microorganisms. Some microbes, such as the yeast Arxula adeninivorans are resistant to these antimicrobial substances and are able to use tannins and gallic acid as carbon sources. In this study, the Arxula gallic acid decarboxylase (Agdc1p) which degrades gallic acid to pyrogallol was characterized and its function in tannin catabolism analyzed. The enzyme has a higher affinity for gallic acid (Km −0.7 ± 0.2 mM, kcat −42.0 ± 8.2 s−1) than to protocatechuic acid (3,4-dihydroxybenzoic acid) (Km −3.2 ± 0.2 mM, kcat −44.0 ± 3.2 s−1). Other hydroxylated aromatic acids, such as 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid are not gallic acid decarboxylase substrates. A. adeninivorans G1212/YRC102-AYNI1-AGDC1, which expresses the AGDC1 gene under the control of the strong nitrate inducible AYNI1 promoter achieved a maximum gallic acid decarboxylase activity of 1064.4 U/l and 97.5 U/g of dry cell weight in yeast grown in minimal medium with nitrate as nitrogen source and glucose as carbon source. In the same medium, gallic acid decarboxylase activity was not detected for the control strain G1212/YRC102 with AGDC1 expression under the control of the endogenous promoter. Gene expression analysis showed that AGDC1 is induced by gallic acid and protocatechuic acid. In contrast to G1212/YRC102-AYNI1-AGDC1 and G1212/YRC102, A. adeninivorans G1234 [Δagdc1] is not able to grow on medium with gallic acid as carbon source but can grow in presence of protocatechuic acid. This confirms that Agdc1p plays an essential role in the tannic acid catabolism and could be useful in the production of catechol and cis,cis-muconic acid. However, the protocatechuic acid catabolism via Agdc1p to catechol seems to be not the only degradation pathway.
Highlights
The non-conventional, non-pathogenic, imperfect, dimorphic yeast Arxula adeninivorans
There are several conserved domains that are fundamental for enzyme catalytic activity and structure were found in the Agdc1p sequence such as subunit B which is homologous with Lactobacillus plantarum WCFS1 gallic acid decarboxylase and putative gallic acid decarboxylase protein sequences from other lactic bacteria described by Jiménez et al (2013) (Figure 1A)
Decarboxylation of hydroxylated aromatic acids has been reported in bacteria and fungi
Summary
The non-conventional, non-pathogenic, imperfect, dimorphic yeast Arxula adeninivorans Blastobotrys adeninivorans) is a fungal organism with great biotechnological potential due to its wide substrate spectrum and robustness against osmotic, salt and temperature stresses (Middelhoven et al, 1984; Wartmann et al, 1995; Malak et al, 2016). It grows in harsh environments, tolerates up to 20% NaCl in the medium, endures temperatures of ≤48◦C. In 2014, the complete genome of A. adeninivorans LS3 was sequenced and genome analysis revealed the presence of two potential tannin acyl hydrolases (Atan1p, Atan2p) as well as gallic acid decarboxylase (Agdc1p) (Kunze et al, 2014)
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