TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus

Abstract

Small peptides formed from non-ribosomal peptide synthetases (NRPS) are bioactive molecules produced by many fungi including the genus Aspergillus. A subset of NRPS utilizes tryptophan and its precursor, the non-proteinogenic amino acid anthranilate, in synthesis of various metabolites such as Aspergillus fumigatus fumiquinazolines (Fqs) produced by the fmq gene cluster. The A. fumigatus genome contains two putative anthranilate synthases - a key enzyme in conversion of anthranilic acid to tryptophan - one beside the fmq cluster and one in a region of co-linearity with other Aspergillus spp. Only the gene found in the co-linear region, trpE, was involved in tryptophan biosynthesis. We found that site-specific mutations of the TrpE feedback domain resulted in significantly increased production of anthranilate, tryptophan, p-aminobenzoate and fumiquinazolines FqF and FqC. Supplementation with tryptophan restored metabolism to near wild type levels in the feedback mutants and suggested that synthesis of the tryptophan degradation product kynurenine could negatively impact Fq synthesis. The second putative anthranilate synthase gene next to the fmq cluster was termed icsA for its considerable identity to isochorismate synthases in bacteria. Although icsA had no impact on A. fumigatus Fq production, deletion and over-expression of icsA increased and decreased respectively aromatic amino acid levels suggesting that IcsA can draw from the cellular chorismate pool.

Keywords: Aspergillus fumigatus; Fumiquinazoline; Metabolic engineering; Metabolic flux; Secondary metabolism; Tryptophan metabolism.

Figure 1. Fumiquinazoline biosynthesis in A. fumigatus

(Modified from that of Ames et al. (2010) and Lim et al.(2014)) (A) Fumiquinazoline gene cluster in A. fumigatus with nearby putative anthranilate synthase gene Afu6g12110 (icsA).* Abbreviation: 12090, Afu6g12090; 12100, Afu6g12100; 12110, Afu6g12110. (B) Biosynthetic route of fumiquinazolines in A. fumigatus. Abbreviations: Ant, anthranilate; l-Trp, l-tryptophan; l-Ala, l-alanine; FqF, fumiquinazoline F; FqA, fumiquinazoline A; FqC, fumiquinazoline C.

Figure 2. Schematic outline of the l-tryptophan metabolism and regulation of enzyme in A. fumigatus

Enzymes are indicated by their gene designations: AroM (Afu1g13740), shikimate kinase (EC:2.7.1.71), EPSP synthase (EC:2.5.1.19); AroB (Afu1g06940), chorismate synthase (EC:4.2.3.5); TrpC (Afu1g13090), TrpE (Afu6g12580), anthranilate synthase (EC:4.1.3.27); TrpD (Afu4g11980), anthranilate phosphoribosyltransferase (EC:2.4.2.18); TrpC (Afu1g13090), phosphoribosylanthranilate isomerase (EC:5.3.1.24), indole-3-glycerol-phosphate synthase (EC:4.1.1.48); TrpB (Afu2g13250), tryptophan synthase (EC:4.2.1.20); PabaA (Afu6g04820), ADC synthetase (EC:2.6.1.85); PabaB (Afu2g01650), ADC lyase (4.1.3.38); AroC (Afu5g13130), chorismate mutase (EC:5.4.99.5); 2g10450 (Afu2g10450), prephenate dehydrogenase (EC:1.3.1.13); 5g05690 (Afu5g05690), prephenate dehydratase (EC:4.2.1.51); AroH (Afu2g13630), aromatic aminotransferase (EC:2.6.1.27; 2.6.1.57; 2.6.1.5); IcsA (Afu6g12110), isochorismate synthetase (EC:5.4.4.2); IdoA (Afu3g14250), IdoB (Afu4g09830), IdoC (Afu7g02010), indoleamine 2,3-dioxygenase (EC:1.13.11.52). Abbreviations: EPSP, enolpyruvylshikimate-3-phosphate; ADC, 4-Amino-4-deoxychorismate.

Figure 3. Maximum likelihood phylogenetic analysis of chorismate-binding domains extracted from 99 characterized protein sequences

Monophyletic leaves of relevant proteins are boxed in grey. A. fumigatus proteins are indicated in bold.

Figure 4. Domain architecture and expression analysis of chorismate-binding domain proteins in A. fumigatus

(A) Domain comparison of A. fumigatus TrpE, PabaA and IcsA. Abbreviations: Ant, Anthranilate; Trp, Tryptophan. (B) Northern expression analysis of trpE and icsA in A. fumigatus wild-type AF293. Ribosomal RNA was visualized by ethidium bromide staining as loading control. A. fumigatus strain AF293 was grown in liquid GMM containing 20mM glutamine as nitrogen source at 37 °C and 250 rpm for 24 h. Then mycelia were collected, transferred into solid GMM and grown in duplicates for the indicated time at 29 °C.

Figure 5. Physiological analysis of A. fumigatus mutants used in this study and the alignment of anthranilate synthases

(A) Radial growth of the A. fumigatus wild-type strain (WT), ΔtrpE, ΔicsA, double deletion mutant ΔicsAΔtrpE, icsA over-expression strain OE::icsA, double mutant ΔtrpE OE::icsA, ΔtrpE complemented strain trpE^C, and site mutants trpE^S77L and trpE^S66R,S77L and on solid GMM and GMM amended with 5mM l-tryptophan (Trp) medium at 37 °C. (B) Alignment of the L*ES*_nS regions of anthranilate synthases from various organisms. The conserved motif is indicated on the bottom line. The sequences shown are: Ec-TrpE, Escherichia coli TrpE (CAA23666); St-TrpE, Salmonella enterica TrpE(WP_001194371); Vc-TrpE, Vibrio cholerae TrpE (WP_001030227); Sc-Trp2, Saccharomyces cerevisiae Trp2 (NP_011014); At-ASA1, Arabidopsis thaliana ASA1 (NP_001190231); At-ASA2, Arabidopsis thaliana ASA2 (NP_180530); An-AN3695, Aspergillus nidulans AN3695 (CBF75591); At-AT03262, Aspergillus terreus AT03262 (XP_001212440); Af-TrpE, Aspergillus fumigatus TrpE (XP_751136). Identical residues among the various proteins are indicated by dark shading, and respective amino acid residue numbers are shown at the C-termini. Arrows mark amino acids of Af-TrpE mutated in this study. (C) Quantification of radial growth on solid media GMM and GMM+5mM l-tryptophan (Trp) at 37 °C, dry weight of mycelia from culture in liquid medium GMM and GMM+5mM l-tryptophan (Trp) at 37 °C, and spore production on solid GMM and GMM+5mM l-tryptophan (Trp) at 37 °C. No phenotype was observed for tryptophan-feedback mutants trpE^S77L and trpE^S66R,S77L compared with trpE^C.

Figure 6. Primary metabolites and fumiquinazoline production in A. fumigatus trpE and icsA mutants

(A) Comparison of primary metabolites and fumiquinazoline production in the complemented control strain trpE^C and the feedback site mutants trpE^S77L and trpE^S66R,S77L grown on GMM medium. (B) Comparison of primary metabolites and fumiquinazoline production in wild-type control strain AF293.1 comp (TJW55.2) and icsA mutants grown on GMM medium. (C) Comparison of primary metabolites and fumiquinazoline production in the complemented control strain trpE^C and the feedback site mutants trpE^S77L and trpE^S66R,S77L grown on GMM supplemented with 5mM l-tryptophan (Trp) medium. The amounts of metabolites were normalized to the relative amount of the complemented control strain trpE^C (100%). Displayed are means ± SEM. Asterisk indicates p < 0.05 using an ANOVA test for statistical significance with Prism 6 software, comparing mutants to the control strain TJW55.2 or trpE^C. Abbreviations: FqF, Fumiquinazoline F; FqC, Fumiquinazoline C; FqA, Fumiquinazoline A; Ala, l-alanine; Ant, Anthranilate; Pab, p-aminobenzoate; Phe, l-phenylalanine; Trp, l-tryptophan; Tyr, l-tyrosine; Knn, l-kynurenine; Idp, Indolepyruvate.

Figure 7. Northern expression analysis of genes related to tryptophan metabolism

Northern expression analysis of indicated genes comparing the A. fumigatus ΔtrpE complemented strain trpE^C, the trpE tryptophan-feedback mutant trpE^S77L, and trpE^S66R,S77L. Ribosomal RNA was visualized by ethidium bromide staining as loading control. The indicated strains were grown in liquid glucose minimal media (GMM) (replacing nitrate with 20 mM glutamine as sole nitrogen source) at 37 °C and 250 rpm for 24 h. After the initial incubation period, 2 g of mycelia were transferred into liquid GMM and GMM supplemented with 5mM l-tryptophan (Trp), respectively. The indicated strains were grown in duplicates for 1 h at 29 °C and 250 rpm.