Overexpression of an LaeA-like Methyltransferase Upregulates Secondary Metabolite Production in Aspergillus nidulans

Abstract

Fungal secondary metabolites (SMs) include medically valuable compounds as well as compounds that are toxic, carcinogenic, and/or contributors to fungal pathogenesis. It is consequently important to understand the regulation of fungal secondary metabolism. McrA is a recently discovered transcription factor that negatively regulates fungal secondary metabolism. Deletion of mcrA (mcrAΔ), the gene encoding McrA, results in upregulation of many SMs and alters the expression of more than 1000 genes. One gene strongly upregulated by the deletion of mcrA is llmG, a putative methyl transferase related to LaeA, a major regulator of secondary metabolism. We artificially upregulated llmG by replacing its promoter with strong constitutive promoters in strains carrying either wild-type mcrA or mcrAΔ. Upregulation of llmG on various media resulted in increased production of the important toxin sterigmatocystin and compounds from at least six major SM pathways. llmG is, thus, a master SM regulator. mcrAΔ generally resulted in greater upregulation of SMs than upregulation of llmG, indicating that the full effects of mcrA on secondary metabolism involve genes in addition to llmG. However, the combination of mcrAΔ and upregulation of llmG generally resulted in greater compound production than mcrAΔ alone (in one case more than 460 times greater than the control). This result indicates that deletion of mcrA and/or upregulation of llmG can likely be combined with other strategies for eliciting SM production to greater levels than can be obtained with any single strategy.

Figure 1.

Replacement of the native llmG promoter with a strong constitutive promoter. A. Four fragments are separately amplified by PCR (primers are given in Supplemental Table 1). The four fragments consisted of a 1080 bp fragment amplified from upstream of the llmG (AN5874) coding sequence (nt minus 1510- to nt minus 430), a 1491 bp fragment containing the Aspergillus terreus pyrG gene (AtpyrG), a fragment containing the constitutive promoter (a 1231 bp fragment in the case of the gpdA promoter and a 502 bp fragment in the case of the hybrid nmtA promoter) and a 1141 bp fragment extending from the start codon of llmG into the llmG coding sequence. B. The four fragments were fused together by fusion PCR using nested primers creating a transforming fragment. B and C. The fusion PCR fragment is used to transform the A. nidulans host strain with AtpyrG as the selectable marker. Double homologous recombination results in llmG transcription being driven by the constitutive promoter.

Figure 2.

HPLC paired profile scans of llmG overexpression and mcrA deletion strains compared to the control strain on A. GMM(s) and B. YAG plates. (i) Control strain (LO11174), (ii) gpdA(p)llmG strain (LO10860), (iii) mcrAΔ strain (LO11177), and (iv) mcrAΔ::gpdA(p)llmG strain (LO10881).

Figure 3.

Chemical structures of compounds that were upregulated in the gpdA(p)llmG (LO10860, LO10864), mcrAΔ (LO11177) and gpdA(p)llmG::mcrAΔ (LO10868, LO10881) strains. Sterigmatocystin (1), terrequinone A (6), emericellin (7), 3’-hydroxyversiconol (11), versiconol (12), nidurufin (13), averufin (14), nidulanin A (18), 2,ω-dihydroxyemodin (21), 3-methylorsellinic acid (22), cichorine intermediate (23), versicolorin B (24), averantin (25), monodictyphenone (26), ω-hydroxyemodin (27), 2-hydroxyemodin (28), emodin (29), chrysophanol (30), O-methyl-3-methylorsellinaldehyde dimer (39), cichorine (40), atrochrysone (44), nidulol (46), trans-emodin-physicon bianthrone (47), cis-emodin-physicon-bianthrone (48).

Figure 4.

Enhanced production of compound 23 observed in a multicluster deletion, mcrAΔ, gpdA(p)llmG strain. A. (i) HPLC profile of a mcrAΔ, gpdA(p)llmG strain (LO10881) on YAG, indicating the production of unknown 23. (ii) HPLC profile, on YAG, of LO11505 in which mcrA was deleted and llmG was upregulated in strain LO8030, that was engineered to have multiple SM gene clusters deleted. Enhanced production of compound 23 was observed in LO11505 compared to LO10881. B. The structure of compound 23. Based on structural similarities observed between compound 23 and cichorine (41), we predict that compound 23 is an intermediate of the cichorine biosynthetic pathway.