Activation of a silent fungal polyketide biosynthesis pathway through regulatory cross talk with a cryptic nonribosomal peptide synthetase gene cluster

Abstract

Filamentous fungi produce numerous natural products that constitute a consistent source of potential drug leads, yet it seems that the majority of natural products are overlooked since most biosynthesis gene clusters are silent under standard cultivation conditions. Screening secondary metabolite genes of the model fungus Aspergillus nidulans, we noted a silent gene cluster on chromosome II comprising two nonribosomal peptide synthetase (NRPS) genes, inpA and inpB, flanked by a regulatory gene that we named scpR for secondary metabolism cross-pathway regulator. The induced expression of the scpR gene using the promoter of the alcohol dehydrogenase AlcA led to the transcriptional activation of both the endogenous scpR gene and the NRPS genes. Surprisingly, metabolic profiling of the supernatant of mycelia overexpressing scpR revealed the production of the polyketide asperfuranone. Through transcriptome analysis we found that another silent secondary metabolite gene cluster located on chromosome VIII coding for asperfuranone biosynthesis was specifically induced. Quantitative reverse transcription-PCR proved the transcription not only of the corresponding polyketide synthase (PKS) biosynthesis genes, afoE and afoG, but also of their activator, afoA, under alcAp-scpR-inducing conditions. To exclude the possibility that the product of the inp cluster induced the asperfuranone gene cluster, a strain carrying a deletion of the NRPS gene inpB and, in addition, the alcAp-scpR overexpression cassette was generated. In this strain, under inducing conditions, transcripts of the biosynthesis genes of both the NRPS-containing gene cluster inp and the asperfuranone gene cluster except gene inpB were detected. Moreover, the existence of the polyketide product asperfuranone indicates that the transcription factor ScpR controls the expression of the asperfuranone biosynthesis gene cluster. This expression as well as the biosynthesis of asperfuranone was abolished after the deletion of the asperfuranone activator gene afoA, indicating that ScpR binds to the afoA promoter. To the best of our knowledge, this is the first report of regulatory cross talk between two biosynthesis gene clusters located on different chromosomes.

FIG. 1.

Northern blot analysis. Total RNA (10 μg) from the A. nidulans wild-type and transformant strains was analyzed. All strains were incubated in AMM under alcAp-repressing (lanes R) and alcAp-inducing (lanes I) conditions. scpR (AN3492) blots were hybridized with an scpR full-length 834-bp DNA fragment obtained by PCR from A. nidulans chromosomal DNA with the oligonucleotides 3492 XmaI for and 3492 XmaI rev (Table 2). (A) A. nidulans wild-type (wt) strain AXB4A and strain SB3.5 alcAp-scpR. The 800-bp inpA (AN3495) probe was obtained by PCR from A. nidulans chromosomal DNA with the oligonucleotides 3495 for and 3495 rev (Table 2). (B) Strain SB3.5 alcAp-scpR. Probes of 800 bp for inpA (AN3495), inpB (AN3496), inpC (AN3490), inpD (AN3491), and AN3497 were obtained by PCR from A. nidulans chromosomal DNA with oligonucleotide pairs 3495 for and 3495 rev, 3496 for and 3496 rev, 3490 for and 3490 rev, 3491 for and 3491 rev, and 3497 for and 3497 rev, respectively (Table 2). (C) Strain SB3.5 alcAp-scpR and strain ALF1.1 alcAp-scpR carrying the afoA deletion. The probes for afoA (AN1029), afoG (AN1036), and inpB (AN3496) were obtained by PCR from A. nidulans chromosomal DNA with oligonucleotide pairs 1029 for and 1029 rev, 1036 for and 1036 rev, and 3496 for and 3496 rev, respectively (Table 2).

FIG. 2.

(A) HPLC profiles of extracts from A. nidulans chemically inducible transformant strains SB3.5 and SB2.7 producing a novel compound. The strains were incubated in AMM under alcAp-inducing (I) and -repressing (R) conditions (the repressing condition profile of strain SB2.7 is identical to the one for SB3.5 [data not shown]). (B) Structure of asperfuranone (8). (C) H,H-COSY and HMBC couplings.

FIG. 3.

Relative quantity of the mRNA steady-state level determined by qRT-PCR of scpR, inpA, inpB, afoA, afoE, and afoG in strains SB3.5, SB2.7, and AXB4A (wild type) under inducing and repressing conditions. Relative quantities are given as the log₁₀ of −ΔΔC_T, where C_T is threshold cycle. Data obtained by cultivation of A. nidulans AXB4A under repressing conditions were set as equal to 1.

FIG. 4.

Model of cross talk between the A. nidulans secondary metabolite gene cluster inp and afo in A. nidulans (modified as described elsewhere [8]). Each arrow indicates the direction of transcription, the relative sizes of the open reading frames, and the intron distribution deduced from analysis of the nucleotide sequences. (A) inp gene cluster on chromosome II. Orange arrows, NRPS genes; black arrows, additional putative cluster genes; red arrow, regulator gene scpR; white arrows, genes not characterized here; inpC, putative AMP binding enzyme; inpD, major facilitator superfamily; inpE, proteasome a type and b type; inpF, serine hydrolase (FSH1). (B) afo (asperfuranone) gene cluster on chromosome VIII. Gray arrows, PKS genes; black arrows, additional cluster genes; blue arrow, regulatory gene afoA; white arrows, genes whose involvement in asperfuranone biosynthesis is unclear.