Global regulator of secondary metabolite biosynthesis and methods of use
The invention provides polypeptides and nucleic acids which identify and encode LaeA, a regulator of fungal secondary metabolite production which exhibits global control over secondary metabolite biosynthetic gene clusters. The invention further provides expression vectors, host cells, methods of increasing the production of secondary metabolites in an organism naturally producing a secondary metabolite or engineered to produce a secondary metabolite, and methods of identifying novel secondary metabolite biosynthesis gene clusters.
1. A method for identifying a novel secondary metabolite biosynthesis gene cluster in a fungus, said method comprising steps of:
a) obtaining a transformed fungus having a disrupted laeA gene wherein said laeA gene before disruption comprises a nucleotide sequence that encodes a nuclear protein methyltransferase and that hybridizes under stringent conditions, comprising overnight hybridization at 42° C. in 50% formamide with 1 mg heparin followed by a 15 minute wash at 65° C. in 0.2x SSC, to either strand of a denatured double-stranded nucleic acid having the nucleotide sequence set forth in SEQ ID NO:2; and wherein expression of the non-disrupted laeA gene in a fungal host cell results in the increased production of a polypeptide capable of regulating a gene cluster specifying the production of at least one secondary metabolite selected from a polyketide, a non-ribosomal peptide and a hyphal pigment and expression of a disrupted laeA gene results in the production of an inactive laeA polypeptide or its absence;
(b) isolating a sample of nucleic acids from the transformed fungus of step (a), said sample of nucleic acids representative of the expressed genes of the transformed fungus;
(c) hybridizing the sample of nucleic acids isolated in step (b) with an array comprising a plurality of nucleic acids representative of the expressed genes of a non-transformed fungus under conditions to form one or more hybridization complexes;
(d) detecting said hybridization complexes:
(e) comparing the levels of the hybridization complexes detected in step (c) with the level of hybridization complexes detected in a sample of nucleic acids isolated from an laeA-expressing fungus and representative of the expressed genes of the laeA-expressing fungus, wherein an altered level of hybridization complexes detected in step (c) compared with a level of hybridization complexes of the sample of the nucleic acids from the laeA-expressing fungus correlates with an identifies at least one gene under regulatory control of the laeA gene product; and
(f) examining genomic nucleotide sequence surrounding said gene identified in step (e) to determine if said gene is clustered with secondary metabolite biosynthesis genes thereby identifying a novel secondary metabolite biosynthesis gene cluster.
2. A method according to claim 1 , wherein said nucleic acids representative of the expressed genes of the non-transformed fungus in step (c) are immobilized on a substrate.
3. A method according to claim 1 , wherein said nucleic acids representative of the expressed genes of the non-transformed fungus in step (c) are hybridizable elements in a microarray.
4. A method according to claim 1 , wherein the sample of nucleic acids isolated from the laeA-expressing fungus and representative of the expressed genes of the laeA-expressing fungus in step (e) are isolated from a transformed fungus overexpressing laeA gene product as compared to the non-transformed fungus.
5. A method according to claim 1 wherein the fungus is an Aspergillus or Fusarium species.