Method for producing terpenes
The present invention relates to a method for producing terpenes in fungi, wherein a terpene biosynthetic gene cluster having terpene biosynthetic genes and regulatory regions operably linked to said genes is activated. The invention relates also to a terpene biosynthetic gene duster and regulatory regions of such terpene biosynthetic gene cluster usable is production of terpenes, use of regulator for regulating the terpene production and use of Aspergillus nidulans FGSC A4 for producing terpenes. The method of invention provides higher yields of enriched terpene product without essential amount of side-products.
1. A method for producing terpenes in fungi, comprising the steps of:
(a) providing a nucleic acid encoding a transcription factor, wherein said transcription factor activates a terpene biosynthetic gene cluster, and wherein said transcription factor comprises a polypeptide having at least 90% sequence identity to SEQ ID NO: 74;
(b) operably linking said nucleic acid to a promoter, to produce an expression construct;
(c) transforming said expression construct into a host cell carrying a terpene biosynthetic gene cluster; and
(d) cultivating said host cell under conditions which allow expression of the transcription factor, and which allow activation of the terpene biosynthetic gene cluster by the transcription factor.
2. The method of claim 1 , wherein said terpene is ent-pimara-8(14),15-diene, or a derivative thereof.
3. The method of claim 1 , wherein said transcription factor comprises a polypeptide having at least 95% sequence identity to SEQ ID NO: 74.
4. The method of claim 1 , wherein the host cell is Aspergillus nidulans.
5. The method of claim 1 , wherein the terpenes are diterpenoids.
6. A host fungus comprising a terpene biosynthetic gene cluster comprising the genes encoding:
(a) a polypeptide having at least 90% identity to SEQ ID NO: 65, a polypeptide having at least 90% identity to SEQ ID NO: 63, a polypeptide having at least 90% identity to SEQ ID NO: 61 and
(b) a polypeptide having at least 90% identity to SEQ ID NO: 67, a polypeptide having at least 94% identity to SEQ ID NO: 73, a polypeptide having at least 90% identity to SEQ ID NO: 69, a polypeptide having at least 90% identity to SEQ ID NO: 71,
and wherein said host fungus has been transformed with an expression construct comprising a nucleic acid encoding a transcription factor, wherein said nucleic acid is operably linked to a promoter, and wherein said transcription factor comprises a polypeptide having at least 90% sequence identity to SEQ ID NO: 74.
7. The production host of claim 6 , wherein the terpene biosynthetic gene cluster further comprises a nucleic acid sequence encoding a polypeptide having at least 90% identity to SEQ ID NO: 59.
8. A method for producing terpenes in fungi, comprising the steps of:
(a) providing a nucleic acid encoding a transcription factor, wherein said transcription factor activates a terpene biosynthetic gene cluster, and wherein said transcription factor comprises a polypeptide having at least 90% sequence identity to SEQ ID NO: 74;
(b) operably linking said nucleic acid to a promoter, to produce an expression construct;
(c) transforming said expression construct into a host cell carrying a terpene biosynthetic gene cluster;
(d) cultivating said host cell under conditions which allow expression of the transcription factor, and which allow activation of the terpene biosynthetic gene cluster by the transcription factor; and
(e) recovering the terpene product.
9. A method for producing terpenes, said method comprising cultivating a host fungus which comprises a terpene biosynthetic gene cluster comprising the genes encoding:
(a) a polypeptide having at least 90% identity to SEQ ID NO: 65, a polypeptide having at least 90% identity to SEQ ID NO: 63, a polypeptide having at least 90% identity to SEQ ID NO: 61 and
(b) a polypeptide having at least 90% identity to SEQ ID NO: 67, a polypeptide having at least 94% identity to SEQ ID NO: 73, a polypeptide having at least 90% identity to SEQ ID NO: 69, a polypeptide having at least 90% identity to SEQ ID NO: 71,
and wherein said host fungus has been transformed with an expression construct comprising a nucleic acid encoding a transcription factor, wherein said nucleic acid is operably linked to a promoter, and wherein said transcription factor comprises a polypeptide having at least 90% sequence identity to SEQ ID NO: 74.
10. The method of claim 9 , wherein the terpene biosynthetic gene cluster further comprises a nucleic acid sequence encoding a polypeptide having at least 90% identity to SEQ ID NO: 59.
11. The method of claim 9 , wherein said method further comprises the step of recovering the terpene product.