Cloning and sequence characterization of a non-reducing polyketide synthase gene from the lichen Xanthoparmelia semiviridis.
Lichens produce a diverse array of secondary metabolites that have shown various biological activities. Of particular interest are the coupled phenolics that originate from polyketide pathways, such as depsides, depsidones and usnic acids, which are produced almost solely by lichens. Based on the presumed catalytic domains required for the synthesis of the key intermediates beta-orsellinic acid and methylphloroacetophenone, two pairs of degenerate primers were designed to target specifically the beta-ketoacylsynthase (KS) and C-methyltransferase (CMeT) domains of fungal non-reducing polyketide synthase (NR-PKS) genes with CMeT domains. These primers were used to explore the genome of the lichen Xanthoparmelia semiviridis, which produces beta-orcinol depsidones and usnic acid. One of the two KS domains amplified from genomic DNA of field-collected X. semiviridis was used as a probe to recover the candidate PKS gene. A 13 kb fragment containing an intact putative PKS gene (xsepks1) of 6555 bp was recovered from a partial genomic library. The inferred amino acid sequence indicated that xsepks1 encodes a protein of 2164 amino acids and contains KS, acyltransferase (AT), acyl carrier protein (ACP) and CMeT domains as expected. This demonstrated a successful strategy for targeting non-reducing PKS genes with CMeT domains. Integration of the 5' fragment of xsepks1, including the native promoter, into Aspergillus nidulans by cotransformation resulted in the transcription of the 5'xsepks1 and the splicing of a 63 bp intron, suggesting that A. nidulans could be a suitable heterologous host for xsepks1 expression.
Chooi
Yit-Heng
YH
School of Applied Sciences, RMIT University, Bundoora Campus, PO Box 71, Bundoora VIC 3083, Australia.
Stalker
David M
DM
Davis
Meryl A
MA
Fujii
Isao
I
Elix
John A
JA
Louwhoff
Simone H J J
SH
Lawrie
Ann C
AC
D001203:Ascomycota / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D001233:Aspergillus nidulans / Q000235:genetics; D001483:Base Sequence; D053898:Biosynthetic Pathways; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008011:Lichens / Q000382:microbiology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D048630:Polyketide Synthases