A gene (pks2) encoding a putative 6-methylsalicylic acid synthase from Glarea lozoyensis.

A gene that encodes for a polyketide synthase (PKS) was cloned from the fungus Glarea lozoyensis and characterized. The gene (pks2) consists of four exons interrupted by three introns of 51, 59, and 65 bp, which are clustered at the 5' end. Its predicted product is a 1791-amino-acid protein containing five catalytic motifs typical of fungal PKSs, including a beta-ketosynthase, an acyltransferase, a dehydratase, a beta-ketoacyl reductase, and an acyl carrier region. The gene is transcribed from an initiation site located 375 bp upstream of the translational start codon and extends to a transcriptional termination site 244 bp downstream of the translational stop codon. The gene function is not required for either vegetative growth of G. lozoyensis or for production of pneumocandin, as shown by Agrobacterium-mediated pks2 gene disruption. Previously reported cluster analysis of ketosynthase motifs from 37 fungal polyketide synthases had grouped the Pks2p from G. lozoyensis with PKSs involved in the biosynthesis of 6-methylsalicylic acid. To verify the function of the gene, it was transferred into Aspergillus nidulans under the control of the trpC promoter. 5'-and 3'-RACE experiments confirmed that it was transcribed in the heterologous host, and was associated with the synthesis of a compound identified as 6-methylsalicylic acid by NMR and mass spectrometry. In G. lozoyensis, pks2 is flanked by a gene that encodes a putative drug resistance efflux pump. The Aspergillus pks2 transformants, which were arginine prototrophs, also exhibited precocious pigmentation and accumulated a benzophenone that appeared to be a precursor of emericellin (variecoxanthone B), a known product of A. nidulans. The buildup of the benzophenone may be related to the use of an alternative splice site for the removal of intron 1 of the pks2 transcript in the heterologous host.

Efficient disruption of a polyketide synthase gene ( pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis.

Glarea lozoyensis produces pneumocandin B(0), a potent inhibitor of fungal glucan synthesis. This industrially important filamentous fungus is slow-growing, is very darkly pigmented, and has not been easy to manipulate genetically. Using a PCR strategy to survey the G. lozoyensis genome for polyketide synthase (PKS) genes, we have identified pks1, a gene that consists of five exons interrupted by four introns of 56, 400, 50 and 341 bp. It encodes a 2124-amino acid protein with five catalytic modules: ketosynthase, acyltransferase, two acyl carrier sites, and thioesterase/Claisen cyclase. The transcriptional initiation and termination sites were found 43 bp upstream of the translational start codon and 295 bp downstream of the translational stop codon, respectively. Cluster analysis of 37 fungal ketosynthase modules grouped the Pks1p with PKSs involved in the biosynthesis of 1,8-dihydroxynaphthalene melanin. Disruption of pks1 yielded knockout mutants that displayed an albino phenotype, suggesting that pks1 encodes a tetrahydroxynaphthalene synthase. Gene replacement was achieved by Agrobacterium-mediated transformation, which proved to be simple and efficient. Loss of pigmentation occurred in more than half the transformants, and examination of six non-pigmented transformants showed that the functional genomic copy of the pks1 gene had been replaced by the disruption cassette in each case. A putative 1215-bp ORF (dsg) devoid of introns was present downstream from pks1. BLAST analysis of the 405-amino acid sequence of its predicted product showed a high degree of similarity with Zn(II)(2)Cys(6) binuclear cluster DNA-binding proteins, a class of fungal transcription factors involved in the regulation of polyketide production and other pathways.