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Analysis of Subunit Interactions in the Iterative Type I Polyketide Synthase ATX from Aspergillus terreus

Aromatic polyketides are biosynthesized by iterative-type polyketide synthases (PKSs). Depending on their architecture, they are classified into three types, type I, II, and III. Type III PKSs are responsible for plant aromatic compounds such as flavonoids and for some bacterial compounds such as 1,3,6,8-tetrahydroxynaphthalene. Most bacterial aromatic polyketides are products of type II PKS systems, in which independent ketosynthase (KS) subunits, a, b, and acyl carrier protein (ACP) combine iteratively to construct polyketide carbon skeletons. In contrast, fungal aromatic polyketides are products of iterative type I PKSs, which consist of KS, acyltransferase (AT), and ACP domains as a minimum, with additional catalytic domains such as ketoreductase (KR) and Claisen cyclase (CYC), all on single polypeptides. These domains are involved in repetitive Claisen condensations to elongate b-polyketomethylene intermediates, which are then cyclized and aromatized by aldol and/or Claisen cyclization. The best studied iterative type I aromatic PKS is the 6-methylsalicylic acid synthase (MSAS). The MSAS from Penicillium patulum was the first PKS to be purified to homogeneity, and was found to be a homotetramer comprised of a 188 kDa subunit. MSAS was also the first fungal PKS to be sequenced. Since then, a number of PKS genes have been cloned from fungi, and sequence information on fungal PKSs is rapidly increasing because of the progress of fungal genome projects. However, there are few examples of fungal PKSs whose functions have been determined by expression and product identification. We reported the expression of the atX gene cloned from Aspergillus terreus using a fungal expression system with an a-amylase promoter, and this experiment identified ATX to be a MSAS. The high productivity of 6-methylsalicylic acid (6MSA) by the ATX expression system led us to study functions of other fungal PKSs such as Aspergillus nidulans WA, Colletotrichum lagenarium PKS1, Aspergillus fumigatus Alb1p, and Alternaria solani PKSN and PKSF. Of those fungal PKSs whose functions have been identified, MSAS is the smallest in size, consisting of KS, AT, dehydratase (DH), KR, and ACP domains within a polypeptide of approximately 190 kDa. The domain organization of MSAS is quite similar to that of mammalian fatty acid synthase (FAS), although MSAS lacks enoyl reductase (ER) and thioesterase (TE) domains. In this study, we report expression of the full-length ATX and its deletion mutants in Saccharomyces cerevisiae. We observed that individual inactive mutants truncated at their N or C termini could complement each other when coexpressed, as judged by 6-MSA production. This intriguing result prompted us to further analyze the subunit–subunit interaction of ATX in detail, and as a result, we identified an interdomain (ID) region required for formation of the active reaction center of ATX. To express the ATX in yeast, its full-length cDNA sequence was first determined as the presence of one intron near the N terminus, predicted to be in the atX gene that was cloned from the genomic DNA library of A. terreus, following the consensus 5’ and 3’ splice-site sequences reported by Kinghorn et al. Reverse transcription was carried out with mRNA obtained from the induction culture of A. nidulans transformant with pTA-ATXF. Sequencing of atX cDNA fragments identified the position of the intron, which was revised to 62 bp-long, running from 124 bp to 185 bp from the start codon. Thus, the full-length atX cDNA was identified to be 5409 bp encoding 1803 amino acids. The predicted architecture of the PKS is shown in Figure 1 along with other typical iterative type I fungal aromatic PKSs. Penicillium patulum MSAS was successfully expressed in S. cerevisiae by Kealey et al. , who used the ADH2 promoter for MSAS and for coexpression of the Bacillus subtilis Sfp, which was necessary for converting the apo-ACP domains of the heterologous PKS to their holoform. To attain easier induction control, we used the GAL10 promoter for ATX expression and the GPD promoter for constitutive expression of Sfp. The full-length atX cDNA was introduced into the pESC-URA vector to construct pESC-ATX. The sfp gene was introduced into the p424 vector with GPD promoter to construct p424-SFP. The yeast INVSc1 transformant with pESC-ATX and p424-SFP cultured in the induction medium containing galactose produced 6-MSA. From 600 mL of the yeast atX transformant induction culture, 16 mg of 6-MSA was obtained as colorless needles and identified by physicochemical analysis. To investigate the role of the N terminus region of ATX, Nterminal deletion mutants were expressed in yeast. ATXN1 lacked the N-terminal region upstream of the intron (44 amino acids) but contained the full-length conserved KS domain, whereas ATXN2 had a longer N-terminal deletion of 81 amino acids, retaining 92% of the conserved KS domain. ATXN1 produced 6-MSA and thus, kept its catalytic activity but ATXN2 did not. (Figure 2) This result indicates that the ATX N terminus region upstream of the KS domain is not essential for 6-MSA synthesis although all of the conserved region of the KS domain is essential, as might be expected. In contrast, an Nterminal deletion mutant of A. nidulans WA, a multi-ring type I aromatic PKS, lost PKS activity completely and no longer produced any polyketide compound. Therefore, N-terminal regions upstream of KS domains, longer in WA and related type I PKSs, could have a critical but as yet unidentified role in PKS activity. [a] T. Moriguchi, Prof. Dr. Y. Ebizuka, Prof. Dr. I. Fujii Graduate School of Pharmaceutical Sciences, The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan) Fax: (+81)3-5841-4744 E-mail : ifujii@mol.f.u-tokyo.ac.jp Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.