Starter unit specificity directs genome mining of polyketide synthase pathways in fungi

Abstract

Search of the protein database with the aflatoxin pathway polyketide synthase (PKS) revealed putative PKSs in the pathogenic fungi Coccidioides immitis and Coccidioides posadasii that could require partnerships with a pair of fatty acid synthase (FAS) subunits for the biosynthesis of fatty acid-polyketide hybrid metabolites. A starter unit:acyl-carrier protein transacylase (SAT) domain was discovered in the nonreducing PKS. This domain is thought to accept the fatty acid product from the FAS to initiate polyketide synthesis. We expressed the C. immitis SAT domain in Escherichia coli and showed that this domain, unlike that from the aflatoxin pathway PKS, transferred octanoyl-CoA four times faster than hexanoyl-CoA. The SAT domain also formed a covalent octanoyl intermediate and transferred this group to a free-standing ACP domain. Our results suggest that C. immitis/posadasii, both human fungal pathogens, contain a FAS/PKS cluster with functional similarity to the aflatoxin cluster found in Aspergillus species. Dissection of the PKS and determination of in vitro SAT domain specificity provides a tool to uncover the growing number of similar sequenced pathways in fungi, and to guide elucidation of the fatty acid-polyketide hybrid metabolites that they produce.

Figure 1. Selected fungal polyketide metabolites

Structures include norsolorinic acid (1), naphthopyrone YWA1 (2), acetyl-1,3,6,8-tetrahydroxynaphthalene (3), cercosporin (4), and bikaverin (5). Hexanoyl or acetyl starter units are bolded.

Figure 2. Phylogenetic divergence of SAT domains

Sequences are listed by their GenBank accession numbers and fungal hosts. PKSs giving rise to known compounds are indicated, and the known hexanoyl acceptors cluster separately from the known acetyl acceptors. Closely-grouping sequences could have the same starter unit specificity. Putative active site motifs are color-coded: Red, GXCXG; green, GXSXG; and blue, GXGXG. (*) indicates a revision in the PKS4 sequence [7].

Figure 3. Phylogenetic alignment of full-length nonreducing PKSs

The tetrahydroxynaphthalene PKSs, PKS1 orthologs, are found closest to the ancestral outgroup Neurospora crassa sequence (XP_960586). Many of the PKSs with SAT sequences containing a GXSXG or GXGXG motif lack the C-terminal TE/CLC domain. The domain organization of nonreducing PKSs is shown. (x) = 1, 2, or 3 ACP domains. The Coccidioides immitis PKS (XP_001241406) contains 2 ACPs.

Figure 4

A: Coccidioides immitis SAT domain specificity. Rates are reported in μmol transfer/min/μmol SAT: malonyl, 0.8 ± 0.9; acetyl, 7.0 ± 2.5; butanoyl, 18.2 ± 0.7; hexanoyl, 23.9 ± 2.9; octanoyl, 98.8 ± 5.2; decanoyl, 1.3 ± 0.7; and palmitoyl, 0.3 ± 0.7. B: Radiochemical transacylase assay. The Coccidioides SAT monodomain covalently accepts a ¹⁴C-labelled octanoyl-group and transfers it to the holo-ACP monodomain from PksA.