Identification of a polyketide synthase required for alternariol (AOH) and alternariol-9-methyl ether (AME) formation in Alternaria alternata

Abstract

Alternaria alternata produces more than 60 secondary metabolites, among which alternariol (AOH) and alternariol-9-methyl ether (AME) are important mycotoxins. Whereas the toxicology of these two polyketide-based compounds has been studied, nothing is known about the genetics of their biosynthesis. One of the postulated core enzymes in the biosynthesis of AOH and AME is polyketide synthase (PKS). In a draft genome sequence of A. alternata we identified 10 putative PKS-encoding genes. The timing of the expression of two PKS genes, pksJ and pksH, correlated with the production of AOH and AME. The PksJ and PksH proteins are predicted to be 2222 and 2821 amino acids in length, respectively. They are both iterative type I reducing polyketide synthases. PksJ harbors a peroxisomal targeting sequence at the C-terminus, suggesting that the biosynthesis occurs at least partly in these organelles. In the vicinity of pksJ we found a transcriptional regulator, altR, involved in pksJ induction and a putative methyl transferase, possibly responsible for AME formation. Downregulation of pksJ and altR caused a large decrease of alternariol formation, suggesting that PksJ is the polyketide synthase required for the postulated Claisen condensations during the biosynthesis. No other enzymes appeared to be required. PksH downregulation affected pksJ expression and thus caused an indirect effect on AOH production.

Figure 1. Structure of alternariol (AOH) and alternariol-9-methyl ether (AME).

Figure 2. Architecture of PKSs of Alternaria alternata.

KS, β-ketoacyl synthase; AT, acyltransferase; DH, dehydratase; MT, methyltransferase; ER, enoyl reductase; KR, ketreductase; ACP, acyl carrier protein, CD, condensation domain; AA, Amino acid adenylation domain; CS:Chalon- and Stilben-Synthase (N)/(C); UDG: Uracil DNA Glycolase Superfamily; NAD, NAD binding domain. The sequences of the PKS loci are deposited under the following accession numbers: pksA (JX103636); pksB (JX103637); pksC (JX103638); pksD (JX103639); pksE (JX103640); pksF (JX103641); pksG (JX103642); pksH (JX103643); pksI (JX103644); pksJ (JX103645). The genbank accession numbers are given in brackets.

Figure 3. Organization of polyketide biosynthesis gene clusters in Alternaria alternata.

Each arrow indicates the direction of transcription deduced from the analysis of the nucleotide sequences. The asterisks indicate the genes of the pksJ and pksH clusters, which have been silenced in addition to pksJ and pksH.

Figure 4. Time-course expression analysis of different PKS genes.

(A) TLC analysis of AOH formation at different time points. Extracts from cultures grown on MCDB agar at 28°C in constant darkness for 3, 5, 7, 10, 12 and 14 days, respectively. For each time point three extractions were performed. The last lane contained the AOH standard. (B) Quantitative real time reverse-transcription polymerase chain reaction (RT-PCR) gene expression analysis of different PKS genes after 7, 12 and 14 days of post inoculation (dpi).

Figure 5. Quantitative PCR analysis of total RNA from the pksJ transformant relative to wildtype, using benA as reference gene.

Both the wildtype and transformants were grown on liquid MCDB medium for 12 days at 28°C in constant darkness. (A) pksJ transcript level of the wildtype and pksJ transformant (ΔJ, heterokaryotic pksJ deletion). (B) Relative expression of selected PKS transcripts in wildtype and transformant (ΔJ). (C) Real time RT-PCR detection of pksJ transcripts in wildtype (WT), empty vector control (E) and the silenced transformants J1, J3, J7.

Figure 6. LC/MS analysis of metabolites produced by Alternaria alternata WT, WT transformed with an empty vector (E), the ΔpksJ strains, the RNAi-pksJ strain, the ΔpksH and the RNAi-pksH strains as detected by UV absorbance.

m/z values are given for the most prominent peaks. Peak no 5 corresponds to alternariol and peak no 11 correspond to alternariol-9-methyl ether.

Figure 7. Quantitative PCR analysis of total RNA from the pksH transformant (ΔH, heterokaryotic pksH deletion) relative to wildtype, using benA as reference gene.

Both the wildtype and transformants were grown on liquid MCDB medium for 12 days at 28°C in constant darkness. (A) pksH transcript level of the wildtype and transformant. (B) Relative expression of selected pks transcripts in wildtype and pksH transformant.

Figure 8. LC/MS analysis of alternariol (AOH) and alternariol-9-methyl ether (AME) formation in wildtype (WT), empty vector (E) and RNAi transformant of selected genes flanked by pksJ and pksH.

H-TF:PksH-transferase; H-CL: PksH-cyclin F-Box; H-cP450: PksH-cytochrome p450; altR ( = alternariol regulator). AOH and AME values are expressed in nmol/10 µl.

Figure 9. Proposed biosynthetic pathway for alternariol and alternariol-9-methyl ether.

The biosynthesis of alternariol and alternariol-9-methyl ether consists of Claisen-type condensations with malonate as building blocks.