Cloning and characterization of novel methylsalicylic acid synthase gene involved in the biosynthesis of isoasperlactone and asperlactone in Aspergillus westerdijkiae

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Abstract

Aspergillus westerdijkiae is the main producer of several biologically active polyketide metabolites including isoasperlactone and asperlactone. A 5298 bp polyketide synthase gene “aomsas” has been cloned in Aspergillus westerdijkiae by using gene walking approach and RACE-PCR. The predicted amino acid sequence of aomsas shows an identity of 40–56% with different methylsalicylic acid synthase genes found in Byssochlamys nivea, P. patulum, A. terreus and Streptomyces viridochromogenes. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aomsas expression was found to be associated with the biosynthesis of isoasperlactone and asperlactone. Moreover an aomsas knockout mutant “aoΔmsas” of A. westerdijkiae, not only lost the capacity to produce isoasperlactone and asperlactone, but also 6-methylsalicylic acid. The genetically complemented mutant ao+msas restored the biosynthesis of all the missing metabolites. Chemical complementation through the addition of 6-methylsalicylic acid, aspyrone and diepoxide to growing culture of aoΔmsas mutant revealed that these compounds play intermediate roles in the biosynthesis of asperlactone and isoasperlactone.

Introduction

Aspergillus westerdijkiae is a producer of several biologically active polyketide metabolites including isoasperlactone and asperlactone (Atoui et al., 2006, Balcells et al., 1995, Gaucher and Shepherd, 1968, Torres et al., 1998). These two metabolites belong to the partially reduced or methylsalicylic acid (MSA) type polyketide group and have same chemical structure but are different stereochemically. Isoasperlactone and asperlactone are on one hand efficient antimicrobial agents (Rosenbrook and Carney, 1970, Torres et al., 1998), and on the other hand they contain ovicidal activities against Nezara viridula (Balcells et al., 1995, Balcells et al., 1998). It has been reported that asperlactone and isoasperlactone presented a strong anti-bacterial and anti-fungal activities when compared with other antimicrobial agents (Balcells et al., 1998).

It has been previously demonstrated that during the biosynthesis of MSA type polyketides, the first step is the formation of 6-methylsalicylic acid (6-MSA) by the condensation of one molecule of acetyl-coA and three molecules of malonyl-coA (David et al., 1995). This reaction is catalyzed by a multifunctional enzyme system known as methylsalicylic acid synthase (MSAS). This enzyme has been reported to catalyze several MSA type polyketide compounds like patuline in Penicillium patulum (Beck et al., 1990) and Byssochlamys fulva (Puel et al., 2007) and avilamycin in Streptomyces viridochromogenes (Gaisser et al., 1997).

Not much is known about the biosynthetic pathways of isoasperlactone and asperlactone, except for a hypothetical scheme proposed by James and Andrew (1991). According to this scheme, the isomeric metabolites aspyrone, isoasperlactone and asperlactone are derived from a common biosynthetic precursor, the diepoxide. James and Andrew (1991) further stated that asperlactone is formed directly from the diepoxide, while isoasperlactone is formed from diepoxide via aspyrone pathway (Fig. 1).

Many PKS genes have been sequenced in filamentous fungi including A. fumigatus and A. niger (Metz et al., 2001, Niermanm et al., 2005, Pel et al., 2007, Sebastian et al., 2007). It is therefore possible to use the most conserved region among these PKS genes (Fig. 2) as primers for a PCR based cloning strategy. For this purpose, mainly the ketosynthase (KS) and acyle transferase (AT) domains, i.e. the most conserved domains among different PKSs (Fig. 2), have been utilized. The previously designed pairs of degenerated primers KS1/KS2, LC1/LC2c and LC3/LC5c (Bingle et al., 1999, Nicholson et al., 2001) has been already utilized in our laboratory to clone eight KS domains from different PKSs (Atoui et al., 2006), including one from MSAS in A. westerdijkiae. Recently one of these PKS genes has been found to be involved in the biosynthesis of ochratoxine A (Bacha et al., 2009).

In this paper, we report sequencing and functional characterization of the first MSAS-type PKS gene “aomsas” identified in A. westerdijkiae. It is involved in the biosynthesis of two pharmacologically important lactonic metabolites, i.e. isoasperlactone and asperlactone. We also demonstrate that 6-methylsalicylic acid (6-MSA), aspyrone and diepoxide are intermediates in their biosynthetic pathway.

Section snippets

Fungal strain and culture conditions

Aspergillus westerdijkiae NRRL 3174 strain was grown for sporulation at 25 °C on potato dextrose agar for 7 days. Spores were collected using a solution of 0.01% (v/v) Tween 80, counted by using Thoma Bright line counting chamber (Optick labor), and stored at −20 °C in 25% (v/v) glycerol before use. Conidia were inoculated (density ∼106/mL) into 250 mL Erlenmeyer flasks containing 100 mL synthetic medium (SAM) at 25 °C for 2–18 days, without shaking. The composition of SAM (per liter of distilled

aomsas is a MSAS-type polyketide gene of A. westerdijkiae

With the intent of extending the 700 bp KS domain fragment of a MSAS-type PKS gene identified by Atoui et al. (2006) in A. westerdijkiae, we adopted the degenerated primers based gene walking and RACE-PCR techniques. The specific primers MS1, MS3, MS5 and MS6 and degenerated primers MS2 and MS4 (Table 1) allowed the sequencing of a complete 5298 bp aomsas gene (Fig. 2). The aomsas gene displayed a unique open reading frame (ORF) of 1766 amino acids. Alignment of the amino acid sequence of aomsas

Discussion

From the analyses of fungal PKS gene sequences, Bingle et al. (1999) suggested that these genes could be divided into two subclasses designated as WA-type and MSAS-type or partially reduced type. They designed two pairs of degenerated primers, i.e. LC1/LC2c and LC3/LC5c for the amplification of fungal PKS genes in each of these subclasses. We have previously utilized LC3/LC5c pair of primer to clone a 700 bp KS domain of aomsas gene in A. westerdijkiae (Atoui et al., 2006). After prolongation of

Acknowledgment

The authors are grateful to the Higher Education Commission of Pakistan for according PhD scholarship to Mr. Nafees Bacha.

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