Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans

Abstract

Genome sequencing has revealed that fungi have the ability to synthesize many more natural products (NPs) than are currently known, but methods for obtaining suitable expression of NPs have been inadequate. We have developed a successful strategy that bypasses normal regulatory mechanisms. By efficient gene targeting, we have replaced, en masse, the promoters of nonreducing polyketide synthase (NR-PKS) genes, key genes in NP biosynthetic pathways, and other genes necessary for NR-PKS product formation or release. This has allowed us to determine the products of eight NR-PKSs of Aspergillus nidulans, including seven novel compounds, as well as the NR-PKS genes required for the synthesis of the toxins alternariol (8) and cichorine (19).

Figure 1

Promoter replacement strategies. A. Single promoter replacement strategy. i. A transforming sequence generated by fusion PCR. It consists of a sequence upstream of the target gene, a selectable marker such as the pyrG gene from Aspergillus fumigatus (AfpyrG), the alcA promoter (alcA(p)) and all or a portion of the target gene. In the present study, the upstream sequence and the portion of the target gene were about 1kb each in length. Transformation with this fragment results in homologous recombination with the chromosomal locus (ii) and replacement of the promoter of the target gene with a selectable marker and the alcA promoter, placing the target gene under the control of the alcA promoter (iii). This strategy was used to replace the promoters of transcription factors, NR-PKS and other genes of NP gene clusters. B. Dual promoter replacement strategy. Fatty acid synthase subunits such as ANID_03381.1 and ANID_03380.1 are generally divergently transcribed in A. nidulans. Two fragments were created by fusion PCR (i). One contained a portion of one target gene (ANID_03381.1) fused to alcA(p) and a portion of the AfpyrG gene. The second contained a second, overlapping, portion of the AfpryG gene and alcA(p) fused to the second target gene (ANID_03380.1). Upon transformation, recombination between the target genes on the transforming fragments and the chromosomal genes (ii) and between the overlapping fragments of AfpyrG results in both target genes being driven by alcA(p) (iii).

Figure 2

The NR-PKSs of A. nidulans, their biosynthetic characteristics and their products. Domain abbreviations: SAT = starter unit-ACP transacylase, KS = ketosynthase, AT = acyl transferase, PT = product template, ACP = acyl carrier protein, TE = thioesterase, TE/CLC = thioesterase/Claisen cyclase, CMeT = C-methyltransferase, R = reductase, β-TE = β-lactamase-type thioesterase. With respect to domain structure, domains encircled with a dotted line are present in some cases and not others. With respect to starter unit, it has been proposed that the Cys or Ser in the GXCXG or GXSXG motif in the SAT domain is responsible for transferring the starter unit by using thioester or oxyester chemistry, respectively. The GXGXG motif in the SAT domain might be inactive or have an unknown function. Most NR-PKSs identified from the A. nidulans genome have GXCXG motif except for AptA and AN07071.1 that have a GXSXG motif, and MdpG and OrsA that have a GXGXG motif in their SAT domains. Release mechanisms: TE cleaves the thioester bond and releases the acid product; TE/CLC releases the product without the acid functional group via intramolecular Claisen cyclization; β-TE cleaves the thioester bond and releases the acid product but cleavage is due to a protein acting in trans, not to a domain of the NR-PKS (the protein acting in trans is listed in each case); R cleaves the product via reductive release such that the product has an aldehyde group. With respect to downstream products, for previously published studies only the final product of the pathway is listed. StcJ (ANID_07815.1) and StcK (ANID_07814.1), and PkiB (ANID_03380.1) and PkiC (ANID_03381.1) are FASs that synthesize starter units for StcA and PkiA, respectively. AfoG (ANID_01036.1) and PkhB (ANID_02035.1) are HR-PKSs that synthesize starter units for AfoE and PkhA, respectively. AptB (ANID_06001.1), MdpF (ANID_00149.1), and PkgB (ANID_07070.1) are β-TE nearby the NR-PKSs, AptA, MdpG, and PkgA, respectively. AptC (ANID_06002.1) is a C2-hydroxylase in the Apt cluster. Note: Induction of ANID_07071.1 resulted in production of compound 9–13. However, compounds 12 and 13 were found in small amounts. These compounds result from incorporation of five Mal-CoAs and they indicate that although ANID_07071.1 is a heptaketide synthase, it is not strict with respect to the number of Mal-CoA molecules incorporated. Compound 18 is a self released shunt product after incorporating three Mal-CoAs and one SAM.

Figure 2

The NR-PKSs of A. nidulans, their biosynthetic characteristics and their products. Domain abbreviations: SAT = starter unit-ACP transacylase, KS = ketosynthase, AT = acyl transferase, PT = product template, ACP = acyl carrier protein, TE = thioesterase, TE/CLC = thioesterase/Claisen cyclase, CMeT = C-methyltransferase, R = reductase, β-TE = β-lactamase-type thioesterase. With respect to domain structure, domains encircled with a dotted line are present in some cases and not others. With respect to starter unit, it has been proposed that the Cys or Ser in the GXCXG or GXSXG motif in the SAT domain is responsible for transferring the starter unit by using thioester or oxyester chemistry, respectively. The GXGXG motif in the SAT domain might be inactive or have an unknown function. Most NR-PKSs identified from the A. nidulans genome have GXCXG motif except for AptA and AN07071.1 that have a GXSXG motif, and MdpG and OrsA that have a GXGXG motif in their SAT domains. Release mechanisms: TE cleaves the thioester bond and releases the acid product; TE/CLC releases the product without the acid functional group via intramolecular Claisen cyclization; β-TE cleaves the thioester bond and releases the acid product but cleavage is due to a protein acting in trans, not to a domain of the NR-PKS (the protein acting in trans is listed in each case); R cleaves the product via reductive release such that the product has an aldehyde group. With respect to downstream products, for previously published studies only the final product of the pathway is listed. StcJ (ANID_07815.1) and StcK (ANID_07814.1), and PkiB (ANID_03380.1) and PkiC (ANID_03381.1) are FASs that synthesize starter units for StcA and PkiA, respectively. AfoG (ANID_01036.1) and PkhB (ANID_02035.1) are HR-PKSs that synthesize starter units for AfoE and PkhA, respectively. AptB (ANID_06001.1), MdpF (ANID_00149.1), and PkgB (ANID_07070.1) are β-TE nearby the NR-PKSs, AptA, MdpG, and PkgA, respectively. AptC (ANID_06002.1) is a C2-hydroxylase in the Apt cluster. Note: Induction of ANID_07071.1 resulted in production of compound 9–13. However, compounds 12 and 13 were found in small amounts. These compounds result from incorporation of five Mal-CoAs and they indicate that although ANID_07071.1 is a heptaketide synthase, it is not strict with respect to the number of Mal-CoA molecules incorporated. Compound 18 is a self released shunt product after incorporating three Mal-CoAs and one SAM.

Figure 3

Total scan HPLC profiles of natural products extracted from the culture medium and mycelium of alcA promoter replaced strains. Peaks that also present in the stcJΔ control strain are indicated with an asterisk (*) and are considered not to be specific to the NR-PKS activated. Peaks that are specifically present in induced NR-PKS promoter replacement strains but are not structurally elucidated due to low yield or poor stability are marked with a dagger (†).

Figure 4

Phylogenetic analysis of all NR-PKSs obtained from the Broad Institute Aspergillus Comparative Database (http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html). NR-PKSs from A. nidulans are labeled in boldface. Domain architectures of protein sequences are list in Table S3. A. terreus MSAS, a PR-PKS, was used as the outgroup. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.