Fungal polyketides (PKs) are one of the largest families of structurally diverse bioactive natural products biosynthesized by multidomain megasynthases, in which thioesterase (TE) domains act as nonequivalent decision gates determining both the shape and the yield of the polyketide intermediate. The endophytic fungus Preussia isomera XL-1326 was discovered to have an excellent capacity for secreting diverse bioactive PKs, i.e., the hot enantiomers (±)-preuisolactone A with antibacterial activity, the single-spiro minimoidione B with α-glucosidase inhibition activity, and the uncommon heptaketide setosol with antifungal activity, which drive us to illustrate how the unique PKs are biosynthesized. In this study, we first reported the genome sequence information of P. isomera. Based on genome mining, we discovered nine transcriptionally active genes encoding polyketide synthases (PKSs), Preu1–Preu9, of which those of Preu3, Preu4, and Preu6 were cloned and functionally characterized due to possessing complete sets of synthetic and release domains. Through heterologous expression in Saccharomyces cerevisiae, Preu3 and Preu6 could release high yields of orsellinic acid (OA) derivatives [3-methylorsellinic acid (3-MOA) and lecanoric acid, respectively]. Correspondingly, we found that Preu3 and Preu6 were clustered into OA derivative synthase groups by phylogenetic analysis. Next, with TE domain swapping, we constructed a novel “non-native” PKS, Preu6-TEPreu3, which shared a very low identity with OA synthase, OrsA, from Aspergillus nidulans but could produce a large amount of OA. In addition, with the use of Preu6-TEPreu3, we synthesized methyl 3-methylorsellinate (synthetic oak moss of great economic value) from 3-MOA as the substrate, and interestingly, 3-MOA exhibited remarkable antibacterial activities, while methyl 3-methylorsellinate displayed broad-spectrum antifungal activity. Taken together, we identified two novel PKSs to biosynthesize 3-MOA and lecanoric acid, respectively, with information on such kinds of PKSs rarely reported, and constructed one novel “non-native” PKS to largely biosynthesize OA. This work is our first step to explore the biosynthesis of the PKs in P. isomera, and it also provides a new platform for high-level environment-friendly production of OA derivatives and the development of new antimicrobial agents.

The in-depth study of fungal secondary metabolites (SMs) over the past few years has led to the discovery of a vast number of novel fungal SMs, some of which possess good biological activity. However, because of the limitations of the traditional natural product mining methods, the discovery of new SMs has become increasingly difficult. In recent years, with the rapid development of gene sequencing technology and bioinformatics, new breakthroughs have been made in the study of fungal SMs, and more fungal biosynthetic gene clusters of SMs have been discovered, which shows that the fungi still have a considerable potential to produce SMs. How to study these gene clusters to obtain a large number of unknown SMs has been a research hotspot. With the continuous breakthrough of molecular biology technology, gene manipulation has reached a mature stage. Methods such as gene knockout and heterologous expression techniques have been widely used in the study of fungal SM biosynthesis and have achieved good effects. In this review, the representative studies on the biosynthesis of fungal SMs by gene knockout and heterologous expression under the fungal genome mining in the last three years were summarized. The techniques and methods used in these studies were also briefly discussed. In addition, the prospect of synthetic biology in the future under this research background was proposed.

Orsellinic acid (OA) derivatives are produced by filamentous fungi using nonreducing polyketide synthases (nrPKSs). The chain-releasing thioesterase (TE) domains of such nrPKSs were proposed to also catalyze dimerization to yield didepsides, such as lecanoric acid. Here, we use combinatorial domain exchanges, domain dissections and reconstitutions to reveal that the TE domain of the lecanoric acid synthase Preu6 of Preussia isomera must collaborate with the starter acyl transferase (SAT) domain from the same nrPKS. We show that artificial SAT-TE fusion proteins are highly effective catalysts and reprogram the ketide homologation chassis to form didepsides. We also demonstrate that dissected SAT and TE domains of Preu6 physically interact, and SAT and TE domains of OA-synthesizing nrPKSs may co-evolve. Our work highlights an unexpected domain-domain interaction in nrPKSs that must be considered for the combinatorial biosynthesis of unnatural didepsides, depsidones, and diphenyl ethers.Derivatives of orsellinic acid (OA, 1) form a large class of structurally diverse polyketides widely distributed in plants, lichens, algae, fungi and bacteria. [1] OA polymers, that is, depsides, depsidones, and diphenyl ethers, feature ester or ether linkages. [2,3] These linkages may form by oxidative coupling or oxidative rearrangements catalyzed by tailoring enzymes after the release of the polyketide core from the nonreducing polyketide synthase (nrPKS) enzyme. [4][5][6] Alter-natively, such linkages result from nrPKS-catalyzed transformations of nascent, acyl carrier protein-bound intermediates. [7,8] Among the ester-linked OA dimers, lecanoric acid (2), a lichen metabolite also produced by filamentous fungi, displays various biological activities, including histidine decarboxylase inhibitory, [9] radical scavenging, [10,11] and antifungal activities. [12] Three nrPKSs from different fungi have been shown to yield the didepside 2, all without the involvement of post-PKS tailoring enzymes. These nrPKSs (AN7909 from Aspergillus nidulans, [7,8] CPUR_07425 from Claviceps purpurea, [13] and Preu6 from Preussia isomera, [12] ) share the same domain architecture, that is, SAT (starter acyl transferase)-KS (ketoacyl synthase)-AT (acyl transferase)-PT (product template)-ACP1 and 2 (acyl carrier protein 1 and 2)-TE (thioesterase) (Figure S1, Table S1).When expressed in Saccharomyces cerevisiae, AN7909 affords the diaryl ether diorcinolic acid (3) with only trace amounts of 2. [7,8] The dissected AN7909-TE efficiently converts the N-acetylcysteamine thioester of 2 (2-SNAC) into 3 and 1-SNAC into 1 in vitro (Figure 1A). [8] This confirms that AN7909-TE not only releases both 3 and 1, but also catalyzes a Smiles rearrangement to the diaryl ether 3. [8] However, AN7909-TE converts 1-SNAC into 3 with low efficiency in vitro; [8] therefore, its role in the in vivo formation of the ester 2 remains to be further elucidated.In contrast, overexpression of CPUR_07425 in C. purpurea yielded 2 and its ethyl ester, with only trace amounts of diaryl et...

Orsellinic acid (OA) derivatives are produced by filamentous fungi using nonreducing polyketide synthases (nrPKSs). The chain-releasing thioesterase (TE) domains of such nrPKSs were proposed to also catalyze dimerization to yield didepsides, such as lecanoric acid. Here, we use combinatorial domain exchanges, domain dissections and reconstitutions to reveal that the TE domain of the lecanoric acid synthase Preu6 of Preussia isomera must collaborate with the starter acyl transferase (SAT) domain from the same nrPKS. We show that artificial SAT-TE fusion proteins are highly effective catalysts and reprogram the ketide homologation chassis to form didepsides. We also demonstrate that dissected SAT and TE domains of Preu6 physically interact, and SAT and TE domains of OA-synthesizing nrPKSs may co-evolve. Our work highlights an unexpected domain-domain interaction in nrPKSs that must be considered for the combinatorial biosynthesis of unnatural didepsides, depsidones, and diphenyl ethers.Derivatives of orsellinic acid (OA, 1) form a large class of structurally diverse polyketides widely distributed in plants, lichens, algae, fungi and bacteria. [1] OA polymers, that is, depsides, depsidones, and diphenyl ethers, feature ester or ether linkages. [2,3] These linkages may form by oxidative coupling or oxidative rearrangements catalyzed by tailoring enzymes after the release of the polyketide core from the nonreducing polyketide synthase (nrPKS) enzyme. [4][5][6] Alter-natively, such linkages result from nrPKS-catalyzed transformations of nascent, acyl carrier protein-bound intermediates. [7,8] Among the ester-linked OA dimers, lecanoric acid (2), a lichen metabolite also produced by filamentous fungi, displays various biological activities, including histidine decarboxylase inhibitory, [9] radical scavenging, [10,11] and antifungal activities. [12] Three nrPKSs from different fungi have been shown to yield the didepside 2, all without the involvement of post-PKS tailoring enzymes. These nrPKSs (AN7909 from Aspergillus nidulans, [7,8] CPUR_07425 from Claviceps purpurea, [13] and Preu6 from Preussia isomera, [12] ) share the same domain architecture, that is, SAT (starter acyl transferase)-KS (ketoacyl synthase)-AT (acyl transferase)-PT (product template)-ACP1 and 2 (acyl carrier protein 1 and 2)-TE (thioesterase) (Figure S1, Table S1).When expressed in Saccharomyces cerevisiae, AN7909 affords the diaryl ether diorcinolic acid (3) with only trace amounts of 2. [7,8] The dissected AN7909-TE efficiently converts the N-acetylcysteamine thioester of 2 (2-SNAC) into 3 and 1-SNAC into 1 in vitro (Figure 1A). [8] This confirms that AN7909-TE not only releases both 3 and 1, but also catalyzes a Smiles rearrangement to the diaryl ether 3. [8] However, AN7909-TE converts 1-SNAC into 3 with low efficiency in vitro; [8] therefore, its role in the in vivo formation of the ester 2 remains to be further elucidated.In contrast, overexpression of CPUR_07425 in C. purpurea yielded 2 and its ethyl ester, with only trace amounts of diaryl et...