Induced biosyntheses of a novel butyrophenone and two aromatic polyketides in the plant pathogen Stagonospora nodorum

Fungal aromatic compounds comprise an important and structurally diverse group of secondary metabolites.Several genome sequencing projects revealed many putative biosynthetic gene clusters of fungal aromatic compounds,but many of these genes seem to be silent under typical laboratory culture conditions.To gain access to this untapped reservoir of natural products,we utilized chemical epigenetic modifiers to induce the expression of dormant biosynthetic genes.As a result,the concomitant supplementation of the histone deacetylase inhibitors suberoylanilide hydroxamic acid(500mM)and nicotinamide(50mM)to the culture medium of a fungal pathogen,Stagonospora nodorum,resulted in the isolation of three aromatic compounds(1-3),including a novel natural butyrophenone,(+)-4'-methoxy-(2S)-methylbutyrophenone(1),and two known polyketides,alternariol(2)and(-)-(3R)-mellein methyl ether(3).

Koninginins N-Q, Polyketides from the Endophytic Fungus Trichoderma koningiopsis Harbored in Panax notoginseng

Four new fungal polyketides named koninginins N-Q(1–4),together with four known analogues(5–8),were isolated from the endophytic fungus Trichoderma koningiopsis YIM PH30002 harbored in Panax notoginseng.Their structures were determined on the basis of spectral data interpretation.These compounds were evaluated for their antifungal activity,nitric oxide inhibition,and anticoagulant activity.

The Combinatorial Biosynthesis of＂Unnatural＂ Products with Polyketides

Polyketides have been widely used clinically due to their significant biological activities, but the needed structural and functional diversity cannot be achieved by common chemical synthetic methods. The tool of combinatorial biosynthesis provides the possibility to produce "unnatural" natural drugs, which has achieved initial success. This paper provides an overview for the strategies of combinatorial biosynthesis in producing the structural and functional diversity of polyketides, including the redesign of metabolic flow, polyketide synthase(PKS) engineering, and PKS post-translational modification. Although encouraging progress has been made in the last decade, challenges still exist regarding the rational combinatorial biosynthesis of polyketides. In this review, the perspectives of polyketide combinatorial biosynthesis are also discussed.

Phylogenetic diversity of TypeⅠpolyketide synthase genes from sediments of Ardley Island in Antarctica

The diversity of modular polyketide synthase （PKS） genes in sediments of Ardley Island in Antarctica, was studied by restriction fragment length polymorphism （RFLP） analysis. Phylogenetic analysis of 14 amino acid （AA） sequences indicates that the identified ketosynthase （KS） domains were clustered with those from diverse bacterial groups, including Cyanobacteria, γ-Proteobacteria, Actinobacteria, Firmicutes, and some unidentified microorganisms from marine sponge, bryozoan and other environmental samples. The obtained KS domains showed 43%–81% similarity at the AA level to reference sequences in GenBank. Six identified KS domains showed diverse sequences of the motif （VQTACSTS） that was used to identify the hybrid PKS/nonribosomal peptide synthetase （NRPS） enzyme complex, and formed a new branch. These results reveal a high diversity and novelty of PKS genes in antarctic sediments.

SQUAMOSTATIN-B, A NEW POLYKETIDE FROM ANNONA SQUAMOSA (ANNONACEAE)

Squamostatin-B (1), a new polyketide or acetogenin, has been isolated from Annona squamosa L. (Annonaceae). Its structure and relative atereochemistry were elucidated on the basis of the spectral analyses of 1 and its derivatives, the acetate (2) and mesitoate (3).

Furan Derivatives and Polyketides from the Fungus Irpex lacteus

Eight new furan derivatives,irpexins A‒H(1‒8),two new polyketides,irpexins I and J(9 and 10),together with nine known compounds were isolated from the fermentation of Irpex lacteus.The structures and absolute configurations were elucidated on the basis of extensive spectroscopic methods and Mosher ester reaction.All compounds shows no cytotoxicity to human MCF-7 and Hela cancer cell lines at the concentration of 10μM.

Rufoolivacin B,a novel polyketide pigment from the fruiting bodies of the fungus Cortinarius rufo-olivaceus(basidiomycetes)

A novel polyketide pigment （1） with the 4＇,10-coupled linkage between 1-naphthalenol and 1,4-anthraquinone, named rufoolivacin B together with the known analog rufoolivacin （2）, has been isolated from the fruiting bodies of the Chinese toadstool Cortinarius rufo-olivaceus （basidiomycetes）. Their structures were characterized by means of analysis of spectroscopic methods, including 2D-NMR experiments and HR-ESI-MS.

Five new polyketides from the basidiomycete Craterellus odoratus

Five new polyketides,craterellones A-E(1-5),were isolated from cultures of basidiomycete Craterellus odoratus,together with five known compounds(6-10).Structures of 1-5 were elucidated on the basis of extensive spectroscopic analysis.All compounds were evaluated for their inhibitory activities against one isozyme of 11β-hydroxysteroid dehydrogenase(11β-HSD1)and cytotoxic activities on five tumor cell lines.Compound 10 exhibited significant cytotoxicity against HL-60,SMMC-7721,A-549,MCF-7,and SW-480,with IC50 values of 0.50,0.69,0.64,1.10,0.54μM,respectively.

Editing function of type Ⅱ thioesterases in the biosynthesis of fungal polyketides

Polyketide synthases(PKSs)are megasynthases with multiple autonomously folding domains,which operate cooperatively in the PKS assemblies to synthesize specific polyketide scaffolds.Any nonreactive intermediates tethered to acyl carrier protein(ACP)domain in the PKS will block the elongation process of polyketide chains.In this study,we systematically elucidate the editing function of fungal typeⅡthioesterases(TEIIs)to hydrolyze ACP domain-bounded nonreactive acyl groups,which are uploaded by substrate promiscuous fungal phosphopantetheinyl transferase.Thereof,the TEIIs encoded in gene clusters of nonreducing PKS with reductase domain exhibit universal editing function.Besides,editing function was also found for TEIIs encoded in gene clusters of highly-reducing PKS with condensation domain.Hence,the editing TEIIs with function of recovery PKS are applied to improve the yield of the fungal polyketides in vivo.Our study provides valuable insights into the editing process of fungal PKSs,highlights the crucial role of TEIIs in enhancing polyketide production and introduces a novel metabolic engineering strategy for fungal polyketide biosynthesis by leveraging the editing function of TEIIs.

(+)/(-)-Mycosphatide A,a pair of highly oxidized polyketides with lipid-lowering activity from the mangrove endophytic fungus Mycosphaerella sp.SYSU-DZG01

(±)-Mycosphatide A(1a/1b),a pair of highly oxidized enantiomeric polyketides featuring a unique5/5/6/5-fused tetracyclic ring system,were isolated from the mangrove endophytic fungus Mycosphaerella sp.SYSU-DZG01.Their structures were established by extensive spectroscopic analyses,single crystal Xray diffraction,and experimental electronic circular dichroism(ECD)spectra comparison.The plausible biosynthetic pathway of 1 was proposed,which involved the generation of a key spiro[4.5]decane scaffold.Compounds(+)-1a and(-)-1b exhibited significant lipid-lowering activity in 3T3-L1 adipocytes model,with EC50values of 7.85±1.56 and 8.87±0.80μmol/L,respectively.

Asperochones A and B,two antimicrobial aromatic polyketides from the endophytic fungus Aspergillus sp.MMC-2

Two novel fungal metabolites,asperochones A and B,were obtained from an Aspergillus sp.Their structures were determined by 1D/2D nuclear magnetic resonance(NMR)spectroscopy,high resolution electrospray ionization mass spectroscopy(HRESIMS),and single-crystal X-ray diffraction analysis.Asperochone A possesses an intriguing skeleton bearing 5/6/6/6/7/5/5/5 octacyclic ring system,and asperochone B also exhibits an unusual carbon skeleton with five stereochiral centers.Their structures were proposed as heterotrimeric and heterodimeric products of aromatic polyketides.In addition,asperochone A exhibited a potential anti-tuberculosis effect since it showed a moderate potency against Mycobacterium smegmatis.

Biosynthesis of trialkyl-substituted aromatic polyketide NFAT-133 involves unusual P450 monooxygenase-mediating aromatization and a putative metallo-beta-lactamase fold hydrolase

The bacterial trialkyl-substituted aromatic polyketides are structurally featured with the unusual aromatic core in the middle of polyketide chain such as TM-123(1),veramycin A(2),NFAT-133(3)and benwamycin I(4),which were discovered from Streptomyces species and demonstrated with antidiabetic and immunosuppressant activities.Though the biosynthetic pathway of 1-3 was reported as a type I polyketide synthase(PKS),the PKS assembly line was interpreted inconsistently,and it remains a mystery how the compound 3 was generated.Herein,the PKS assembly logic of 1-4 was revised by site-mutagenetic analysis of the PKS dehydratase domains.Based on gene deletion and complementation,the putative P450 monooxygenase nftE1 and metallo-beta-lactamase(MBL)fold hydrolase nftF1 were verified as essential genes for the biosynthesis of 1-4.The absence of nftE1 led to abolishment of 1-4 and accumulation of new products(5-8).Structural elucidation reveals 5-8 as the non-aromatic analogs of 1,suggesting the NftE1-catalyzed aromatic core formation.Deletion of nftF1 resulted in disappearance of 3 and 4 with the compounds 1 and 2 unaffected.As a rare MBL-fold hydrolase from type I PKSs,NftF1 potentially generates the compound 3 through two strategies:catalyze premature chain-offloading as a trans-acting thioesterase or hydrolyze the lactone-bond of compound 1 as an esterase.

Global-scale analysis reveals distinct patterns of non-ribosomal peptide and polyketide synthase encoding genes in root and soil bacterial communities

Secondary metabolites(SMs)produced by soil bacteria,for instance antimicrobials and siderophores,play a vital role in bacterial adaptation to soil and root ecosystems and can contribute to plant health.Many SMs are non-ribosomal peptides and polyketides,assembled by non-ribosomal peptides synthetase(NRPS)and polyketide synthase(PKS)and encoded by biosynthetic gene clusters(BGCs).Despite their ecological importance,little is known about the occurrence and diversity of NRPs and PKs in soil.We extracted NRPS-and PKS-encodiing BGCs from 20 publicly available soil and root-associated metagenomes and annotated them using antiSMASH-DB.We found that the overall abundance of NRPSs and PKSs is similar in both environments,however NRPSs and PKSs were significantly clustered between soil and root samples.Moreover,the majority of identified sequences were unique to either soil-or root-associated datasets and had low identity to known BGCs,suggesting their novelty.Overall,this study illuminates the huge untapped diversity of predicted SMs in soil and root microbiomes,and indicates presence of specific SMs,which may play a role in inter-and intra-bacteriial interactions in root ecosystems.

Discovery of angucyclinone polyketides from marine actinomycetes with a genomic DNA-based PCR assay targeting type Ⅱ polyketide synthase

Angucyclinones are aromatic polyketides produced by type Ⅱ polyketide synthases（PKS） and are mainly found in terrestrial actinomycetes. To discover more angucyclinones from marine actinomycetes, a genomic DNA-based PCR assay targeting type Ⅱ polyketide synthases was performed. Among the 167 marine actinomycetes strains screened, twelve strains were identified as the ＂positive＂ strains possessing type Ⅱ PKS-encoding genes based on the sequencing of PCR products. One of the 12 ＂positive＂ strains, Streptomyces sp. PKU-MA00218 was selected for the large-scale fermentation based on the HPLC and TLC analysis. Four angucyclinones, 6-deoxy-8-O-methylrabelomycin（1）, 8-O-methylrabelomycin（2）, 8-O-methyltetrangulol（3）, C-ring cleavage product of angucyclinone C（4）, were isolated and their structures were elucidated based on spectroscopic analyses. The isolation of angucyclinones 1–4 highlights the power of genome mining technologies based on biosynthetic knowledge in natural products discovery.

Two terpenoids and a polyketide from the endophytic fungus Trichoderma sp. Xy24 isolated from mangrove plant Xylocarpus granatum

Three known compounds were isolated from the endophytic fungus Trichoderma sp Xy24 from a mangrove plant Xylocarpus granatum by using various column chromatography techniques. Their structures were identified as harzianone （1）, trichoacorenol （2）, and trichodimerol （3） by extensive spectroscopic analysis. Among them, 1 was a harziane diterpene, 2 was a sesquiterpene alcohol, and 3 was a polyketide with a completely symmetric configuration. Compound 3 exhibited medium inhibitory activity with an IC50 value of 74.6 μM using a NA （H7N9）/MUNANA model.

Engineered polyketides:Synergy between protein and host level engineering

Metabolic engineering efforts toward rewiring metabolism of cells to produce new compounds often require the utilization of non-native enzymatic machinery that is capable of producing a broad range of chemical functionalities.Polyketides encompass one of the largest classes of chemically diverse natural products.With thousands of known polyketides,modular polyketide synthases(PKSs)share a particularly attractive biosynthetic logic for generating chemical diversity.The engineering of modular PKSs could open access to the deliberate production of both existing and novel compounds.In this review,we discuss PKS engineering efforts applied at both the protein and cellular level for the generation of a diverse range of chemical structures,and we examine future applications of PKSs in the production of medicines,fuels and other industrially relevant chemicals.

Rational Engineering of Secondary Metabolic Pathways in a Heterologous Host to Enable the Biosynthesis of Hibarimicin Derivatives with Enhanced Anti-Melanomic Activity

A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which generated a trace of the target products but accumulated a large amount of shunt products.Based on rational analysis of the relevant secondary metabolism,directed engineering of the biosynthetic pathways resulted in the high production of HBM B,as well as new HBM derivates with improved antitumor activity.These results not only establish a biosynthetic system to effectively synthesize HBMs-a class of the largest and most complex Type-Ⅱpolyketides,with a unique pseudo-dimeric structure-but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.

Increasing the heterologous production of spinosad in Streptomyces albus J1074 by regulating biosynthesis of its polyketide skeleton

Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete,Saccharopolyspora spinosa.However,their large-scale overproduction is hindered by poorly understood bottlenecks in optimizing the original strain,and poor adaptability of the heterologous strain to the production of spinosyn.In this study,we genetically engineered heterologous spinosyn-producer Streptomyces albus J1074 and optimized the fermentation to improve the production of spinosad(spinosyn A and spinosyn D)based on our previous work.We systematically investigated the result of overexpressing polyketide synthase genes(spnA,B,C,D,E)using a constitutive promoter on the spinosad titer in S.albus J1074.The supply of polyketide synthase precursors was then increased to further improve spinosad production.Finally,increasing or replacing the carbon source of the culture medium resulted in a final spinosad titer of~70 mg/L,which is the highest titer of spinosad achieved in heterologous Streptomyces species.This research provides useful strategies for efficient heterologous production of natural products.

Programmable polyketide biosynthesis platform for production of aromatic compounds in yeast

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms,development of new,versatile,bio-based production platforms is required.Here we present a novel yeast-based platform for biosynthesis of bacterial aromatic polyketides.The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host.

The polyketide synthase OsPKS2 is essential for pollen exine and Ubisch body patterning in rice

Lipid and phenolic metabolism are important for pollen exine formation. In Arabidopsis, polyketide synthases （PKSs） are essential for both sporopollenin biosynthesis and exine formation. Here, we characterized the role of a polyketide synthase （OsPKS2） in male reproduction of rice （Oryza sativa）. Recombinant OsPKS2 catalyzed the condensation of fatty acyl-CoA with malonyl- CoA to generate triketide and tetraketide α-pyrones, the main components of pollen exine. Indeed, the ospks2 mutant had defective exine patterning and was male sterile. However, the mutant showed no significant reduction in sporopollenin accumulation. Compared with the WT （wild type）, ospks2 displayed unconfined and amorphous tectum and nexine layers in the exine, and less organized Ubisch bodies. Like the pksb/lap5 mutant of the Arabidopsis ortholog, ospks2 showed broad alterations in the profiles of anther-related phenolic compounds. However, unlike pksb/laps, in which most detected phenolics were substantially decreased, ospks2 accumu- lated higher levels of phenolics. Based on these results and our observation that OsPKS2 is unable to fully restore the exine defects in the pksb/laps, we propose that PKS proteins have functionally diversified during evolution. Collectively, our results suggest that PKSs represent a conserved and diversified biochemical pathway for anther and pollen development in higher plants.