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.

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.

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.

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.

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.

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.

Diversified Polyketides and Nitrogenous Compounds from the Mangrove Endophytic Fungus Penicillium steckii SCSIO 41025

Four new polyketide decalin derivatives,penicisteck acids A-D(1-4),and three new nitrogenous compounds(7-9)z together with eight previously reported compounds,were isolated from the mangrove endophytic fungus Penicillium steckii SCSIO 41025.

Unleashing the power of energy storage: Engineering β-oxidation pathways for polyketide production

Overproduction of polyketides has been a challenge for metabolic engineering for decades.However,recent studies have demonstrated that in both native host and heterologous host,engineeringβ-oxidation pathways can lead to dramatic improvement of polyketide production.

Peptides and polyketides isolated from the marine sponge-derived fungus Aspergillus terreus SCSIO 41008

Two new isomeric modified tripeptides,aspergillamides C and D(compounds 1 and 2),together with fifteen known compounds(compounds 3-17),were obtained from the marine sponge-derived fungus Aspergillus terreus SCSIO 41008.The structures of the new compounds,including absolute configurations,were determined by extensive analyses of spectroscopic data(NMR,MS,UV,and IR)and comparisons between the calculated and experimental electronic circular dichroism(ECD)spectra.Butyrolactone I(compound 11)exhibited strong inhibitory effects against Mycobacterium tuberculosis protein tyrosine phosphatase B(MptpB)with the IC_(50) being 5.11±0.53μmol·L^(–1),and acted as a noncompetitive inhibitor based on kinetic analysis.

Functional characterization of key polyketide synthases by integrated metabolome and transcriptome analysis on curcuminoid biosynthesis in Curcuma wenyujin

Leaf and tuber extracts of Curcuma wenyujin contain a mixture of curcuminoids.However,the curcuminoid constituents and their molecular mechanisms are poorly understood,and the relevant curcumin synthases remain unclear.In this study,we comprehensively compared the metabolite profiles of the leaf and tuber tissues of C.wenyujin.A total of 11 curcuminoid metabolites were identified and exhibited differentially changed contents in the leaf and tuber tissues.An integrated analysis of metabolomic and transcriptomic data revealed the proposed biosynthesis pathway of curcuminoid.Two candidate type III polyketide synthases(PKSs)were identified in the metabolically engineering yeasts,indicating that CwPKS1 and CwPKS2 maintained substrate and product specificities.Especially,CwPKS1 is the first type III PKS identified to synthesize hydrogenated derivatives of curcuminoid,dihydrocurcumin and tetrehydrocurcumin.Interestingly,the substitution of the glycine at position 219 with aspartic acid(G219D mutant)resulted in the complete inactivation of CwPKS1.Our results provide the first comparative metabolome analysis of C.wenyujin and functionally identified type III PKSs,giving valuable information for curcuminoids biosynthesis.

Tripodalsporormielones A-C,unprecedented cage-like polyketides with complex polyvdent bridged and fused ring systems

A chemical investigation on Sporormiella sp.led to the isolation and structural elucidation of tripodalsporormielones Ae C(1-3),a new class of polyketide possessing unprecedented cage-like skeletons with polyvdent bridged and fused ring systems.These polyketides with cage-like skeletons were characterized as a high non-protonated carbon-containing system,which resulted in few HMBC correlations observed and made the accurate structures hard to be obtained by NMR.Especially,some signals of non-protonated sp;carbons are weak and even unobservable in compound 1.In order to establish the structure of 1,the calculated NMR with DP4 evaluation was applied to determine the structure from the plausible structure candidates obtained from the detailed NMR analysis.Based on NMR experiments and calculated NMR,the structures of isolated compounds were established and confirmed by X-ray technology.Through chiral isolation,the optically pure enantiomers of 1 and 3 were obtained,and their absolute configurations were determined based on ECD quantum chemical calculation.Based on the isolated compounds and our previous work,1-3 would be derived from 3-methylorcinaldehyde,and their plausible biosynthetic mechanism was proposed.Furthermore,1 exhibited obvious short-term memory improvement activity on an Alzheimer’s disease fly model.

Linear polyketides produced by co-culture of Penicillium crustosum and Penicillium fellutanum

Two new polyketides, penifellutins A (1) and B (2), possessing a 22 carbon linear skeleton, were isolated from a co-culture of the deep-sea-derived fungi Penicillium crustosum PRB-2 and Penicillium fellutanum HDN14-323. Meanwhile, two esterification products of 1, penifellutins C (3) and D (4), were obtained because compound 1 could be esterified spontaneously when stored in methanol. Their configurations were difficult to determine because of chiral central crowdedness, structural flexibility and instability. As such, we solved this issue by comprehensively using Mo2(OAc)4-based CD experiments, density functional theory calculation of 13C NMR, DP4 + probability analysis and many chemical reactions, including making acetonide derivative, Mosher’s method, PGME method, etc. Compounds 1 and 2 show obvious inhibitory activity on the liver hyperplasia of zebrafish larvae at a concentration of 10 μmol/L, while 3 and 4 show no activity, indicating that two carboxyls in the structure are important active sites.

Site directed mutagenesis as a precision tool to enable synthetic biology with engineered modular polyketide synthases

Modular polyketide synthases(PKSs)are a multidomain megasynthase class of biosynthetic enzymes that have great promise for the development of new compounds,from new pharmaceuticals to high value commodity and specialty chemicals.Their colinear biosynthetic logic has been viewed as a promising platform for synthetic biology for decades.Due to this colinearity,domain swapping has long been used as a strategy to introduce molecular diversity.However,domain swapping often fails because it perturbs critical protein-protein interactions within the PKS.With our increased level of structural elucidation of PKSs,using judicious targeted mutations of individual residues is a more precise way to introduce molecular diversity with less potential for global disruption of the protein architecture.Here we review examples of targeted point mutagenesis to one or a few residues harbored within the PKS that alter domain specificity or selectivity,affect protein stability and interdomain communication,and promote more complex catalytic reactivity.

Fragment antigen binding domains (F_(ab)s) as tools to study assembly-line polyketide synthases

The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions.Therefore,discovering tools that aid crystallization is crucial.In this review,the versatility of fragment-antigen binding domains(F_(ab)s)as protein crystallization chaperones is discussed.F_(ab)s have aided the crystallization of membrane-bound and soluble proteins as well as RNA.The ability to bind three F_(ab)s onto a single protein target has demonstrated their potential for crystallization of challenging proteins.We describe a high-throughput workflow for identifying F_(ab)s to aid the crystallization of a protein of interest(POI)by leveraging phage display technologies and differential scanning fluorimetry(DSF).This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases(PKSs),which harbor flexible domains and assume transient conformations.PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds.Despite years of research studying these megasynthases,their overall topology has remained elusive.One F ab in particular,1B2,has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic(cryoEM)analyses of multiple modules from distinct assembly-line PKSs.Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM,thereby enabling the visualization of intact PKS modules at near-atomic(3–5Å)resolution.The identification of PKS-binding F_(ab)s can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.

Three New Compounds from the Actinomycete Actinocorallia aurantiaca

Aurantiadioic acids A(1)and B(2),two new furan-containing polyketides,and aurantoic acid A(3),a new natural product,were isolated from the liquid fermentation of the sika deer dung-derived actinomycete Actinocorallia aurantiaca.The structures of the new compounds were established by extensive spectroscopic methods,including 1D&2D NMR,HRESIMS spectroscopic analysis.The absolute configuration of 3 was assigned by comparison of the specific optical rotations with the reported derivatives.Biological activity evaluations suggested that compounds 1-3 showed weak inhibition on NO production in the murine monocytic RAW 264.7 macrophages with IC_(50)values of 35.8,41.8,45.2μM,respectively.Compound 3 showed weak inhibition on influenza A virus(A/PuertoRico/8/1934,H1N1)with an EC_(50)value of 35.9μM,and a selective index higher than 13.3.

Characterization of the depsidone gene cluster reveals etherification,decarboxylation and multiple halogenations as tailoring steps in depsidone assembly

Depsides and depsidones have attracted attention for biosynthetic studies due to their broad biological activities and structural diversity.Previous structure-activity relationships indicated that triple halogenated depsidones display the best anti-pathogenic activity.However,the gene cluster and the tailoring steps responsible for halogenated depsidone nornidulin(3)remain enigmatic.In this study,we disclosed the complete biosynthetic pathway of the halogenated depsidone through in vivo gene disruption,heterologous expression and in vitro biochemical experiments.We demonstrated an unusual depside skeleton biosynthesis process mediated by both highly-reducing polyketide synthase and nonreducing polyketide synthase,which is distinct from the common depside skeleton biosynthesis.This skeleton was subsequently modified by two in-cluster enzymes DepG and DepF for the ether bond formation and decarboxylation,respectively.In addition,the decarboxylase DepF exhibited substrate promiscuity for different scaffold substrates.Finally,and interestingly,we discovered a halogenase encoded remotely from the biosynthetic gene cluster,which catalyzes triple-halogenation to produce the active end product nornidulin(3).These discoveries provide new insights for further understanding the biosynthesis of depsidones and their derivatives.

Gaining access to acetyl-CoA by peroxisomal surface display

Synthetic biology is constantly making progress for producing compounds on demand.Recently,Yocum and collaborators have developed an outstanding approach based on the anchoring of biosynthetic enzymes to the peroxisomal membrane.This allowed access to an untapped resource of acetyl-CoA and stimulated the synthesis of a valuable polyketide.