In Vitro Reconstitution of a PKS Pathway for the Biosynthesis of Galbonolides in Streptomyces sp. LZ35

The galbonolides are 14‐membered macrolide antibiotics with a macrocyclic backbone similar to that of erythromycins. Galbonolides exhibit broad‐spectrum antifungal activities. Retro‐biosynthetic analysis suggests that the backbone of galbonolides is assembled by a type I modular polyketide synthase (PKS). Unexpectedly, the galbonolide biosynthetic gene cluster, gbn, in Streptomyces sp. LZ35 encodes a hybrid fatty acid synthase (FAS)‐PKS pathway. In vitro reconstitution revealed the functions of GbnA (an AT‐ACP didomain protein), GbnC (a FabH‐like enzyme), and GbnB (a novel multidomain PKS module without AT and ACP domains) responsible for assembling the backbone of galbonolides, respectively. To our knowledge, this study is the first biochemical characterization of a hybrid FAS‐PKS pathway for the biosynthesis of 14‐membered macrolides. The identification of this pathway provides insights into the evolution of PKSs and could facilitate the design of modular pools for synthetic biology.     

Parallel Post‐Polyketide Synthase Modification Mechanism Involved in FD‐891 Biosynthesis in Streptomyces graminofaciens A‐8890

To isolate a key polyketide biosynthetic intermediate for the 16‐membered macrolide FD‐891 ( 1 ), we inactivated two biosynthetic genes coding for post‐polyketide synthase (PKS) modification enzymes: a methyltransferase (GfsG) and a cytochrome P450 (GfsF). Consequently, FD‐892 ( 2 ), which lacks the epoxide moiety at C8–C9, the hydroxy group at C10, and the O‐methyl group at O‐25 of FD‐891, was isolated from the gfsF/gfsG double‐knockout mutant. In addition, 25‐O‐methyl‐FD‐892 ( 3 ) and 25‐O‐demethyl‐FD‐891 ( 4 ) were isolated from the gfsF and gfsG mutants, respectively. We also confirmed that GfsG efficiently catalyzes the methylation of 2 and 4 in vitro. Further, GfsF catalyzed the epoxidation of the double bond at C8‐C9 of 2 and 3 and subsequent hydroxylation at C10, to afford 4 and 1 , respectively. These results suggest that a parallel post‐PKS modification mechanism is involved in FD‐891 biosynthesis.     

Identification of the Biosynthetic Gene Cluster for Himeic Acid A: A Ubiquitin‐Activating Enzyme (E1) Inhibitor in Aspergillus japonicus MF275

Himeic acid A, which is produced by the marine fungus Aspergillus japonicus MF275, is a specific inhibitor of the ubiquitin‐activating enzyme E1 in the ubiquitin–proteasome system. To elucidate the mechanism of himeic acid biosynthesis, feeding experiments with labeled precursors have been performed. The long fatty acyl side chain attached to the pyrone ring is of polyketide origin, whereas the amide substituent is derived from leucine. These results suggest that a polyketide synthase–nonribosomal peptide synthase (PKS‐NRPS) is involved in himeic acid biosynthesis. A candidate gene cluster was selected from the results of genome sequencing analysis. Disruption of the PKS‐NRPS gene by Agrobacterium‐mediated transformation confirms that HimA PKS‐NRPS is involved in himeic acid biosynthesis. Thus, the him biosynthetic gene cluster for himeic acid in A. japonicus MF275 has been identified.     

Comparison of 10,11‐Dehydrocurvularin Polyketide Synthases from Alternaria cinerariae and Aspergillus terreus Highlights Key Structural Motifs

Iterative type I polyketide synthases (PKSs) from fungi are multifunctional enzymes that use their active sites repeatedly in a highly ordered sequence to assemble complex natural products. A phytotoxic macrolide with anticancer properties, 10,11‐dehydrocurvularin (DHC), is produced by cooperation of a highly reducing (HR) iterative PKS and a non‐reducing (NR) iterative PKS. We have identified the DHC gene cluster in Alternaria cinerariae, heterologously expressed the active HR PKS (Dhc3) and NR PKS (Dhc5) in yeast, and compared them to corresponding proteins that make DHC in Aspergillus terreus. Phylogenetic analysis and homology modeling of these enzymes identified variable surfaces and conserved motifs that are implicated in product formation.     

Biosynthetic Code for Divergolide Assembly in a Bacterial Mangrove Endophyte

Divergolides are structurally diverse ansamycins produced by a bacterial endophyte (Streptomyces sp.) of the mangrove tree Bruguiera gymnorrhiza. By genomic analyses a gene locus coding for the divergolide pathway was detected. The div gene cluster encodes genes for the biosynthesis of 3‐amino‐5‐hydroxybenzoate and the rare extender units ethylmalonyl‐CoA and isobutylmalonyl‐CoA, polyketide assembly by a modular type I polyketide synthase (PKS), and enzymes involved in tailoring reactions, such as a Baeyer–Villiger oxygenase. A detailed PKS domain analysis confirmed the stereochemical integrity of the divergolides and provided valuable new insights into the formation of the diverse aromatic chromophores. The bioinformatic analyses and the isolation and full structural elucidation of four new divergolide congeners led to a revised biosynthetic model that illustrates the formation of four different types of ansamycin chromophores from a single polyketide precursor.     

In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line

Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl‐CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl‐CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self‐malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self‐acylation. This lack of self‐malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein–protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.     

Cloning and Characterization of the Biosynthetic Gene Cluster of 16‐Membered Macrolide Antibiotic FD‐891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

FD‐891 is a 16‐membered cytotoxic antibiotic macrolide that is especially active against human leukemia such as HL‐60 and Jurkat cells. We identified the FD‐891 biosynthetic (gfs) gene cluster from the producer Streptomyces graminofaciens A‐8890 by using typical modular type I polyketide synthase (PKS) genes as probes. The gfs gene cluster contained five typical modular type I PKS genes (gfsA, B, C, D, and E), a cytochrome P450 gene (gfsF), a methyltransferase gene (gfsG), and a regulator gene (gfsR). The gene organization of PKSs agreed well with the basic polyketide skeleton of FD‐891 including the oxidation states and α‐alkyl substituent determined by the substrate specificities of the acyltransferase (AT) domains. To clarify the involvement of the gfs genes in the FD‐891 biosynthesis, the P450 gfsF gene was inactivated; this resulted in the loss of FD‐891 production. Instead, the gfsF gene‐disrupted mutant accumulated a novel FD‐891 analogue 25‐O‐methyl‐FD‐892, which lacked the epoxide and the hydroxyl group of FD‐891. Furthermore, the recombinant GfsF enzyme coexpressed with putidaredoxin and putidaredoxin reductase converted 25‐O‐methyl‐FD‐892 into FD‐891. In the course of the GfsF reaction, 10‐deoxy‐FD‐891 was isolated as an enzymatic reaction intermediate, which was also converted into FD‐891 by GfsF. Therefore, it was clearly found that the cytochrome P450 GfsF catalyzes epoxidation and hydroxylation in a stepwise manner in the FD‐891 biosynthesis. These results clearly confirmed that the identified gfs genes are responsible for the biosynthesis of FD‐891 in S. graminofaciens.     

The Phormidolide Biosynthetic Gene Cluster: A trans‐AT PKS Pathway Encoding a Toxic Macrocyclic Polyketide

Phormidolide is a polyketide produced by a cultured filamentous marine cyanobacterium and incorporates a 16‐membered macrolactone. Its complex structure is recognizably derived from a polyketide synthase pathway, but possesses unique and intriguing structural features that prompted interest in investigating its biosynthetic origin. Stable isotope incorporation experiments confirmed the polyketide nature of this compound. We further characterized the phormidolide gene cluster (phm) through genome sequencing followed by bioinformatic analysis. Two discrete trans‐type acyltransferase (trans‐AT) ORFs along with KS‐AT adaptor regions (ATd) within the polyketide synthase (PKS) megasynthases, suggest that the phormidolide gene cluster is a trans‐AT PKS. Insights gained from analysis of the mode of acetate incorporation and ensuing keto reduction prompted our reevaluation of the stereochemistry of phormidolide hydroxy groups located along the linear polyketide chain.     

Identification of the Biosynthetic Gene Cluster and Regulatory Cascade for the Synergistic Antibacterial Antibiotics Griseoviridin and Viridogrisein in Streptomyces griseoviridis

Griseoviridin (GV) and viridogrisein (VG, also referred to as etamycin), produced by Streptomyces griseoviridis, are two chemically unrelated compounds belonging to the streptogramin family. Both of these natural products demonstrate broad‐spectrum antibacterial activity and constitute excellent candidates for future drug development. To elucidate the biosynthetic machinery associated with production of these two unique antibiotics, the gene cluster responsible for both GV and VG production was identified within the Streptomyces griseoviridis genome and characterized, and its function in GV and VG biosynthesis was confirmed by inactivation of 30 genes and complementation experiments. This sgv gene cluster is localized to a 105 kb DNA region that consists of 36 open reading frames (ORFs), including four nonribosomal peptide synthetases (NRPSs) for VG biosynthesis and a set of hybrid polyketide synthases (PKS)‐NRPSs with a discrete acyltransferase (AT), SgvQ, to assemble the GV backbone. The enzyme encoding genes for VG versus GV biosynthesis are separated into distinct “halves” of the cluster. A series of four genes: sgvA, sgvB, sgvC, and sgvK, were found downstream of the PKS‐NRPS; these likely code for construction of a γ‐butyrolactone (GBL)‐like molecule. GBLs and the corresponding GBL receptor systems are the highest ranked regulators that are able to coordinate the two streptomyces antibiotic regulatory protein (SARP) family positive regulators SgvR2 and SgvR3; both are key biosynthetic activators. Models of GV, VG, and GBL biosynthesis were proposed by using functional gene assignments, determined on the basis of bioinformatics analysis and further supported by in vivo gene inactivation experiments. Overall, this work provides new insights into the biosyntheses of the GV and VG streptogramins that are potentially applicable to a host of combinatorial biosynthetic scenarios.     

Cloning and Characterization of the Ravidomycin and Chrysomycin Biosynthetic Gene Clusters

The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1‐1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D ‐ravidosamine and D ‐virenose biosynthetic genes, post‐PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross‐complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino‐ and neutral sugars, exemplified through the structurally distinct 6‐membered D ‐ravidosamine and 5‐membered D ‐fucofuranose, to the coumarin‐based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.     

Analysis of Specific Mutants in the Lasalocid Gene Cluster: Evidence for Enzymatic Catalysis of a Disfavoured Polyether Ring Closure

Lasalocid is a highly atypical polyether ionophoric antibiotic, firstly because it contains a type of aromatic ring normally associated with fungal polyketides, and secondly because the formation of its tetrahydropyran ring appears to contravene Baldwin's rules, which predict the kinetically preferred routes for cyclisation reactions in organic chemistry. The lasalocid biosynthetic gene cluster has been cloned from Streptomyces lasaliensis, and the las locus (73 533 bp) was found to contain seven modular polyketide synthase (PKS) genes, including all the activities necessary for the synthesis of the aromatic moiety. Specific deletion from the gene cluster of the flanking lasC gene, which is predicted to encode a flavin‐linked epoxidase, abolished production both of lasalocid and of the minor cometabolite iso‐lasalocid without leading to accumulation of an identifiable intermediate; this suggests that oxidative cyclisation to form the polyether rings takes place on the PKS before release of the full‐length polyketide product. Meanwhile, a mutant in which the adjacent epoxide hydrolase lasB had been deleted produced iso‐lasalocid only. Iso‐lasalocid differs from lasalocid in the replacement of the tetrahydropyran ring by a tetrohydrofuran ring and represents the kinetically favoured product of cyclisation. The LasB epoxide hydrolase is therefore directly implicated in control of the stereochemical course of polyether ring formation during lasalocid biosynthesis.     

Evolutionary Imprint of Catalytic Domains in Fungal PKS–NRPS Hybrids

Fungal hybrid enzymes consisting of a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) module are involved in the biosynthesis of a vast array of ecologically and medicinally relevant natural products. Whereas a dozen gene clusters could be assigned to the requisite PKS–NRPS pathways, the programming of the multifunctional enzymes is still enigmatic. Through engineering and heterologously expressing a chimera of PKS (lovastatin synthase, LovB) and NRPS (cytochalasin synthase, CheA) in Aspergillus terreus, we noted the potential incompatibility of a fungal highly reducing PKS (hrPKS) with the NRPS component of fungal PKS–NRPS hybrids. To rationalize the unexpected outcome of the gene fusion experiments, we conducted extensive bioinformatic analyses of fungal PKS–NRPS hybrids and LovB‐type PKS. From motif studies and the function of the engineered chimeras, a noncanonical function of C‐terminal condensation (C) domains in truncated PKS–NRPS homologues was inferred. More importantly, sequence alignments and phylogenetic trees revealed an evolutionary imprint of the PKS–NRPS domains, which reflect the evolutionary history of the entire megasynthase. Furthermore, a detailed investigation of C and adenylation (A) domains provides support for a scenario in which not only the A domain but also the C domain participates in amino acid selection. These findings shed new light on the complex code of this emerging class of multifunctional enzymes and will greatly facilitate future combinatorial biosynthesis and pathway engineering approaches towards natural product analogues.     

Functional analysis of the aureothin iterative type I polyketide synthase

The modular-type polyketide synthase (PKS) that is involved in aureothin (aur) biosynthesis represents one of the first examples in which a single PKS module (AurA) is used in an iterative fashion. Here we report on the heterologous expression of an engineered AurAB fusion protein that unequivocally proves the iterative nature of AurA. In addition, point mutations reveal that aur PKS module 4 participates in polyketide biosynthesis despite its aberrant acyltransferase domain.     

Involvement of β-Alkylation Machinery and Two Sets of Ketosynthase-Chain-Length Factors in the Biosynthesis of Fogacin Polyketides in Actinoplanes missouriensis

Fogacin and two novel fogacin derivatives, fogacins B and C, were isolated from the rare actinomycete Actinoplanes missouriensis. Biosynthesis of fogacin C apparently requires β alkylation of a polyketide chain. The fogacin biosynthetic type II polyketide synthase (PKS) gene cluster contains a hydroxymethylglutaryl-coenzyme A synthase (HCS) cassette, which is usually responsible for β alkylation in the type I PKS system. Another characteristic of the fog cluster is that it encodes two sets of ketosynthase (KS) and chain-length factor (CLF). Inactivation of either of the two KS genes in A. missouriensis and heterologous expression of the HCS cassette with either of the two KS-CLF genes in Streptomyces albus indicated that each KS-CLF had a different starter substrate specificity: one preferred an unusual β-alkylated starter and the other preferred a normal acetyl starter. This study expands knowledge of HCS cassette-dependent β alkylation into the type II PKS system and provides a natural example of combinatorial biosynthesis for producing diverse polyketides from different starter substrates.     

Structural Diversification of Macrolactones by Substrate‐Flexible Cytochrome P450 Monooxygenases

The substrate flexibilities of several cytochrome P450 monooxygenases involved in macrolide biosynthesis were investigated to test their potential for the generation of novel macrolides. PikC hydroxylase in the pikromycin producer Streptomyces venezuelae accepted oleandomycin as an alternative substrate and introduced a hydroxy group at the C‐4 position, which is different from the intrinsic C‐12 hydroxylation position in the natural substrate. This is the first report of C‐4 hydroxylation activity of cytochrome P450 monooxygenase involved in the biosynthesis of 14‐membered macrolides. EryF hydroxylase from the erythromycin biosynthetic pathway of Saccharopolyspora erythraea and OleP oxidase from the oleandomycin biosynthetic pathway of Streptomyces antibioticus also showed a certain degree of plasticity towards alternative substrates. In particular, EryF and OleP were found to oxidize a 12‐membered macrolactone as an alternative substrate. These results demonstrate the potential usefulness of these enzymes to diversify macrolactones by post‐PKS oxidations.     

Synthesis of C‐Glucosylated Octaketide Anthraquinones in Nicotiana benthamiana by Using a Multispecies‐Based Biosynthetic Pathway

Carminic acid is a C‐glucosylated octaketide anthraquinone and the main constituent of the natural dye carmine (E120), possessing unique coloring, stability, and solubility properties. Despite being used since ancient times, longstanding efforts to elucidate its route of biosynthesis have been unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C‐glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway, which consists of AaOKS, StZhuI, StZhuJ, and DcUGT2, presents an alternative biosynthetic approach for the production of polyketides by using a type III polyketide synthase (PKS) and tailoring enzymes originating from a type II PKS system. The current study showcases the power of using transient expression in Nicotiana benthamiana for efficient and rapid identification of functional biosynthetic pathways, including both soluble and membrane‐bound enzymes.     

Aromatic Polyketide GTRI‐02 is a Previously Unidentified Product of the act Gene Cluster in Streptomyces coelicolor A3(2)

The biosynthesis of aromatic polyketides derived from type II polyketide synthases (PKSs) is complex, and it is not uncommon that highly similar gene clusters give rise to diverse structural architectures. The act biosynthetic gene cluster (BGC) of the model actinomycete Streptomyces coelicolor A3(2) is an archetypal type II PKS. Here we show that the act BGC also specifies the aromatic polyketide GTRI‐02 ( 1 ) and propose a mechanism for the biogenesis of its 3,4‐dihydronaphthalen‐1(2H)‐one backbone. Polyketide 1 was also produced by Streptomyces sp. MBT76 after activation of the act‐like qin gene cluster by overexpression of the pathway‐specific activator. Mining of this strain also identified dehydroxy‐GTRI‐02 ( 2 ), which most likely originated from dehydration of 1 during the isolation process. This work shows that even extensively studied model gene clusters such as act of S. coelicolor can still produce new chemistry, offering new perspectives for drug discovery.     

Characterization of an Orphan Type III Polyketide Synthase Conserved in Uncultivated “Entotheonella” Sponge Symbionts

Uncultivated bacterial symbionts from the candidate genus “Entotheonella” have been shown to produce diverse natural products previously attributed to their sponge hosts. In addition to these known compounds, “Entotheonella” genomes contain rich sets of biosynthetic gene clusters that lack identified natural products. Among these is a small type III polyketide synthase (PKS) cluster, one of only three clusters present in all known “Entotheonella” genomes. This conserved “Entotheonella” PKS (cep) cluster encodes the type III PKS CepA and the putative methyltransferase CepB. Herein, the characterization of CepA as an enzyme involved in phenolic lipid biosynthesis is reported. In vitro analysis showed a specificity for alkyl starter substrates and the production of tri- and tetraketide pyrones and tetraketide resorcinols. The conserved distribution of the cep cluster suggests an important role for the phenolic lipid polyketides produced in “Entotheonella” variants.     

Non-colinear polyketide biosynthesis in the aureothin and neoaureothin pathways: an evolutionary perspective

Aureothin and neoaureothin (spectinabilin) represent rare nitroaryl-substituted polyketide metabolites from Streptomyces thioluteus and Streptomyces orinoci, respectively, which only differ in the lengths of the polyene backbones. Cloning and sequencing of the 39 kb neoaureothin (nor) biosynthesis gene cluster and its comparison with the aureothin (aur) pathway genes revealed that both polyketide synthase (PKS) assembly lines are remarkably similar. In both cases the module architecture breaks with the principle of colinearity, as individual PKS modules are used in an iterative fashion. Parsimony and neighbour-joining phylogenetic studies provided insights into the evolutionary process that led to the programming of these unusual type I PKS systems and to prediction of which modules act iteratively. The iterative function of the first module in the neoaureothin pathway, NorA, was confirmed by a successful cross-complementation.