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.     

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.     

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.     

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.     

Salinipyrone and Pacificanone Are Biosynthetic By‐products of the Rosamicin Polyketide Synthase

Salinipyrones and pacificanones are structurally related polyketides from Salinispora pacifica CNS‐237 that are proposed to arise from the same modular polyketide synthase (PKS) assembly line. Genome sequencing revealed a large macrolide PKS gene cluster that codes for the biosynthesis of rosamicin A and a series of new macrolide antibiotics. Mutagenesis experiments unexpectedly correlated salinipyrone and pacificanone biosynthesis to the rosamicin octamodule Spr PKS. Remarkably, this bifurcated polyketide pathway illuminates a series of enzymatic domain‐ and module‐skipping reactions that give rise to natural polyketide product diversity. Our findings enlarge the growing knowledge of polyketide biochemistry and illuminate potential challenges in PKS bioengineering.     

Uncovering a Glycosyltransferase Provides Insights into the Glycosylation Step during Macrolactin and Bacillaene Biosynthesis

Macrolactins (MLNs) have unique structural patterns containing a 24‐membered ring lactone and diverse bioactivities. The MLN skeleton is biosynthesized via a trans‐acyl transferase (AT) type I polyketide synthase (PKS) pathway, but the tailoring steps are still unknown. Herein, we report the identification of a glycosyltransferase (GT) gene bmmGT1, which is located at different locus from the MLN gene cluster in the genome of marine‐derived Bacillus marinus B‐9987, and its functional characterization as an MLN GT, thus affording five novel MLNs analogues. Surprisingly, this GT is also capable of catalyzing the glycosylation of bacillaenes (BAEs), which are the prototypes of trans‐AT polyketides, thus suggesting broad substrate flexibility. These results provide the first significant insights into the glycosylation step in MLN and BAE biosynthetic pathways.     

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.     

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.     

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.     

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.     

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.     

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.     

Biosynthesis of the Myxobacterial Antibiotic Corallopyronin A

Corallopyronin A is a myxobacterial compound with potent antibacterial activity. Feeding experiments with labelled precursors resulted in the deduction of all biosynthetic building blocks for corallopyronin A and revealed an unusual feature of this metabolite: its biosynthesis from two chains, one solely PKS‐derived and the other NRPS/PKS‐derived. The starter molecule is believed to be carbonic acid or its monomethyl ester. The putative corallopyronin A biosynthetic gene cluster is a trans‐AT‐type mixed PKS/NRPS gene cluster, containing a β‐branching cassette. Striking features of this gene cluster are a NRPS‐like adenylation domain that is part of a PKS‐type module and is believed to be responsible for glycine incorporation, as well as split modules with individual domains occurring on different genes. It is suggested that CorB is a trans‐acting ketosynthase and it is proposed that it catalyses the Claisen condensation responsible for the interconnection of the two chains. Additionally, the stereochemistry of corallopyronin A was deduced by a combination of a modified Mosher's method and ozonolysis with subsequent chiral GC analyses.     

The AT2 Domain of KirCI Loads Malonyl Extender Units to the ACPs of the Kirromycin PKS

The antibiotic kirromycin is assembled by a hybrid modular polyketide synthases (PKSs)/nonribosomal peptide synthetases (NRPSs). Five of six PKSs of this complex assembly line do not have acyltransferase (AT) and have to recruit this activity from discrete AT enzymes. Here, we show that KirCI is a discrete AT which is involved in kirromycin production and displays a rarely found three‐domain architecture (AT₁‐AT₂‐ER). We demonstrate that the second AT domain, KirCI‐AT₂, but not KirCI‐AT₁, is the malonyl‐CoA‐specific AT which utilizes this precursor for loading the acyl carrier proteins (ACPs) of the trans‐AT PKS in vitro. In the kirromycin biosynthetic pathway, ACP5 is exclusively loaded with ethylmalonate by the enzyme KirCII and is not recognized as a substrate by KirCI. Interestingly, the excised KirCI‐AT₂ can also transfer malonate to ACP5 and thus has a relaxed ACP‐specificity compared to the entire KirCI protein. The ability of KirCI‐AT₂ to load different ACPs provides opportunities for AT engineering as a potential strategy for polyketide diversification.     

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.     

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.     

Genome Mining of the Hitachimycin Biosynthetic Gene Cluster: Involvement of a Phenylalanine‐2,3‐aminomutase in Biosynthesis

Hitachimycin is a macrolactam antibiotic with (S)‐β‐phenylalanine (β‐Phe) at the starter position of its polyketide skeleton. To understand the incorporation mechanism of β‐Phe and the modification mechanism of the unique polyketide skeleton, the biosynthetic gene cluster for hitachimycin in Streptomyces scabrisporus was identified by genome mining. The identified gene cluster contains a putative phenylalanine‐2,3‐aminomutase (PAM), five polyketide synthases, four β‐amino‐acid‐carrying enzymes, and a characteristic amidohydrolase. A hitA knockout mutant showed no hitachimycin production, but antibiotic production was restored by feeding with (S)‐β‐Phe. We also confirmed the enzymatic activity of the HitA PAM. The results suggest that the identified gene cluster is responsible for the biosynthesis of hitachimycin. A plausible biosynthetic pathway for hitachimycin, including a unique polyketide skeletal transformation mechanism, is proposed.     

4‐Hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐one Synthesis by a Type III Polyketide Synthase from Rhodospirillum centenum

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl‐CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C_10–14 fatty acyl‐CoA unit, one malonyl‐CoA unit and two methylmalonyl‐CoA units. We identified these products as 4‐hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl‐CoA as the extender substrate. In addition, in vitro reactions with ¹³C‐labeled malonyl‐CoA revealed that RpsA produced tetraketide 6‐alkyl‐4‐hydroxy‐1,5‐dimethyl‐2‐oxocyclohexa‐3,5‐diene‐1‐carboxylic acids from C_14–20 fatty acyl‐CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.     

Identification and Characterization of the Cuevaene A Biosynthetic Gene Cluster in Streptomyces sp. LZ35

Genome sequence analysis of Streptomyces sp. LZ35 has revealed a large number of secondary metabolite pathways, including one encoded in an orphan type I polyketide synthase gene cluster that contains a putative chorismatase/3‐hydroxybenzoate synthase gene. Mutagenesis and comparative metabolic profiling led to the identification of cuevaene A as the metabolic product of the gene cluster, thus making it the first 3‐HBA containing polyketide biosynthetic gene cluster described to date. Cuv10 was proven to be responsible for the conversion of chorismate into 3‐HBA; Cuv18 is speculated to be responsible for the 6‐hydroxylation of 3‐HBA during polyketide chain elongation. Additionally, several pathway‐specific regulatory factors that affect the production of cuevaene A were identified. Our results indicate that targeted inactivation of a gene followed by comparative metabolic profiling is a useful approach to identify and characterize cryptic biosynthetic gene clusters.