Protein-Protein Recognition Involved in the Intermodular Transacylation Reaction in Modular Polyketide Synthase in the Biosynthesis of Vicenistatin

The ketosynthase (KS) domain is a core domain found in modular polyketide synthases (PKSs). To maintain the polyketide biosynthetic fidelity, the KS domain must only accept an acyl group from the acyl carrier protein (ACP) domain of the immediate upstream module even when they are separated into different polypeptides. Although it was reported that both the docking domain-based interactions and KS-ACP compatibility are important for the interpolypeptide transacylation reaction in 6-deoxyerythronolide B synthase, it is not clear whether these findings are broadly applied to other modular PKSs. Herein, we describe the importance of protein-protein recognition in the intermodular transacylation between VinP1 module 3 and VinP2 module 4 in vicenistatin biosynthesis. We compared the transacylation activity and crosslinking efficiency of VinP2 KS₄ against the cognate VinP1 ACP₃ with the noncognate one. As a result, it appeared that VinP2 KS₄ distinguishes the cognate ACP₃ from other ACPs.     

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.     

Structural and Biochemical Insight into the Recruitment of Acyl Carrier Protein-Linked Extender Units in Ansamitocin Biosynthesis

A few acyltransferase (AT) domains of modular polyketide synthases (PKSs) recruit acyl carrier protein (ACP)-linked extender units with unusual C2 substituents to confer functionalities that are not available in coenzyme A (CoA)-linked ones. In this study, an AT specific for methoxymalonyl (MOM)-ACP in the third module of the ansamitocin PKS was structurally and biochemically characterized. The AT uses a conserved tryptophan residue at the entrance of the substrate binding tunnel to discriminate between different carriers. A W275R mutation switches its carrier specificity from the ACP to the CoA molecule. The acyl-AT complex structures clearly show that the MOM-ACP accepted by the AT has the 2S instead of the opposite 2R stereochemistry that is predicted according to the biosynthetic derivation from a d -glycolytic intermediate. Together, these results reveal the structural basis of ATs recognizing ACP-linked extender units in polyketide biosynthesis.     

Phosphopantetheinylation and Specificity of Acyl Carrier Proteins in the Mupirocin Biosynthetic Cluster

Acyl carrier proteins are vital for the biosynthesis of fatty acids and polyketides. The mupirocin biosynthetic cluster of Pseudomonas fluorescens encodes eleven type I ACPs embedded in its multifunctional polyketide synthase (PKS) proteins plus five predicted type II ACPs (mAcpA‐E) that are known to be essential for mupirocin biosynthesis by deletion and complementation analysis. MupN is a putative Sfp‐type phosphopantetheinyl transferase. Overexpression of three type I and three type II mupirocin ACPs in Escherichia coli, with or without mupN, followed by mass spectroscopy revealed that MupN can modify both mupirocin type I and type II ACPs to their holo‐form. The endogenous phosphopantetheinyl transferase of E. coli modified mAcpA but not mAcpC or D. Overexpression of the type II ACPs in macp deletion mutants of the mupirocin producer P. fluorescens 10586 showed that they cannot substitute for each other while hybrids between mAcpA and mAcpB indicated that, at least for mAcpB, the C‐terminal domain determines functional specificity. Amino acid alignments identified mACPs A and D as having C‐terminal extensions. Mutation of these regions generated defective ACPs, the activity of which could be restored by overexpression of the macp genes on separate plasmids.     

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.     

Bifunctionality of ActIV as a Cyclase‐Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

Type II polyketide synthases iteratively generate a nascent polyketide thioester of the acyl carrier protein (ACP); this is structurally modified to produce an ACP‐free intermediate towards the final metabolite. However, the timing of ACP off‐loading is not well defined because of the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess TE activity in vitro with a model substrate, anthraquinone‐2‐carboxylic acid‐N‐acetylcysteamine. In order to investigate its function further, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was reconstituted in vitro in a stepwise fashion up to (S)‐DNPA, and the product of ActIV reaction was characterized as an ACP‐free bicyclic intermediate. These findings indicate that ActIV is a bifunctional CYC‐TE and provide clear evidence for the release timing of the intermediate from the ACP anchor.     

Fatty acyl-AMP ligase involvement in the production of alkylresorcylic acid by a Myxococcus xanthus type III polyketide synthase

Fatty acyl-AMP ligases (FAALs) activate fatty acids as acyladenylates, and subsequently catalyze their transfer onto the acyl carrier proteins (ACPs) of polyketide synthases (PKSs) or nonribosomal peptide synthetases to produce lipidic metabolites. Myxococcus xanthus contains a polyketide biosynthesis gene cluster in which putative FAAL (FtpD) and ACP (FtpC) genes are located close to a type III PKS (FtpA) gene. Here we describe the characterization of these three proteins in vitro. FtpD adenylated stearic acid and produced stearoyl-FtpC. The stearoyl moiety was then transferred to FtpA. When extender substrates (malonyl-CoA and methylmalonyl-CoA) were added to the reaction, the alkylresorcinol 5-heptadecyl-4-methyl-benzene-1,3-diol was synthesized. Further in vitro analysis indicated that FtpA produces an alkylresorcylic acid as the direct product, and that this decarboxylates to alkylresorcinol nonenzymatically. This is the first report of a FAAL supplying a long-chain fatty acyl-ACP starter substrate to a type III PKS.     

Acyltransferase Domain Exchange between Two Independent Type I Polyketide Synthases in the Same Producer Strain of Macrolide Antibiotics

Streptomyces graminofaciens A-8890 produces two macrolide antibiotics, FD-891 and virustomycin A, both of which show significant biological activity. In this study, we identified the virustomycin A biosynthetic gene cluster, which encodes type I polyketide synthases (PKSs), ethylmalonyl-CoA biosynthetic enzymes, methoxymalony-acyl carrier protein biosynthetic enzymes, and post-PKS modification enzymes. Next, we demonstrated that the acyltransferase domain can be exchanged between the Vsm PKSs and the PKSs involved in FD-891 biosynthesis (Gfs PKSs), without any supply problems of the unique extender units. We exchanged the malonyltransferase domain in the loading module of Gfs PKS with the ethylmalonyltransferase domain and the methoxymalonyltransferase domain of Vsm PKSs. Consequently, the expected two-carbon-elongated analog 26-ethyl-FD-891 was successfully produced with a titer comparable to FD-891 production by the wild type; however, exchange with the methoxymalonyltransferase domain did not produce any FD-891 analogs. Furthermore, 26-ethyl-FD-891 showed potent cytotoxic activity against HeLa cells, like natural FD-891.     

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.     

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.     

Predicted Incorporation of Non‐native Substrates by a Polyketide Synthase Yields Bioactive Natural Product Derivatives

The polyether ionophore monensin is biosynthesized by a polyketide synthase that delivers a mixture of monensins A and B by the incorporation of ethyl‐ or methyl‐malonyl‐CoA at its fifth module. Here we present the first computational model of the fifth acyltransferase domain (AT5_mon) of this polyketide synthase, thus affording an investigation of the basis of the relaxed specificity in AT5_mon, insights into the activation for the nucleophilic attack on the substrate, and prediction of the incorporation of synthetic malonic acid building blocks by this enzyme. Our predictions are supported by experimental studies, including the isolation of a predicted derivative of the monensin precursor premonensin. The incorporation of non‐native building blocks was found to alter the ratio of premonensins A and B. The bioactivity of the natural product derivatives was investigated and revealed binding to prenyl‐binding protein. We thus show the potential of engineered biosynthetic polyketides as a source of ligands for biological macromolecules.     

Glyceryl-S-acyl carrier protein as an intermediate in the biosynthesis of tetronate antibiotics

The biosynthetic pathway to the unusual tetronate ring of certain polyketide natural products, including the antibiotics abyssomicin and tetronomycin (TMN) and the antitumour compound chlorothricin (CHL), is presently unknown. The gene clusters governing chlorothricin and tetronomycin biosynthesis both contain a gene encoding an atypical member of the FkbH family of enzymes, which has previously been shown to synthesise glyceryl-S-acyl carrier protein (ACP) as the first step in production of unusual extender units for modular polyketide biosynthesis. We show here that purified recombinant FkbH-like protein, Tmn16, from the TMN gene cluster catalyses the efficient transfer of a glyceryl moiety from D-1,3-bisphosphoglycerate (1,3-BPG) to either of the dedicated ACPs, Tmn7a and ChlD2, to form glyceryl-S-ACP, which directly implicates this compound as an intermediate in tetronate biosynthesis as well. Neither Tmn16 nor Tmn7a produced glyceryl-S-ACP when incubated, respectively, with analogous ACP and FkbH-like proteins from a known extender-unit pathway; this indicates a highly selective channelling of glycolytic metabolites into tetronate biosynthesis.     

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.     

Base‐Pairing Behavior of a Carbocyclic Janus‐AT Nucleoside Analogue Capable of Recognizing A and T within a DNA Duplex

Janus‐type nucleosides are heterocycles with two faces, each of which is designed to complement the H‐bonding interactions of natural nucleosides comprising a canonical Watson–Crick base pair. By intercepting all of the hydrogen bonds contained within the base pair, oligomeric Janus nucleosides are expected to achieve sequence‐specific DNA recognition through the formation of J‐loops that will be more stable than D‐loops, which simply replaces one base‐pair with another. Herein, we report the synthesis of a novel Janus‐AT nucleoside analogue, J_AT, affixed on a carbocyclic analogue of deoxyribose that was converted to the corresponding phosphoramidite. A single J_AT was successfully incorporated into a DNA strand by solid phase for targeting both A and T bases, and characterized through biophysical and computational methods. Experimental UV‐melting and circular dichroism data demonstrated that within the context of a standard duplex, J_AT associates preferentially with T over A, and much more poorly with C and G. Density functional theory calculations confirm that the J_AT structure is well suited to associate only with A and T thereby highlighting the importance of the electronic structure in terms of H‐bonding. Finally, molecular dynamics simulations validated the observation that J_AT can substitute more effectively as an A‐analogue than as a T‐analogue without substantial distortion of the B‐helix. Overall, this new Janus nucleotide is a promising tool for the targeting of A–T base pairs in DNA, and will lead to the development of oligo‐Janus‐nucleotide strands for sequence‐specific DNA recognition.     

Biosynthesis of Colabomycin E,a New Manumycin‐Family Metabolite,Involves an Unusual Chain‐Length Factor

Colabomycin E is a new member of the manumycin‐type metabolites produced by the strain Streptomyces aureus SOK1/5‐04 and identified by genetic screening from a library of streptomycete strains. The structures of colabomycin E and accompanying congeners were resolved. The entire biosynthetic gene cluster was cloned and expressed in Streptomyces lividans. Bioinformatic analysis and mutagenic studies identified components of the biosynthetic pathway that are involved in the formation of both polyketide chains. Recombinant polyketide synthases (PKSs) assembled from the components of colabomycin E and asukamycin biosynthetic routes catalyzing the biosynthesis of “lower” carbon chains were constructed and expressed in S. aureus SOK1/5‐04 ΔcolC11–14 deletion mutant. Analysis of the metabolites produced by recombinant strains provided evidence that in both biosynthetic pathways the length of the lower carbon chain is controlled by an unusual chain‐length factor supporting biosynthesis either of a triketide in asukamycin or of a tetraketide in colabomycin E. Biological activity assays indicated that colabomycin E significantly inhibited IL‐1β release from THP‐1 cells and might thus potentially act as an anti‐inflammatory agent.     

真菌芳香聚酮合酶研究进展

真菌芳香聚酮化合物是由真菌非还原聚酮合酶（NR-PKS）催化合成的一大类具有生物活性的天然产物。真菌芳香聚酮合酶是一个包含酮基合酶（KS）、酰基载体蛋白（ACP）、起始单元：ACP酰基转移酶（SAT）、丙二酰：ACP酰基转移酶（MAT）、产物模板（PT）和硫酯-环化酶（TE-CLC）6个结构域的巨大蛋白,负责催化真菌芳香聚酮的生物合成。在催化循环中如何选择起始单元,如何催化链的起始与延伸,如何进行中间体的折叠与环化,以及如何控制产物的正确解离,是真菌芳香聚酮合酶催化机制中的核心问题。对这些问题的理解为从真菌中筛选天然产物合成途径和利用组合生物合成手段产生新型活性分子奠定了基础。     

Catalytic relationships between type I and type II iterative polyketide synthases: The Aspergillus parasiticus norsolorinic acid synthase

Norsolorinic acid synthase (NSAS) is a type I iterative polyketide synthase that occurs in the filamentous fungus Aspergillus parasiticus. PCR was used to clone fragments of NSAS corresponding to the acyl carrier protein (ACP), acyl transferase (AT) and beta-ketoacyl-ACP synthase (KS) catalytic domains. Expression of these gene fragments in Escherichia coli led to the production of soluble ACP and AT proteins. Coexpression of ACP with E. coli holo-ACP synthase (ACPS) let to production of NSAS holo-ACP, which could also be formed in vitro by using Streptomyces coelicolor ACPS. Analysis by mass spectrometry showed that, as with other type I carrier proteins, self-malonylation is not observed in the presence of malonyl CoA alone. However, the NSAS holo-ACP serves as substrate for S. coelicolor MCAT, S. coelicolor actinorhodin holo-ACP and NSAS AT domain-catalysed malonate transfer from malonyl CoA. The AT domain could transfer malonate from malonyl CoA to NSAS holo-ACP, but not hexanoate or acetate from either the cognate CoA or FAS ACP species to NSAS holo-ACP. The NSAS holo-ACP was also active in actinorhodin minimal PKS assays, but only in the presence of exogenous malonyl transferases.     

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.