Yuhui Sun

A Complete Gene Cluster from Streptomyces nanchangensis NS3226 Encoding Biosynthesis of the Polyether Ionophore Nanchangmycin

The PKS genes for biosynthesis of the polyether nanchangmycin are organized to encode two sets of proteins (six and seven ORFs, respectively), but are separated by independent ORFs that encode an epimerase, epoxidase, and epoxide hydrolase, and, notably, an independent ACP. One of the PKS modules lacks a corresponding ACP. We propose that the process of oxidative cyclization to form the polyether structure occurs when the polyketide chain is still anchored on the independent ACP before release. 4-O-methyl-L-rhodinose biosynthesis and its transglycosylation involve four putative genes, and regulation of nanchangmycin biosynthesis seems to involve activation as well as repression. In-frame deletion of a KR6 domain generated the nanchangmycin aglycone with loss of 4-O-methyl-L-rhodinose and antibacterial activity, in agreement with the assignments of the PKS domains catalyzing specific biosynthetic steps.

Prediction and Manipulation of the Stereochemistry of Enoylreduction in Modular Polyketide Synthases

When an enoylreductase enzyme of a modular polyketide synthase reduces a propionate extender unit that has been newly added to the growing polyketide chain, the resulting methyl branch may have either S or R configuration. We have uncovered a correlation between the presence or absence of a unique tyrosine residue in the ER active site and the chirality of the methyl branch that is introduced. When this position in the active site is occupied by a tyrosine residue, the methyl branch has S configuration, otherwise it has R configuration. In a model PKS in vivo, a mutation (Tyr to Val) in an erythromycin PKS-derived ER caused a switch in the methyl branch configuration in the product from S to R. In contrast, alteration (Val to Tyr) at this position in a rapamycin-derived PKS ER was insufficient to achieve a switch from R to S, showing that additional residues also participate in stereocontrol of enoylreduction.

Analysis of the Tetronomycin Gene Cluster: Insights into the Biosynthesis of a Polyether Tetronate Antibiotic

The biosynthetic gene cluster for tetronomycin (TMN), a polyether ionophoric antibiotic that contains four different types of ring, including the distinctive tetronic acid moiety, has been cloned from Streptomyces sp. NRRL11266. The sequenced tmn locus (113 234 bp) contains six modular polyketide synthase (PKS) genes and a further 27 open‐reading frames. Based on sequence comparison to related biosynthetic gene clusters, the majority of these can be assigned a plausible role in TMN biosynthesis. The identity of the cluster, and the requirement for a number of individual genes, especially those hypothesised to contribute a glycerate unit to the formation of the tetronate ring, were confirmed by specific gene disruption. However, two large genes that are predicted to encode together a multifunctional PKS of a highly unusual type seem not to be involved in this pathway since deletion of one of them did not alter tetronomycin production. Unlike previously characterised polyether PKS systems, oxidative cyclisation appears to take place on the modular PKS rather than after transfer to a separate carrier protein, while tetronate ring formation and concomitant chain release share common mechanistic features with spirotetronate biosynthesis.

In vitro reconstruction of tetronate RK-682 biosynthesis

The protein phosphatase inhibitor RK-682 is one of a number of potentially valuable tetronate polyketide natural products. Understanding how the tetronate ring is formed has been frustrated by the inaccessibility of the putative substrates. We report the heterologous expression of rk genes in Saccharopolyspora erythraea and reconstitution of the RK-682 pathway using recombinant enzymes, and show that RkD is the enzyme required for RK-682 formation from acyl carrier protein-bound substrates.

Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system

The current diminishing returns in finding useful antibiotics and the occurrence of drug-resistant bacteria call for the need to find new antibiotics. Moreover, the whole genome sequencing revealed that the biosynthetic potential of Streptomyces, which has produced the highest numbers of approved and clinical-trial drugs, has been greatly underestimated. Considering the known gene editing toolkits were arduous and inefficient, novel and efficient gene editing system are desirable. Here, we developed an engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) combined with the counterselection system CodA(sm), the D314A mutant of cytosine deaminase, to rapidly and effectively edit Streptomyces genomes. In-frame deletion of the actinorhodin polyketide chain length factor gene actI-ORF2 was created in Streptomyces coelicolor M145 as an illustration. This CRISPR/Cas9-CodA(sm) combined system strikingly increased the frequency of unmarked mutants and shortened the time required to generate them. We foresee the system becoming a routine laboratory technique for genome editing to exploit the great biosynthetic potential of Streptomyces and perhaps for other medically and economically important actinomycetes.

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.

A Late-Stage Intermediate in Salinomycin Biosynthesis Is Revealed by Specific Mutation in the Biosynthetic Gene Cluster

Salinomycin (1) from Streptomyces albus DSM 41398 is an antibiotic polyether ionophore with a complex tricyclic bispiroacetal core structure that selectively binds K ions and transports them across cell membranes, thus leading to cell death. The therapeutic use of salinomycin is limited by its toxicity, but it is widely used in animal husbandry as a coccidiostat. Salinomycin has attracted strong renewed interest owing to its potent and selective activity against cancer stem cells and cancer cell lines. Engineering the biosynthetic pathway to salinomycin offers an attractive route to novel and potentially useful analogues of this complex molecule. We have cloned and analysed the salinomycin gene cluster from S. albus DSM 41398, and found that the polyketide chain is synthesised on an assembly line of nine polyketide synthase (PKS) multienzymes. We have also initiated targeted deletion of the genes that control oxidative cyclisation so as to probe the mechanism of polyether ring formation. One such mutant produces a novel polyketide diene whose structure provides the first evidence for the likely order of key steps in the biosynthesis. Polyether ionophores make up a particularly numerous subclass of complex polyketide antibiotics. They are synthesised on canonical modular PKS assembly-line multienzymes, in which each module houses fatty acid synthase-like enzyme domains, and there is colinearity between the order (and enzyme domain content) of successive modules and the chemistry of the product. The antibiotic polyethers adopt a characteristic conformation in which multiple oxygen atoms provide ligands for a centrally held specific cation, whereas the external surface is exclusively nonpolar. A general model for the biosynthesis of polyethers has been proposed in which the PKS produces a linear polyketide chain containing two or more trans or E double bonds. Stereoselective epoxidation of these double bonds leads to a polyepoxide, whose ring opening (in a series of controlled SN2-like reactions) generates the characteristic rings of the polyether. This model is strongly supported by the results of extensive genetic studies on the cloned and characterised biosynthetic gene clusters for monensin A and several other polyethers. Comparison of these gene clusters has revealed the presence of a conserved set of genes that are a hallmark of polyether biosynthesis: the monC family b] that encode a flavin-linked epoxidase, and the monB family c] that encode novel epoxide hydrolase/cyclase enzymes. It also appears that all stages of the biosynthesis take place while the intermediates are tethered either to a discrete acyl carrier protein (ACP) or to an ACP domain within the PKS. 11] However, important aspects of the oxidative cyclisation process remain undefined, especially the molecular basis for the exquisite stereospecificity and stereoselectivity of the process. Salinomycin, and related polyether metabolites produced by S. albus DSM 41398, possess an unusual and particularly complex tricyclic

Analysis of functions in plasmid pHZ1358 influencing its genetic and structural stability in Streptomyces lividans 1326

The complete DNA sequence of plasmid pHZ1358, a widely used vector for targeted gene disruption and replacement experiments in many Streptomyces hosts, has been determined. This has allowed a detailed analysis of the basis of its structural and segregational instability, compared to the high copy number plasmid pIJ101 of Streptomyces lividans 1326 from which it was derived. A 574-bp DNA region containing sti (strong incompatibility locus) was found to be a determinant for segregational instability in its original S. lividans 1326 host, while the structural instability was found to be related to the facile deletion of the entire Escherichia coli-derived part of pHZ1358, mediated by recombination between 36-bp direct repeats. A point mutation removing the BamHI site inside the rep gene encoding a replication protein (rep*) and/or a spontaneous deletion of the 694-bp region located between rep and sti including the uncharacterized ORF85 (orf85−) produced little or no effect on stability. A pHZ1358 derivative (pJTU412, sti−, rep*, orf85−) was then constructed which additionally lacked one of the 36-bp direct repeats. pJTU412 was demonstrated to be structurally stable but segregationally unstable and, in contrast to sti+ pHZ1358, allowed efficient targeted gene replacement in S. lividans 1326.

Unusual Acetylation–Elimination in the Formation of Tetronate Antibiotics

Tetronate antibiotics comprise an important and growing family of polyketide natural products possessing a characteristic tetronate (4-hydroxy-[5H]furan-2-one) ring system. They have been isolated from both terrestrial and marine bacteria, and show a diverse range of biological activities. They include the tetronate polyethers tetronomycin and tetronasin, the fatty acyltetronate antibiotic agglomerin, and the structurally closely related protein phosphatase inhibitor RK682 (Figure 1). Of particular interest are the structurally intriguing spirotetronates (Figure 1a), including the antibacterial compounds chlorothricin and abyssomicin, the antiviral compound quartromicin, and the antitumour compounds tetrocarcin and kijanimicin. These compounds appear to arise through an enzyme-catalyzed Diels– Alder reaction after specific dehydration of an initially formed tetronate precursor, as shown for atrop-abyssomicin C in Figure 1b. Analysis of the biosynthetic gene clusters for several of these natural products 5–10] has highlighted the presence of a set of highly conserved genes unique to tetronate biosynthesis, whose predicted products include candidate enzymes that might catalyze formation of the tetronate ring C C and C O bonds. Reconstitution of RK-682 biosynthesis in vitro has been used to show that RkE is a glyceryl-S-acyl carrier protein (ACP) synthase, and that the ketoacyl-S-ACP synthase FabH-like RkD is necessary and sufficient to catalyze formation of the tetronate ring in vitro starting from a 3-ketoacyl thioester and glyceryl-S-ACP. Similar results were recently obtained for the FabH-like QmnD5 in quartromicin biosynthesis and it seems highly likely that most (if not all) tetronates follow an analogous biosynthetic pathway. Until now, the course of the dehydration step in spirotetronate biosynthesis, which provides the dienophile for the ensuing Diels–Alder-like reaction, has remained obscure. We show here, by cloning and analysis of the gene cluster for biosynthesis of agglomerins A–D in Pantoea agglomerans (formerly Enterobacter agglomerans) PB-6042, its heterologous expression in Escherichia coli, and the total reconstitution of agglomerin biosynthesis in vitro, that the mechanism of dehydration actually involves two steps after formation of the tetronate ring: O-acetylation catalyzed by Agg4, followed by elimination of acetic acid to form the exocyclic double bond catalyzed by Agg5. We propose that the biosynthesis of spirotetronates involves the same two-step reaction sequence, catalyzed by enzymes homologous to Agg4 and Agg5. The agglomerin biosynthetic pathway of P. agglomerans provided an attractive system in which to study these key steps, given the relative simplicity of the structures of agglomerin A and its congeners agglomerins B–D, which differ from each other only in the nature of the fatty acyl sidechain (Figure 1). Cosmid and whole-genome sequencing of P. agglomerans PB-6042 was used to reveal a circular chromosome of approximately 4.3 Mbp (bp = base pairs), within which the agglomerin cluster was readily identified through its similarity to that of RK-682 (Supporting Information, Figure S1). A 12 kbp DNA sequence encodes seven ORFs that could be plausibly assigned to the cluster. As well as the expected high homology between several genes in the agg and rk clusters, key differences in enzymology could also be inferred from the comparison of these clusters: whereas RK-682 obtains its linear precursor from palmitic acid, which is activated and then elongated on a modular polyketide synthase (PKS) to give 3-oxo-stearoyl-S-ACP, no counterpart of the rkC PKS could be found anywhere on the P. agglomerans chromosome. The precursors for agglomerins A–D appear to be taken directly from primary metabolism, probably as the corresponding 3-oxoacyl-CoA thioesters. In support of this, when the seven genes of the putative [*] C. Kanchanabanca, Dr. H. Hong, Dr. M. Samborskyy, Prof. Dr. P. F. Leadlay Department of Biochemistry, University of Cambridge 80 Tennis Court Road, Cambridge CB2 1GA (UK) E-mail: pfl10@cam.ac.uk Dr. W. Tao, Y. Liu, Prof. Dr. Z. Deng, Prof. Dr. Y. Sun Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and School of Pharmaceutical Sciences, Wuhan University 185 East Lake Road, Wuchan 430071 (P.R.China) E-mail: yhsun@whu.edu.cn

Cloning of Separate Meilingmycin Biosynthesis Gene Clusters by Use of Acyltransferase-Ketoreductase Didomain PCR Amplification

ABSTRACT Five meilingmycins, A to E, with A as the major component, were isolated from Streptomyces nanchangensis NS3226. Through nuclear magnetic resonance (NMR) characterization, meilingmycins A to E proved to be identical to reported milbemycins α11, α13, α14, β1, and β9, respectively. Sequencing of a previously cloned 103-kb region identified three modular type I polyketide synthase genes putatively encoding the last 11 elongation steps, three modification proteins, and one transcriptional regulatory protein for meilingmycin biosynthesis. However, the expected loading module and the first two elongation modules were missing. In meilingmycin, the presence of a methyl group at C-24 and a hydroxyl group at C-25 suggests that the elongation module 1 contains a methylmalonyl-coenzyme A (CoA)-specific acyltransferase (ATp) domain and a ketoreductase (KR) domain. Based on the conserved motifs of the ATp and KR domains, a pair of primers was designed for PCR amplification, and a 1.40-kb expected fragment was amplified, whose sequence shows significant homology with the elongation module 1 of the aveA1-encoded enzyme AVES1. A polyketide synthase (PKS) gene encoding one loading and two elongation modules, with a downstream C-5-O-methyltransferase gene, meiD, was subsequently localized 55 kb apart from the previously sequenced region, and its deletion abolishes meilingmycin production. A series of deletions within the 55-kb intercluster region rules out its involvement in meilingmycin biosynthesis. Furthermore, gene deletion of meiD eliminates meilingmycins D and E, with methyls at C-5. Our work provides a more specific strategy for the cloning of modular type I PKS gene clusters. The cloning of the meilingmycin gene clusters paves the way for its pathway engineering.