Policing starter unit selection of the enterocin type II polyketide synthase by the type II thioesterase EncL

Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete 'Streptomyces maritimus'. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-β-ketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here, we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates.     

Substrate tolerance of module 6 of the epothilone synthetase

The epothilone synthetase is a decamodular megasynthase responsible for the biosynthesis of a class of polyketide natural products with clinically promising antitumor activity. Recently, we developed a system comprised of modules 6-9 of the epothilone synthetase for the precursor-directed biosynthesis of epothilones in Escherichia coli [Boddy, C. N., Hotta, K., Tse, M. L., Watts, R. E., and Khosla, C. (2004) J. Am. Chem. Soc. 126, 7436-7437]. To systematically explore the biosynthetic potential of this system, we have now investigated the ability of the crucial first module in this engineered pathway, EpoD-M6, to accept, elongate, and process unnatural substrates. EpoD-M6 was expressed, purified, and demonstrated to accept both acyl-CoA and acylSNAC substrates. Of the substrates that were tested, octanoylSNAC and 3-octenoylSNAC proved to be excellent substrates in addition to the more complex natural substrate. Thus, this polyketide synthase module showed considerable tolerance, a feature that bodes well for the precursor-directed biosynthesis of epothilone analogues and related complex polyketides.     

Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase

Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.     

Identification of a gene cluster responsible for the biosynthesis of cyclic lipopeptide verlamelin

Only limited studies are available on the molecular-level biosynthesis of cyclic lipopeptides (cyclic and hybrid molecules consisting of peptide and fatty acid moieties) in filamentous fungi. Here, we identified and characterized biosynthetic genes of the cyclic lipopeptides, known as verlamelins. Only four genes, coding for non-ribosomal peptide synthetase (NRPS), fatty acid hydroxylase, thioesterase, and AMP-dependent ligase, were found to be involved in verlamelin biosynthesis by the analysis of corresponding gene knockouts. Surprisingly, no gene(s) coding for fatty acid synthase or polyketide synthase was present in the cluster, while verlamelin A/B contained a 5-hydroxytetradecanoic acid moiety. Precursor feeding experiment indicated that both fatty acid hydroxylase and thioesterase are involved to supply 5-hydroxytetradecanoic acid. The results suggested that 5-hydroxytetradecanoic acid was supplied from primary metabolism via fatty acid hydroxylase and loaded onto NRPS. Elongation of the peptide and final cyclization were accomplished by NRPS. The knowledge obtained through this study should provide new insight into fungal lipopeptide biosynthesis.     

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.     

Isolation and characterization of a non-reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

Usnea longissima has long been used as a traditional medicine in China, India, Turkey, Canada and Europe. This lichen can produce several bioactive compounds that primarily belong to the polyketide family. The enzymes responsible for the production of these compounds are the polyketide synthases, but the biosynthetic processes in lichens are still unclear. In this study, a cultured mycobiont of Usnea longissima was used to isolate and characterize a polyketide synthase gene (UlPKS1). Complete sequence information regarding UlPKS1 (6,468 bp) was obtained by screening a Fosmid genomic library using a 512-bp fragment corresponding to part of the ketosynthase (KS) domain. Sequence analysis of UlPKS1 suggested that it contained features of a non-reducing fungal type I PKS with a starter unit of ACP transacylase (SAT), ketosynthase (KS), product template (PT), acyl carrier protein (ACP) transacylase, acyltransferase (AT) and thioesterase (TE) domain, and had five intervening introns. The domain organization of UlPKS1 (SAT-KS-AT-PT-ACP-ACP-TE) was quite similar to that of aromatic PKSs, and phylogenetic analysis showed that UlPKS1 belonged to the clade of lichenized fungal non-reducing PKS. RT-PCR analyses revealed that the expression of UlPKS1 was down-regulated by glycine and high concentrations of sorbitol, inositol and fructose and up-regulated by sucrose and glucose. Here, we introduce a non-reducing PKS gene in the lichen-forming fungus U. longissima, with a domain structure similar to the structure of orsellinic acid synthase A (OrsA) which is required for orsellinic acid biosynthesis in Aspergillus nidulans.     

Synthetic biology enabling access to designer polyketides

The full potential of polyketide discovery has yet to be reached owing to a lack of suitable technologies and knowledge required to advance engineering of polyketide biosynthesis. Recent investigations on the discovery, enhancement, and non-natural use of these biosynthetic gene clusters via computational biology, metabolic engineering, structural biology, and enzymology-guided approaches have facilitated improved access to designer polyketides. Here, we discuss recent successes in gene cluster discovery, host strain engineering, precursor-directed biosynthesis, combinatorial biosynthesis, polyketide tailoring, and high-throughput synthetic biology, as well as challenges and outlooks for rapidly generating useful target polyketides.     

Type III polyketide synthases in natural product biosynthesis

Polyketides represent an important class of biologically active and structurally diverse compounds in nature. They are synthesized from acyl-coenzyme A substrates by polyketide synthases (PKSs). PKSs are classified into three groups: types I, II, and III. This article introduces recent studies on type III PKSs identified from plants, bacteria, and fungi, and describes the catalytic functions of these enzymes in detail. Plant type III PKSs have been widely studied, as exemplified by chalcone synthase, which plays an important role in the synthesis of plant metabolites. Bacterial type III PKSs fall into five groups, many of which were identified from Streptomyces, a genus that has been well known for its production of bioactive molecules and genetic alterability. Although it was believed that type III PKSs exist exclusively in plants and bacteria, recent fungal genome sequencing projects and biochemical studies revealed the presence of type III PKSs in filamentous fungi, which provides a new chance to study fungal secondary metabolism and synthesize "unnatural" natural products. Type III PKSs have been used for the biosynthesis of novel molecules through precursor-directed and structure-based mutagenesis approaches.     

Evidence for a novel phosphopantetheinyl transferase domain in the polyketide synthase for enediyne biosynthesis

The polyketide synthase associated with the biosynthesis of enediyne-containing calicheamicin contains a putative phosphopantetheinyl transferase (PPTase) domain. By cloning and expressing the C-terminal region of the polyketide synthase and in vitro phosphopantetheinylation assay, we found that the PPTase domain exhibits preferred substrate specificity towards acyl and peptidyl carrier proteins in fatty acid and non-ribosomal peptide synthesis over its cognate partner. We also found evidence suggesting that the PPTase domain adopts a pseudo-trimeric structure, distinct from the pseudo-dimeric structure of type II PPTases. The results revealed a novel type of PPTase with unique structure and substrate specificity, and suggested that the polyketide synthase probably acquired the PPTase domain from a primary metabolic pathway in evolution.     

Genes for the Biosynthesis of the Fungal Polyketides Hypothemycin from Hypomyces subiculosus and Radicicol from Pochonia chlamydosporia

Gene clusters for biosynthesis of the fungal polyketides hypothemycin and radicicol from Hypomyces subiculosus and Pochonia chlamydosporia, respectively, were sequenced. Both clusters encode a reducing polyketide synthase (PKS) and a nonreducing PKS like those in the zearalenone cluster of Gibberella zeae, plus enzymes with putative post-PKS functions. Introduction of an O-methyltransferase (OMT) knockout construct into H. subiculosus resulted in a strain with increased production of 4-O-desmethylhypothemycin, but because transformation of H. subiculosus was very difficult, we opted to characterize hypothemycin biosynthesis using heterologous gene expression. In vitro, the OMT could methylate various substrates lacking a 4-O-methyl group, and the flavin-dependent monooxygenase (FMO) could epoxidate substrates with a 1′,2′ double bond. The glutathione S-transferase catalyzed cis-trans isomerization of the 7′,8′ double bond of hypothemycin. Expression of both hypothemycin PKS genes (but neither gene alone) in yeast resulted in production of trans-7′,8′-dehydrozearalenol (DHZ). Adding expression of OMT, expression of FMO, and expression of cytochrome P450 to the strain resulted in methylation, 1′,2′-epoxidation, and hydroxylation of DHZ, respectively. The radicicol gene cluster encodes halogenase and cytochrome P450 homologues that are presumed to catalyze chlorination and epoxidation, respectively. Schemes for biosynthesis of hypothemycin and radicicol are proposed. The PKSs encoded by the two clusters described above and those encoded by the zearalenone cluster all synthesize different products, yet they have significant sequence identity. These PKSs may provide a useful system for probing the mechanisms of fungal PKS programming.     

Biosynthesis of sphinganine-analog mycotoxins

Sphinganine-analog mycotoxins (SAMT) are polyketide-derived natural products produced by a number of plant pathogenic fungi and are among the most economically important mycotoxins. The toxins are structurally similar to sphinganine, a key intermediate in the biosynthesis of ceramides and sphingolipids, and competitive inhibitors for ceramide synthase. The inhibition of ceramide and sphingolipid biosynthesis is associated with several fatal diseases in domestic animals and esophageal cancer and neural tube defects in humans. SAMT contains a highly reduced, acyclic polyketide carbon backbone, which is assembled by a single module polyketide synthase. The biosynthesis of SAMT involves a unique polyketide chain-releasing mechanism, in which a pyridoxal 5'-phosphate-dependent enzyme catalyzes the termination, offloading and elongation of the polyketide chain. This leads to the introduction of a new carbon-carbon bond and an amino group to the polyketide chain. The mechanism is fundamentally different from the thioesterase/cyclase-catalyzed polyketide chain releasing found in bacterial and other fungal polyketide biosynthesis. Genetic data suggest that the ketosynthase domain of the polyketide synthase and the chain-releasing enzyme are important for controlling the final product structure. In addition, several post-polyketide modifications have to take place before SAMT become mature toxins.     

Polyketide-nonribosomal peptide epothilone antitumor agents: the EpoA,B, C subunits

The epothilones are a family of macrolactone natural products from the myxobacterial species Sorangium cellulosum. Similar to taxol, they are of current clinical interest as anticancer agents. Sequence analysis of the epothilone gene cluster allowed the identification of polyketide synthase and nonribosomal peptide synthetase modules involved in catalyzing epothilone biosynthesis. Given this information, it has been possible to test the predicted functions of several modules to date. EpoA ACP, EpoB, and EpoC have been overproduced in Escherichia coli, allowing in vitro reconstitution of the EpoA/B/C interface and production of the expected epothilone precursor. Further experiments probed the tolerance of EpoB and EpoC for unnatural substrates. These studies of the first three modules of the epothilone biosynthetic cluster suggest that combinatorial biosynthesis may lead to the production of a variety of epothilone analogs that incorporate diversity into the heterocycle starter unit. Additional efforts with the remaining modules, coupled with increased understanding of the macrocyclizing thioesterase domain, may lead to the production of epothilone variants with improved clinical properties.