Hybrid Nonribosomal Peptide Synthetase Research Articles

Enzymatic α-Ketothioester Decarbonylation Occurs in the Assembly Line of Barbamide for Skeleton Editing.

The decarbonylation reaction has been developed significantly in organic chemistry as an effective approach to various synthetic applications, but enzymatic precedents for this reaction are rare. Based on investigations into the hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line of barbamide, we report an on-line α-ketothioester decarbonylation reaction that leads to one-carbon truncation of the elongating skeleton. This enzymatic editing reaction occurs in the first round of lipopeptide extension and modification involving the multienzymes BarE and BarF, which successively house an NRPS module to initiate the biosynthesis and a PKS module to catalyze the first round of chain extension. Starting with processing a leucine-derived α-ketoacyl starter, the ketosynthase domain in BarE displays an unusual dual activity that results in net one-carbon chain elongation. It extrudes carbon monoxide from α-keto-isocaproyl thioester and then mediates decarboxylative condenses of the resultant isovaleryl thioester with malonyl thioester to form a diketide intermediate, followed by BarF-based O-methylation to stabilize the enol form of the β-carbonyl and afford an unusual E-double bond. Biochemical characterization, chemical synthesis, computational analysis, and the experimental outcome of site-directed mutagenesis illustrate the extraordinary catalytic capability of this ketosynthase domain. This work furthers the appreciation of assembly line chemistry and opens the door to new approaches for skeleton editing/engineering of related molecules using synthetic biology approaches.

Transcriptomic analysis of polyketide synthesis in dinoflagellate, Prorocentrum lima

The benthic dinoflagellate Prorocentrum lima is among the most common toxic morphospecies with a cosmopolitan distribution. P. lima can produce polyketide compounds, such as okadaic acid (OA), dinophysistoxin (DTX) and their analogues, which are responsible for diarrhetic shellfish poisoning (DSP). Studying the molecular mechanism of DSP toxin biosynthesis is crucial for understanding the environmental driver influencing toxin biosynthesis as well as for better monitoring of marine ecosystems. Commonly, polyketides are produced by polyketide synthases (PKS). However, no gene has been confirmatively assigned to DSP toxin production. Here, we assembled a transcriptome from 94,730,858 Illumina RNAseq reads using Trinity, resulting in 147,527 unigenes with average sequence length of 1035 nt. Using bioinformatics analysis methods, we found 210 unigenes encoding single-domain PKS with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 15 transcripts encoding multi-domain PKS (forming typical type I PKSs modules) and 5 transcripts encoding hybrid nonribosomal peptide synthetase (NRPS)/PKS were found. Using comparative transcriptome and differential expression analysis, a total of 16 PKS genes were identified to be up-regulated in phosphorus-limited cultures, which was related to the up regulation of toxin expression. In concert with other recent transcriptome analyses, this study contributes to the building consensus that dinoflagellates may utilize a combination of Type I multi-domain and single-domain PKS proteins, in an as yet undefined manner, to synthesize polyketides. Our study provides valuable genomic resource for future research in order to understand the complex mechanism of toxin production in this dinoflagellate.

Genomic Comparative Analysis of Cordyceps pseudotenuipes with Other Species from Cordyceps.

The whole genome of Cordyceps pseudotenuipes was sequenced, annotated, and compared with three related species to characterize the genome. The antibiotics and Secondary Metabolites Analysis Shell (antiSMASH) and local BLAST analysis were used to explore the secondary metabolites (SMs) and biosynthesis gene clusters (BGCs) of the genus Cordyceps. The genome-wide basic characteristics of C. pseudotenuipes, C. tenuipes, C. cicadae, and C. militaris revealed unequal genome size, with C. cicadae as the largest (34.11 Mb), followed by C. militaris (32.27 Mb). However, the total gene lengths of C. pseudotenuipes and C. tenuipes were similar (30.1 Mb and 30.06 Mb). The GC contents of C. pseudotenuipes, C. tenuipes, C. cicadae, and C. militaris genomes differed slightly (51.40% to 54.11%). AntiSMASH and local BLAST analysis showed that C. pseudotenuipes, C. tenuipes, C. cicadae, and C. militaris had 31, 28, 31, and 29 putative SM BGCs, respectively. The SM BGCs contained different quantities of polyketide synthetase (PKS), nonribosomal peptide synthetase (NRPS), terpene, hybrid PKS + NRPS, and hybrid NRPS + Other. Moreover, C. pseudotenuipes, C. tenuipes, C. cicadae, and C. militaris had BGCs for the synthesis of dimethylcoprogen. C. pseudotenuipes, C. tenuipes, and C. cicadae had BGCs for the synthesis of leucinostatin A/B, neosartorin, dimethylcoprogen, wortmanamide A/B, and beauvericin. In addition, the SM BGCs unique to C. pseudotenuipes were clavaric acid, communesin, and deoxynivalenol. Synteny analysis indicated that the scaffolds where the SM BGC was located were divided into more than 70 collinear blocks, and there might be rearrangements. Altogether, these findings improved our understanding of the molecular biology of the genus Cordyceps and will facilitate the discovery of new biologically active SMs from the genus Cordyceps using heterologous expression and gene knockdown methods.

Genome Features and AntiSMASH Analysis of an Endophytic Strain Fusarium sp. R1.

Endophytic fungi are one of the most prolific sources of functional biomolecules with therapeutic potential. Besides playing an important role in serious plant diseases, Fusarium strains possess the powerful capability to produce a diverse array of bioactive secondary metabolites (SMs). In order to in-depth mine gene clusters for SM biosynthesis of the genus Fusarium, an endophytic strain Fusarium sp. R1 isolated from Rumex madaio Makino was extensively investigated by whole-genome sequencing and in-depth bioinformatic analysis, as well as antiSMASH annotation. The results displayed that strain R1 harbors a total of 51.8 Mb genome, which consists of 542 contigs with an N50 scaffold length of 3.21 Mb and 50.4% GC content. Meanwhile, 19,333 functional protein-coding genes, 338 tRNA and 111 rRNA were comprehensively predicted and highly annotated using various BLAST databases including non-redundant (Nr) protein sequence, nucleotide (Nt) sequence, Swiss-Prot, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups (COG), as well as Pathogen Host Interactions (PHI) and Carbohydrate-Active enzymes (CAZy) databases. Antibiotics and Secondary Metabolites Analysis Shell (AntiSMASH) results showed that strain R1 has 37 SM biosynthetic gene clusters (BGCs), including 17 nonribosomal peptide synthetases (NRPSs), 13 polyketide synthetases (PKSs), 3 terpene synthases (Ts), 3 hybrid NRPS + PKS and 1 hybrid indole + NRPS. These findings improve our knowledge of the molecular biology of the genus Fusarium and would promote the discovery of new bioactive SMs from strain R1 using gene mining strategies including gene knockout and heteroexpression.

Genomic insights into the biosynthesis and physiology of the cyanobacterial neurotoxin 2,4-diaminobutanoic acid (2,4-DAB)

Cyanobacteria are an ancient clade of photosynthetic prokaryotes, whose worldwide occurrence, especially in water, presents health hazards to humans and animals due to the production of a range of toxins (cyanotoxins). These include the sometimes co-occurring, non-encoded diaminoacid neurotoxins 2,4-diaminobutanoic acid (2,4-DAB) and its structural analogue β-N-methylaminoalanine (BMAA). Knowledge of the biosynthetic pathway for 2,4-DAB, and its role in cyanobacteria, is lacking. The aspartate 4-phosphate pathway is a known route of 2,4-DAB biosynthesis in other bacteria and in some plant species. Another pathway to 2,4-DAB has been described in Lathyrus species. Here, we use bioinformatics analyses to investigate hypotheses concerning 2,4-DAB biosynthesis in cyanobacteria. We assessed the presence or absence of each enzyme in candidate biosynthesis routes, the aspartate 4-phosphate pathway and a pathway to 2,4-DAB derived from S-adenosyl-L-methionine (SAM), in 130 cyanobacterial genomes using sequence alignment, profile hidden Markov models, substrate specificity/active site identification and the reconstruction of gene phylogenies. In the aspartate 4-phosphate pathway, for the 18 species encoding diaminobutanoate-2-oxo-glutarate transaminase, the co-localisation of genes encoding the transaminase with the downstream decarboxylase or ectoine synthase – often within hybrid non-ribosomal peptide synthetase (NRPS)-polyketide synthases (PKS) clusters, NRPS-independent siderophore (NIS) clusters and incomplete ectoine clusters – is compatible with the hypothesis that some cyanobacteria use the aspartate 4-phosphate pathway for 2,4-DAB production. Through this route, in cyanobacteria, 2,4-DAB may be functionally associated with environmental iron-scavenging, via the production of siderophores of the schizokinen/synechobactin type and of some polyamines. In the pathway to 2,4-DAB derived from SAM, eight cyanobacterial species encode homologs of SAM-dependent 3-amino-3-carboxypropyl transferases. Other enzymes in this pathway have not yet been purified or sequenced. Ultimately, the biosynthesis of 2,4-DAB appears to be either restricted to some cyanobacterial species, or there may be multiple and additional routes, and roles, for the synthesis of this neurotoxin.

Thiocysteine lyases as polyketide synthase domains installing hydropersulfide into natural products and a hydropersulfide methyltransferase

Nature forms S-S bonds by oxidizing two sulfhydryl groups, and no enzyme installing an intact hydropersulfide (-SSH) group into a natural product has been identified to date. The leinamycin (LNM) family of natural products features intact S-S bonds, and previously we reported an SH domain (LnmJ-SH) within the LNM hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line as a cysteine lyase that plays a role in sulfur incorporation. Here we report the characterization of an S-adenosyl methionine (SAM)-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin (GNM) biosynthesis, discovery of hydropersulfides as the nascent products of the GNM and LNM hybrid NRPS-PKS assembly lines, and revelation of three SH domains (GnmT-SH, LnmJ-SH, and WsmR-SH) within the GNM, LNM, and weishanmycin (WSM) hybrid NRPS-PKS assembly lines as thiocysteine lyases. Based on these findings, we propose a biosynthetic model for the LNM family of natural products, featuring thiocysteine lyases as PKS domains that directly install a -SSH group into the GNM, LNM, or WSM polyketide scaffold. Genome mining reveals that SH domains are widespread in Nature, extending beyond the LNM family of natural products. The SH domains could also be leveraged as biocatalysts to install an -SSH group into other biologically relevant scaffolds.

Genomic insights into the potent antifungal activity of B. ambifaria T16

Burkholderia ambifaria T16 is a bacterial strain isolated from the rhizosphere of barley plants, which showed a potent antifungal activity against several Fusarium species. In this work, the construction of a mini-Tn5 insertional library in this strain allowed us the identification of several genes involved in the antifungal activity. In particular, several mutants with mini-Tn5 insertions located in a biosynthetic gene cluster (BGC) involved in the production of cyclic glycolipopeptides known as occidiofungins (Ocfs) were completely unable to inhibit the in vitro growth of F. oxysporum. Tandem mass spectrometry analysis of lipopeptide extracts obtained from culture supernatants of the wild-type strain allowed the identification of eight Ocfs structural variants, that were not detected in extracts of the mutants with insertions in the ocf gene cluster. These results indicate that the antifungal activity of B. ambifaria T16 mainly relies on Ocfs production. Moreover, an insertion in a BGC predicted to be involved in the biosynthesis of a membrane-associated lipopeptide designated AFC-BC11 reduced the antifungal activity of B. ambifaria T16 by approximately 50%. Accordingly, membrane extracts from the insertional mutant in the afc-BC11 cluster showed a marked reduction in the antifungal activity against several fungi. In contrast, the antifungal activity of the membrane fraction of mutants unable to produce Ocfs was not affected. Furthermore, a detailed bioinformatic analysis of the proteins encoded by the ocf and afc-BC11 gene clusters revealed that the biosynthesis of these lipopeptides would be initiated by fatty acyl-AMP ligase (FAAL) domain containing enzymes and continued by a hybrid non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) or a type III PKS, respectively. These results provide new insights into lipopeptide biosynthesis, which may be valuable in the field of novel antifungal compound design.

Understanding the early stages of peptide formation during the biosynthesis of teicoplanin and related glycopeptide antibiotics.

The biosynthesis of the glycopeptide antibiotics (GPAs) demonstrates the exceptional ability of nonribosomal peptide (NRP) synthesis to generate diverse and complex structures from an expanded array of amino acid precursors. Whilst the heptapeptide cores of GPAs share a conserved C terminus, including the aromatic residues involved cross-linking and that are essential for the antibiotic activity of GPAs, most structural diversity is found within the N terminus of the peptide. Furthermore, the origin of the (D)-stereochemistry of residue 1 of all GPAs is currently unclear, despite its importance for antibiotic activity. Given these important features, we have now reconstituted modules (M) 1-4 of the NRP synthetase (NRPS) assembly lines that synthesise the clinically relevant type IV GPA teicoplanin and the related compound A40926. Our results show that important roles in amino acid modification during the NRPS-mediated biosynthesis of GPAs can be ascribed to the actions of condensation domains present within these modules, including the incorporation of (D)-amino acids at position 1 of the peptide. Our results also indicate that hybrid NRPS assembly lines can be generated in a facile manner by mixing NRPS proteins from different systems and that uncoupling of peptide formation due to different rates of activity seen for NRPS modules can be controlled by varying the ratio of NRPS modules. Taken together, this indicates that NRPS assembly lines function as dynamic peptide assembly lines and not static megaenzyme complexes, which has significant implications for biosynthetic redesign of these important biosynthetic systems.

Characterization of a Hybrid Nonribosomal Peptide-Carbohydrate Biosynthetic Pathway in Photorhabdus luminescens.

Advances in genome sequencing and analysis have afforded a trove of "orphan" bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)-carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydro-β-d- N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathway-targeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new "glycoamino acid" metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl unit derived from the NRPS machinery onto the free 1,6-anhydro-β-d- N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.

An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases in Myxococcus xanthus DK1622.

One mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 from Myxococcus xanthus DK1622 for characterization. The M. xanthus DK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination of in vivo and in vitro assays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping of M. xanthus DK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a "universal" MLP for generating functional hybrid NRPSs.IMPORTANCE MbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP from Myxococcus xanthus DK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a "universal" MLP during the construction of functional hybrid NRPSs.

P450-Catalyzed Tailoring Steps in Leinamycin Biosynthesis Featuring Regio- and Stereoselective Hydroxylations and Substrate Promiscuities.

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown. Based on gene inactivation and chemical investigation of three mutant strains, we investigated the tailoring steps catalyzed by two cytochromes P450 (P450s), LnmA and LnmZ, in LNM biosynthesis. Our studies revealed that (i) LnmA and LnmZ regio- and stereoselectively hydroxylate the C-8 and C-4' positions, respectively, on the scaffold of LNM; (ii) both LnmA and LnmZ exhibit substrate promiscuity, resulting in multiple LNM analogs from several shunt pathways; and (iii) the C-8 and C-4' hydroxyl groups play important roles in the cytotoxicity of LNM analogs against different cancer cell lines, shedding light on the structure-activity relationships of the LNM scaffold and the LNM-type natural products in general. These studies set the stage for future biosynthetic pathway engineering and combinatorial biosynthesis of the LNM family of natural products for structure diversity and drug discovery.

Gene Deletion Leads to Improved Valinomycin Production by Streptomyces sp. CBMAI 2042

The genus Streptomyces represents one of the largest producers of molecules with antibiotic activity. The whole genome sequencing of the endophytic microorganism Streptomyces sp. CBMAI 2042 revealed 35 gene clusters encoding for secondary metabolism including 3 non-ribosomal peptide synthetases (NRPS) and 7 NRPS-hybrids. Combining genome mining and cultivation profile analysis, the depsipeptide ionophore valinomycin was identified as one of the main metabolites produced by this strain. To better understand the metabolic machinery codified in CBMAI 2042 genome an adenylation domain from a hybrid NRPS cluster was deleted through a double crossing knockout experiment. Though the deletion was not plentiful to elucidate the encoded NRP metabolite related to the adenilation domain, an astounding increase of 10.5-fold in valinomycin production was observed in the mutant. These results suggest a metabolic flux redistribution of common substrates as an outcome of a gene target deletion.

Both modular and single-domain Type I polyketide synthases are expressed in the brevetoxin-producing dinoflagellate, Karenia brevis (Dinophyceae).

Dinoflagellates are prolific producers of polyketide compounds, many of which are potent toxins with adverse impacts on human and marine animal health. To identify polyketide synthase (PKS) genes in the brevetoxin‐producing dinoflagellate, Karenia brevis, we assembled a transcriptome from 595 million Illumina reads, sampled under different growth conditions. The assembly included 125,687 transcripts greater than 300 nt in length, with over half having >100× coverage. We found 121 transcripts encoding Type I ketosynthase (KS) domains, of which 99 encoded single KS domains, while 22 contained multiple KS domains arranged in 1–3 protein modules. Phylogenetic analysis placed all single domain and a majority of multidomain KSs within a monophyletic clade of protist PKSs. In contrast with the highly amplified single‐domain KSs, only eight single‐domain ketoreductase transcripts were found in the assembly, suggesting that they are more evolutionarily conserved. The multidomain PKSs were dominated by trans‐acyltransferase architectures, which were recently shown to be prevalent in other algal protists. Karenia brevis also expressed several hybrid nonribosomal peptide synthetase (NRPS)/PKS sequences, including a burA‐like sequence previously reported in a wide variety of dinoflagellates. This contrasts with a similarly deep transcriptome of Gambierdiscus polynesiensis, which lacked NRPS/PKS other than the burA‐like transcript, and may reflect the presence of amide‐containing polyketides in K. brevis and their absence from G. polynesiensis. In concert with other recent transcriptome analyses, this study provides evidence for both single domain and multidomain PKSs in the synthesis of polyketide compounds in dinoflagellates.

Characterization of the Functional Variance in MbtH-like Protein Interactions with a Nonribosomal Peptide Synthetase.

Many nonribosomal peptide synthetases (NRPSs) require MbtH-like proteins (MLPs) for solubility or for activation of amino acid substrate by the adenylation domain. MLPs are capable of functional crosstalk with noncognate NRPSs at varying levels. Using enterobactin biosynthesis in Escherichia coli as a model MLP-dependent NRPS system, we use in vivo and in vitro techniques to characterize how seven noncognate MLPs influence the function of the enterobactin NRPS EntF when the cognate MLP, YbdZ, is absent. Using a series of in vitro assays to analyze EntF solubility, adenylation, aminoacylation, and in vitro enterobactin production, we show that interactions between MLPs and NRPSs are multifaceted and more complex than previously appreciated. We separate MLP influence on solubility and function in a manner that shows altered solubility is not indicative of a functional MLP/NRPS pair. Although much of the functional variation among these noncognates can be explained by differences in EntF affinity for an MLP or the extent an MLP alters EntF l-Ser affinity, we demonstrate that MLPs can have a broader impact beyond solubility and adenylation. First, we show that a noncognate MLP can affect formation of l-Ser-S-EntF. Second, under in vitro conditions saturating for substrate and MLP, enterobactin production remains compromised in the absence of an appropriate MLP partner. These data suggest that we expand our investigations into how the MLPs influence NRPS enzymology. A more detailed understanding of these influences will be essential for downstream engineering of hybrid NRPS systems.

Characterization of the Ketosynthase and Acyl Carrier Protein Domains at the LnmI Nonribosomal Peptide Synthetase-Polyketide Synthase Interface for Leinamycin Biosynthesis.

Leinamycin (LNM) is biosynthesized by a hybrid nonribosomal peptide synthetase (NRPS)–acyltransferase (AT)-less type I polyketide synthase (PKS). Characterization of LnmI revealed ketosynthase (KS)–acyl carrier protein (ACP)–KS domains at the NRPS–PKS interface. Inactivation of the KS domain or ACP domain in vivo abolished LNM production, and the ACP domain can be phosphopantetheinylated in vitro. The LnmI KS–ACP–KS architecture represents a new mechanism for functional crosstalk between NRPS and AT-less type I PKS in the biosynthesis of hybrid peptide–polyketide natural products.

Antifungal activity improved by coproduction of cyclodextrins and anabaenolysins in Cyanobacteria

Cyclodextrins are cyclic oligosaccharides widely used in the pharmaceutical industry to improve drug delivery and to increase the solubility of hydrophobic compounds. Anabaenolysins are lipopeptides produced by cyanobacteria with potent lytic activity in cholesterol-containing membranes. Here, we identified the 23- to 24-kb gene clusters responsible for the production of the lipopeptide anabaenolysin. The hybrid nonribosomal peptide synthetase and polyketide synthase biosynthetic gene cluster is encoded in the genomes of three anabaenolysin-producing strains of Anabaena. We detected previously unidentified strains producing known anabaenolysins A and B and discovered the production of new variants of anabaenolysins C and D. Bioassays demonstrated that anabaenolysins have weak antifungal activity against Candida albicans. Surprisingly, addition of the hydrophilic fraction of the whole-cell extracts increased the antifungal activity of the hydrophobic anabaenolysins. The fraction contained compounds identified by NMR as α-, β-, and γ-cyclodextrins, which undergo acetylation. Cyclodextrins have been used for decades to improve the solubility and bioavailability of many drugs including antifungal compounds. This study shows a natural example of cyclodextrins improving the solubility and efficacy of an antifungal compound in an ancient lineage of photosynthetic bacteria.

C-S bond cleavage by a polyketide synthase domain

Leinamycin (LNM) is a sulfur-containing antitumor antibiotic featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2-dithiolane moiety is essential for LNM's antitumor activity, by virtue of its ability to generate an episulfonium ion intermediate capable of alkylating DNA. We have previously cloned and sequenced the lnm gene cluster from Streptomyces atroolivaceus S-140. In vivo and in vitro characterizations of the LNM biosynthetic machinery have since established that: (i) the 18-membered macrolactam backbone is synthesized by LnmP, LnmQ, LnmJ, LnmI, and LnmG, (ii) the alkyl branch at C-3 of LNM is installed by LnmK, LnmL, LnmM, and LnmF, and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the nascent product of the LNM hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Sulfur incorporation at C-3 of LNM E1, however, has not been addressed. Here we report that: (i) the bioinformatics analysis reveals a pyridoxal phosphate (PLP)-dependent domain, we termed cysteine lyase (SH) domain (LnmJ-SH), within PKS module-8 of LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by using l-cysteine and l-cysteine S-modified analogs as substrates through a PLP-dependent β-elimination reaction, establishing l-cysteine as the origin of sulfur at C-3 of LNM; and (iii) the LnmJ-SH domain, sharing no sequence homology with any other enzymes catalyzing C-S bond cleavage, represents a new family of PKS domains that expands the chemistry and enzymology of PKSs and might be exploited to incorporate sulfur into polyketide natural products by PKS engineering.

Biosynthesis of antimycins with a reconstituted 3-formamidosalicylate pharmacophore in Escherichia coli.

Antimycins are a family of natural products generated from a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line. Although they possess an array of useful biological activities, their structural complexity makes chemical synthesis challenging, and their biosynthesis has thus far been dependent on slow-growing source organisms. Here, we reconstituted the biosynthesis of antimycins in Escherichia coli, a versatile host that is robust and easy to manipulate genetically. Along with Streptomyces genetic studies, the heterologous expression of different combinations of ant genes enabled us to systematically confirm the functions of the modification enzymes, AntHIJKL and AntO, in the biosynthesis of the 3-formamidosalicylate pharmacophore of antimycins. Our E. coli-based antimycin production system can not only be used to engineer the increased production of these bioactive compounds, but it also paves the way for the facile generation of novel and diverse antimycin analogues through combinatorial biosynthesis.

Chemical variation from the neoantimycin depsipeptide assembly line

Here we report the biosynthetic pathway for the neoantimycin and present three novel neoantimycin analogues, neoantimycin D (1), E (2) and F (3), from this assembly system from Streptoverticillium orinoci. Identification of these novel neoantimycin variants was achieved by selective MS/MS interrogation of natural product extracts using diagnostic fragments of the known neoantimycins. Their structures, including the absolute configurations, were elucidated using a combination of NMR experiments, detailed MS/MS experiments and the advanced Marfey’s method. The biosynthetic pathway of neoantimycin was dissected by genome sequencing data analysis for the first time, which includes a hybrid nonribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) assembly lines.

Structures and biosynthesis of 12-membered macrocyclic depsipeptides from Streptomyces sp. ML55

Two novel depsipeptides (1–2) were isolated from Streptomyces sp. ML55 together with two known analogues (3–4). Their structures were elucidated using a combination of NMR experiments, as well as detailed MS/MS experiments. The biosynthetic pathway of isolated compounds was dissected by genome sequencing data analysis for a hybrid nonribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) assembly line.