Biosynthesis of a new skyllamycin in Streptomyces nodosus: a cytochrome P450 forms an epoxide in the cinnamoyl chain.

Activation of a silent gene cluster in Streptomyces nodosus leads to synthesis of a cinnamoyl-containing non-ribosomal peptide (CCNP) that is related to skyllamycins. This novel CCNP was isolated and its structure was interrogated using mass spectrometry and nuclear magnetic resonance spectroscopy. The isolated compound is an oxidised skyllamycin A in which an additional oxygen atom is incorporated in the cinnamoyl side-chain in the form of an epoxide. The gene for the epoxide-forming cytochrome P450 was identified by targeted disruption. The enzyme was overproduced in Escherichia coli and a 1.43 Å high-resolution crystal structure was determined. This is the first crystal structure for a P450 that forms an epoxide in a substituted cinnamoyl chain of a lipopeptide. These results confirm the proposed functions of P450s encoded by biosynthetic gene clusters for other epoxidized CCNPs and will assist investigation of how epoxide stereochemistry is determined in these natural products.

Characterisation of Modular Polyketide Synthases Designed to Make Pentaene Analogues of Amphotericin B.

Glycosylated polyene macrolides are important antifungal agents that are produced by many actinomycete species. Development of new polyenes may deliver improved antibiotics. Here, Streptomyces nodosus was genetically re-programmed to synthesise pentaene analogues of the heptaene amphotericin B. These pentaenes are of interest as surrogate substrates for enzymes catalysing unusual, late-stage biosynthetic modifications. The previous deletion of amphotericin polyketide synthase modules 5 and 6 generated S. nodosus M57, which produces an inactive pentaene. Here, the chain-terminating thioesterase was fused to module 16 to generate strain M57-16TE, in which cycles 5, 6, 17 and 18 are eliminated from the biosynthetic pathway. Another variant of M57 was obtained by replacing modules 15, 16 and 17 with a single 15-17 hybrid module. This gave strain M57-1517, in which cycles 5, 6, 15 and 16 are deleted. M57-16TE and M57-1517 gave reduced pentaene yields. Only M57-1517 delivered its predicted full-length pentaene macrolactone in low amounts. For both mutants, the major pentaenes were intermediates released from modules 10, 11 and 12. Longer pentaene chains were unstable. The novel pentaenes were not glycosylated and were not active against Candida albicans. However, random mutagenesis and screening may yet deliver new antifungal producers from the M57-16TE and M57-1517 strains.

Structural analysis of P450 AmphL from Streptomyces nodosus provides insights into substrate selectivity of polyene macrolide antibiotic biosynthetic P450s.

AmphL is a cytochrome P450 enzyme that catalyzes the C8 oxidation of 8-deoxyamphotericin B to the polyene macrolide antibiotic, amphotericin B. To understand this substrate selectivity, we solved the crystal structure of AmphL to a resolution of 2.0 Å in complex with amphotericin B and performed molecular dynamics (MD) simulations. A detailed comparison with the closely related P450, PimD, which catalyzes the epoxidation of 4,5-desepoxypimaricin to the macrolide antibiotic, pimaricin, reveals key catalytic structural features responsible for stereo- and regio-selective oxidation. Both P450s have a similar access channel that runs parallel to the active site I helix over the surface of the heme. Molecular dynamics simulations of substrate binding reveal PimD can "pull" substrates further into the P450 access channel owing to additional electrostatic interactions between the protein and the carboxyl group attached to the hemiketal ring of 4,5-desepoxypimaricin. This substrate interaction is absent in AmphL although the additional substrate -OH groups in 8-deoxyamphotericin B help to correctly position the substrate for C8 oxidation. Simulations of the oxy-complex indicates that these -OH groups may also participate in a proton relay network required for O2 activation as has been suggested for two other macrolide P450s, PimD and P450eryF. These findings provide experimentally testable models that can potentially contribute to a new generation of novel macrolide antibiotics with enhanced antifungal and/or antiprotozoal efficacy.

Engineered biosynthesis and characterisation of disaccharide-modified 8-deoxyamphoteronolides.

Several polyene macrolides are potent antifungal agents that have severe side effects. Increased glycosylation of these compounds can improve water solubility and reduce toxicity. Three extending glycosyltransferases are known to add hexoses to the mycosaminyl sugar residues of polyenes. The Actinoplanes caeruleus PegA enzyme catalyses attachment of a D-mannosyl residue in a ß-1,4 linkage to the mycosamine of the aromatic heptaene 67-121A to form 67-121C. NppY from Pseudonocardia autotrophica adds an N-acetyl-D-glucosamine to the mycosamine of 10-deoxynystatin. NypY from Pseudonocardia sp. P1 adds an extra hexose to a nystatin, but the identity of the sugar is unknown. Here, we express the nypY gene in Streptomyces nodosus amphL and show that NypY modifies 8-deoxyamphotericins more efficiently than C-8 hydroxylated forms. The modified heptaene was purified and shown to be mannosyl-8-deoxyamphotericin B. This had the same antifungal activity as amphotericin B but was slightly less haemolytic. Chemical modification of this new disaccharide polyene could give better antifungal antibiotics.

Polyene macrolide biosynthesis in streptomycetes and related bacteria: recent advances from genome sequencing and experimental studies.

The polyene macrolide group includes important antifungal drugs, to which resistance does not arise readily. Chemical and biological methods have been used in attempts to make polyene antibiotics with fewer toxic side effects. Genome sequencing of producer organisms is contributing to this endeavour, by providing access to new compounds and by enabling yield improvement for polyene analogues obtained by engineered biosynthesis. This recent work is also enhancing bioinformatic methods for deducing the structures of cryptic natural products from their biosynthetic enzymes. The stereostructure of candicidin D has recently been determined by NMR spectroscopy. Genes for the corresponding polyketide synthase have been uncovered in several different genomes. Analysis of this new information strengthens the view that protein sequence motifs can be used to predict double bond geometry in many polyketides.Chemical studies have shown that improved polyenes can be obtained by modifying the mycosamine sugar that is common to most of these compounds. Glycoengineered analogues might be produced by biosynthetic methods, but polyene glycosyltransferases show little tolerance for donors other than GDP-α-D-mycosamine. Genome sequencing has revealed extending glycosyltransferases that add a second sugar to the mycosamine of some polyenes. NppY of Pseudonocardia autotrophica uses UDP-N-acetyl-α-D-glucosamine as donor whereas PegA from Actinoplanes caeruleus uses GDP-α-D-mannose. These two enzymes show 51 % sequence identity and are also closely related to mycosaminyltransferases. These findings will assist attempts to construct glycosyltransferases that transfer alternative UDP- or (d)TDP-linked sugars to polyene macrolactones.

Exploiting the genome sequence of Streptomyces nodosus for enhanced antibiotic production.

The genome of the amphotericin producer Streptomyces nodosus was sequenced. A single scaffold of 7,714,110 bp was obtained. Biosynthetic genes were identified for several natural products including polyketides, peptides, siderophores and terpenes. The majority of these clusters specified known compounds. Most were silent or expressed at low levels and unlikely to compete with amphotericin production. Biosynthesis of a skyllamycin analogue was activated by introducing expression plasmids containing either a gene for a LuxR transcriptional regulator or genes for synthesis of the acyl moiety of the lipopeptide. In an attempt to boost amphotericin production, genes for acyl CoA carboxylases, a phosphopantetheinyl transferase and the AmphRIV transcriptional activator were overexpressed, and the effects on yields were investigated. This study provides the groundwork for metabolic engineering of S. nodosus strains to produce high yields of amphotericin analogues.

Role of polyol moiety of amphotericin B in ion channel formation and sterol selectivity in bilayer membrane.

Amphotericin B (AmB) is a polyene macrolide antibiotic widely used to treat mycotic infections. In this paper, we focus on the role of the polyol moiety of AmB in sterol selectivity using 7-oxo-AmB, 7α-OH-AmB, and 7ß-OH-AmB. The 7-OH analogs were prepared from 7-oxo-AmB. Their K(+) flux activity in liposomes showed that introduction of an additional ketone or hydroxy group on the polyol moiety reduces the original activity. Conformational analyses of these derivatives indicated that intramolecular hydrogen-bonding network possibly influenced the conformational rigidity of the macrolactone ring, and stabilized the active conformation in the membrane. Additionally, the flexible polyol leads to destabilization of the whole macrolactone ring conformation, resulting in a loss of sterol selectivity.

Engineered biosynthesis of disaccharide-modified polyene macrolides.

Recent work has uncovered genes for two glycosyltransferases that are thought to catalyze mannosylation of mycosaminyl sugars of polyene macrolides. These two genes are nypY from Pseudonocardia sp. strain P1 and pegA from Actinoplanes caeruleus. Here we analyze these genes by heterologous expression in various strains of Streptomyces nodosus, producer of amphotericins, and in Streptomyces albidoflavus, which produces candicidins. The NypY glycosyltransferase converted amphotericins A and B and 7-oxo-amphotericin B to disaccharide-modified forms in vivo. The enzyme did not act on amphotericin analogs lacking exocyclic carboxyl or mycosamine amino groups. Both NypY and PegA acted on candicidins. This work confirms the functions of these glycosyltransferases and provides insights into their acceptor substrate tolerance. Disaccharide-modified polyenes may have potential as less toxic antibiotics.

Versatility of enzymes catalyzing late steps in polyene 67-121C biosynthesis.

Actinoplanes caeruleus produces 67-121C, a heptaene macrolide modified with a D-mannosyl-D-mycosaminyl disaccharide. Draft genome sequencing revealed genes encoding mycosaminyltransferase, mycosamine synthase, a cytochrome P450 that modifies the macrolactone core, and the extending mannosyltransferase. Only the mycosamine synthase and P450 were active in the biosynthesis of amphotericins in Streptomyces nodosus, the amphotericin producer.

A Biomimetic Polymer for the Extraction and Purification of Superior Analogues of Amphotericin B.

Amphotericin B has been an essential drug in the fight against leishmaniasis and fungal pathogens for decades, and has more recently gained attention for the very limited microbial resistance displayed against it. However, its toxicity has restricted its use to only the most severe cases of disease, and attempts to reduce these ill effects via formulation have had only minor success. Genetic engineering has allowed the development of superior amphotericin analogues, notably 16-descarboxyl-16-methyl amphotericin B (MeAmB), which shows a ten-fold reduction in toxicity in addition to a slight improvement in therapeutic activity. However, MeAmB is difficult to extract from its bacterial source and purify. Presented here is an alternative method of MeAmB purification. A biomimetic polymer with a high affinity for MeAmB was designed via computational modelling and synthesised. Prepared as a separation column, the polymer was able to retain the target MeAmB whilst allowing the removal of cell debris from the bacterial extract. Starting with a simple bacterial extract, the relatively simple process allowed the purification of an MeAmB salt complex at approximately 70% MeAmB, and likely higher purification from further extraction. The mean MeAmB recovery between the pre-purification extract sample and the final product was 81%. This is the first successful demonstration of extraction or purification of any amphotericin molecule with any polymeric material. The biomimetic polymer was additionally reusable and simple to fabricate, giving this technique significant advantages over traditional methods of extraction and purification of valuable compounds.

Streptomyces nodosus host strains optimized for polyene glycosylation engineering.

The AmphDI glycosyltransferase transfers a mycosaminyl sugar residue from GDP onto 8-deoxyamphoteronolide B, the aglycone of the antifungal amphotericin B. In this study the amphDI gene was inactivated in Streptomyces nodosus strains lacking the AmphN cytochrome P450. The new mutants produced 8-deoxy-16-methyl-16-descarboxyl amphoteronolides in high yield. These strains and aglycones should prove valuable for in vivo and in vitro glycosylation engineering.

New Glycosylated Polyene Macrolides: Refining the Ore from Genome Mining.

Glycosylated polyene macrolides include effective antifungal agents, such as pimaricin, nystatin, candicidin, and amphotericin B. For the treatment of systemic mycoses, amphotericin B has been described as a gold-standard antibiotic because of its potent activity against a broad spectrum of fungal pathogens, which do not readily become resistant. However, amphotericin B has severe toxic side effects, and the development of safer alternatives remains an important objective. One approach towards obtaining such compounds is to discover new related natural products. Advances in next-generation sequencing have delivered a wealth of microbial genome sequences containing polyene biosynthetic gene clusters. These typically encode a modular polyketide synthase that catalyzes the assembly of the aglycone core, a cytochrome P450 that oxidizes a methyl branch to a carboxyl group, and additional enzymes for synthesis and attachment of a single mycosamine sugar residue. In some cases, further P450s catalyze epoxide formation or hydroxylation within the macrolactone. Bioinformatic analyses have identified over 250 of these clusters. Some are predicted to encode potentially valuable new polyenes that have not been uncovered by traditional screening methods. Recent experimental studies have characterized polyenes with new polyketide backbones, previously unknown late oxygenations, and additional sugar residues that increase water-solubility and reduce hemolytic activity. Here we review these studies and assess how this new knowledge can help to prioritize silent polyene clusters for further investigation. This approach should improve the chances of discovering better antifungal antibiotics.

A labile point in mutant amphotericin polyketide synthases.

Streptomyces nodosus produces the antifungal polyene amphotericin B. Numerous modifications of the amphotericin polyketide synthase have yielded new analogues. However, previous inactivation of the ketoreductase in module 10 resulted in biosynthesis of truncated polyketides. Here we show that modules downstream of this domain remain intact. Therefore, loss of ketoreductase-10 activity is sufficient to cause early chain termination. This modification creates a labile point in cycle 11 of the polyketide biosynthetic pathway. Non-extendable intermediates are released to accumulate as polyenyl-pyrones.

Isolation and characterisation of amphotericin B analogues and truncated polyketide intermediates produced by genetic engineering of Streptomyces nodosus.

Amphotericin B is a powerful but toxic drug used against fungal infections and leishmaniases. These diseases would be treated more effectively if non-toxic amphotericin derivatives could be produced on a large scale at low cost. Genetic manipulation of the amphotericin B producer, Streptomyces nodosus, has previously led to the detection and partial characterisation of 8-deoxyamphotericin B, 16-descarboxyl-16-methyl-amphotericin B, 15-deoxy-16-descarboxyl-16-methyl-15-oxo-amphotericin B, 7-oxo-amphotericin B and pentaene analogues. Here we report improved production and purification protocols that have allowed detailed chemical analyses of these compounds. The polyketide synthase product 8-deoxy-16-descarboxyl-16-methyl-amphoteronolide B was identified for the first time. In addition, the ketoreductase 10 domain of the polyketide synthase was specifically inactivated by targeted gene replacement. The resulting mutants produced truncated polyketide intermediates as linear polyenyl-pyrones.

Effects of new amphotericin analogues on the scrapie isoform of the prion protein.

Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative disorders associated with the conversion of a normal host prion protein (PrP(C)) into a pathogenic isoform (PrP(Sc)). Despite years of research, there is still no known cure for TSEs. Amphotericin B (AmB), an anti-fungal antibiotic, has antiprion activity but its usage is limited by its toxicity. This study assessed the antiprion properties of new amphotericin analogues in which the exocyclic carboxyl groups were replaced by methyl groups. These analogues reduced levels of the abnormal PrP(Sc) isoform of the mouse prion protein in cultured cells. 16-descarboxyl-16-methyl-amphotericin B (16B) had antiprion activity equivalent to that of amphotericin B and was significantly less toxic to cells as determined by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide dye reduction assay. A non-anti-fungal analogue, 16-descarboxyl-16-methyl-19-O-(6-deoxyhexosyl)-19-O-desmycosaminyl-amphotericin (16-19B) had higher antiprion activity and significantly lower toxicity than AmB. Some of the new amphotericin analogues may have potential as antiprion drugs.

Phosphomannose isomerase and phosphomannomutase gene disruptions in Streptomyces nodosus: impact on amphotericin biosynthesis and implications for glycosylation engineering.

Streptomycetes synthesise several bioactive natural products that are modified with sugar residues derived from GDP-mannose. These include the antifungal polyenes, the antibacterial antibiotics hygromycin A and mannopeptimycins, and the anticancer agent bleomycin. Three enzymes function in biosynthesis of GDP-mannose from the glycolytic intermediate fructose 6-phosphate: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMPP). Synthesis of GDP-mannose from exogenous mannose requires hexokinase or phosphotransferase enzymes together with PMM and GMPP. In this study, a region containing genes for PMI, PMM and GMPP was cloned from Streptomyces nodosus, producer of the polyenes amphotericins A and B. Inactivation of the manA gene for PMI resulted in production of amphotericins and their aglycones, 8-deoxyamphoteronolides. A double mutant lacking the PMI and PMM genes produced 8-deoxyamphoteronolides in good yields along with trace levels of glycosylated amphotericins. With further genetic engineering these mutants may activate alternative hexoses as GDP-sugars for transfer to aglycones in vivo.

Engineered synthesis of 7-oxo- and 15-deoxy-15-oxo-amphotericins: insights into structure-activity relationships in polyene antibiotics.

Site-directed mutagenesis and gene replacement were used to inactivate two ketoreductase (KR) domains within the amphotericin polyketide synthase in Streptomyces nodosus. The KR12 domain was inactivated in the DeltaamphNM strain, which produces 16-descarboxyl-16-methyl-amphotericins. The resulting mutant produced low levels of the expected 15-deoxy-15-oxo analogs that retained antifungal activity. These compounds can be useful for further chemical modification. Inactivation of the KR16 domain in the wild-type strain led to production of 7-oxo-amphotericin A and 7-oxo-amphotericin B in good yield. 7-oxo-amphotericin B was isolated, purified, and characterized as the N-acetyl methyl ester derivative. 7-oxo-amphotericin B had good antifungal activity and was less hemolytic than amphotericin B. These results indicate that modification at the C-7 position can improve the therapeutic index of amphotericin B.

The stereochemistry of ketoreduction.

In this issue of Chemistry & Biology, Leadlay and coworkers report overproduction of a number of ketoreductase domains from modular polyketide synthases. These discrete enzymes allow the stereochemistry of polyketide ketoreduction to be studied in isolation.

Corrigendum to "Dissecting complex polyketide biosynthesis" [Comput Struct Biotechnol J 3(4) (2012) 1-8].

[This corrects the article DOI: 10.5936/csbj.201210010.].

New start and finish for complex polyketide biosynthesis.

The polyketide vicenistatin has significant anticancer activity. In the January issue of Chemistry & Biology, Kakinuma and coworkers reported on the vicenistatin biosynthetic gene cluster and demonstrated in vitro glycosylation of its aglycone core, raising the possibility of producing analogs with altered sugar residues.