Occurrence of two 5-aminolevulinate biosynthetic pathways in Streptomyces nodosus subsp. asukaensis is linked with the production of asukamycin

We report the results of cloning genes for two key biosynthetic enzymes of different 5-aminolevulinic acid (ALA) biosynthetic routes from Streptomyces. The genes encode the glutamyl-tRNAGlu reductase (GluTR) of the C5 pathway and the ALA synthase (ALAS) of the Shemin pathway. While Streptomyces coelicolor A3(2) synthesizes ALA via the C5 route, both pathways are operational in Streptomyces nodosus subsp. asukaensis, a producer of asukamycin. In this strain, the C5 route produces ALA for tetrapyrrole biosynthesis; the ALA formed by the Shemin pathway serves as a precursor of the 2-amino-3-hydroxycyclopent-2-enone moiety (C5N unit), an antibiotic component. The growth of S. nodosus and S. coelicolor strains deficient in the GluTR genes (gtr) is strictly dependent on ALA or heme supplementation, whereas the defect in the ALAS-encoding gene (hemA-asuA) abolishes the asukamycin production in S. nodosus. The recombinant hemA-asuA gene was expressed in Escherichia coli and in Streptomyces, and the encoded enzyme activity was demonstrated both in vivo and in vitro. The hemA-asuA gene is situated within a putative cluster of asukamycin biosynthetic genes. This is the first report about the cloning of genes for two different ALA biosynthetic routes from a single bacterium.     

A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum

A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.     

High titer production of tetracenomycins by heterologous expression of the pathway in a Streptomyces cinnamonensis industrial monensin producer strain

Streptomyces cinnamonensis C730.1 and C730.7, are industrially mutagenized strains that produce moderate and high levels of the polyketide polyether antibiotic monensin A, respectively, in an oil-based fermentation medium. The possibility that these strains could be used for high titer production of a heterologous polyketide product was investigated by expression of the entire tetracenomycin (TCM) biosynthetic pathway using an integrative plasmid, pSET154. Expression in C730.1 led to stable production of ~0.44 g/l TCM C (the final biosynthetic product) and ~2.69 g/l TCM A2 (the penultimate biosynthetic product), and resulted in a 40% decrease in monensin production. Expression in the C730.7 led to higher levels of TCMs, ~0.6 g/l TCM C and ~4.35 g/l TCM A2, without any detectable decrease in the higher titer monensin production. Abrogation of monensin production in this strain through deletion of the corresponding biosynthetic genes did not lead to higher levels of TCM products. In the case of the C730.7 host, 85% of the TCM C and virtually all of the TCM A2 were intracellular, suggesting feedback inhibition leads to the accumulation of the final pathway intermediate. These observations contrast those made for the native producer Streptomyces glaucescens where the predominant product is TCM C and TCM titers are significantly lower levels (~0.3 g/l), and demonstrate the potential utility of S. cinnamonensis strains as heterologous hosts for high level expression of a variety of polyketide synthase derived products.     

Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters.Bacterial natural products are excellent lead compounds for drug discovery and have played major roles in the development of pharmaceutical agents in nearly all therapeutic areas (1, 7, 9). Unfortunately, the rate of discovery of new bacterial natural products has decreased, due in part to frequent rediscovery of known compounds (7). An enormous and currently inaccessible reservoir of new natural products is located in the biosynthetic pathways found in the genomes of uncultivated bacteria (18). Heterologous expression of these biosynthetic gene clusters represents a powerful tool for discovering new natural products (20, 21). Herein, we demonstrate that the deltaproteobacterium Myxococcus xanthus is an effective host for heterologous expression of aromatic polyketide biosynthetic pathways. This work expands the scope of polyketide biosynthetic pathways which can be heterologously expressed in M. xanthus and suggests that M. xanthus may be a suitable general host for heterologous expression.Molecular phylogenetic studies have shown that bacterial diversity is enormous, and the vast majority of the diversity is found in uncultivated bacterial species (18). Estimates suggest that 99% of bacteria from the environment are uncultivatable using standard techniques (2, 15, 16). Culture-independent analyses of metagenomic DNA libraries from soil and marine environments indicate that there is a wealth of natural product diversity in these uncultivated strains. For example, analysis of a soil metagenome for a highly conserved region of polyketide synthase genes showed that none of the sequences found were present in the known public databases (5). Polyketide synthases are key enzymes responsible for the production of the polyketide family of natural products in proteobacteria, actinobacteria, and “low-G+C Gram-positive bacteria” (4, 12, 19). Polyketide natural products have been developed into antibiotic, anticancer, and immunosuppressant clinical agents (1, 6, 8). Based on these observations, metagenomic DNA libraries are expected to possess a large number of new polyketide biosynthetic pathways, representing substantial new chemical diversity for drug discovery.Heterologous expression of biosynthetic pathways can play a major role in interrogating metagenomic DNA libraries for new polyketide biosynthetic pathways. Heterologous production of polyketides in hosts such as Streptomyces coelicolor and Streptomyces lividans is an important tool in the identification and characterization of these pathways (6, 8, 17). Results from these studies have shown that Streptomyces strains are good hosts for heterologous production of many polyketides, particularly those from actinomycetes. However, Streptomyces strains have proved to be poor hosts for expression of deltaproteobacterial polyketide biosynthetic pathways, such as those in myxobacteria (10, 17). As polyketide biosynthetic pathways in metagenomic DNA libraries contain both actinomycete- and deltaproteobacterium-derived pathways, a heterologous expression host competent to express pathways of both origins is needed.We examined the ability of the deltaproteobacterium M. xanthus to act as a general heterologous expression host. M. xanthus is a predatory bacterium that undergoes multicellular development in response to nutrient starvation. During development, M. xanthus is known to be an effective host for the heterologous expression of the deltaproteobacterium-derived epothilone D biosynthetic pathway and has been used for the production of epothilone D for clinical trials (17). M. xanthus has also been shown to be an excellent host for the heterologous expression of several other myxobacterial metabolites, including myxothiazol and myxochromide S (3, 11, 22). We demonstrate that M. xanthus can also heterologously express the Streptomyces rimosus oxytetracycline biosynthetic pathway, producing oxytetracycline. This is the first example of a polyketide from a nonmyxobacterial species heterologously expressed in a myxobacterium.To generate an M. xanthus strain capable of heterologously expressing oxytetracycline, the Streptomyces rimosus oxytetracycline biosynthetic pathway (Fig. ​(Fig.1)1) was inserted via homologous recombination into the asgE locus of M. xanthus. The asgE locus of M. xanthus was amplified and inserted into the BglII site of pET28b (Novagen) to produce pMRH02. The oligonucleotides used for the amplification of the asgE locus were 5′-GACGAGATCTGTTGGAAGGTCGGCAACTGG-3′ and 5′-CTTAAGATCTTCCGTGAAGTACTGGCGCAC-3′. The asgE locus provides a chromosomal region for single-crossover homologous recombination into the M. xanthus chromosome. The 32-kb oxytetracycline pathway in S. rimosus was excised from pYT264 (24) and cloned into the EcoRI site of pMRH02 to produce pMRH08. M. xanthus DK1622 was electroporated under standard conditions (13) with pMRH08 to provide an M. xanthus ΔasgE Kan^r mutant. Positive selection for the chromosomal insertion was maintained throughout all experiments by use of kanamycin supplementation (40 μg/ml). This large genomic insertion significantly increased the doubling time for the strain (doubling time, ≈10 h).Open in a separate windowFIG. 1.Oxytetracycline biosynthetic pathway. (A) Enzymatic pathway responsible for formation of oxytetracycline. (B) Oxytetracycline biosynthesis gene cluster from S. rimosus.Oxytetracycline was heterologously produced in M. xanthus under standard rich medium culture conditions and detected in culture broth by liquid chromatography-mass spectrometry (LC-MS). A liquid culture of the mutant strain containing the oxytetracycline gene cluster was cultured for 10 days at 33°C in CTTYE (1.0% Casitone, 0.5% yeast extract, 10.0 mM Tris-HCl, 1.0 mM KH₂PO₄, and 8.0 mM MgSO₄; 100 ml). Acetone (10%, vol/vol) was added to the culture and vigorously mixed. The resulting mixture was extracted with 3 volumes of ethyl acetate to remove the organic soluble materials, including oxytetracycline. The organic extracts were concentrated in vacuo and resuspended in methanol (100 μl). LC-MS analyses were carried out using an Altima Hypersil C₁₈ column (3-μm particle size; 150 mm by 2.1 mm) with a linear gradient of water-acetonitrile (5 to 95%) with 0.05% formic acid over 90 min (0.20 ml/min), followed by positive-ion electrospray ionization (5,500 V) and analysis with a Shimadzu 2010A single quadrupole mass spectrometer. LC-MS analysis indicated that oxytetracycline was present in the fermentation broth (Fig. ​(Fig.2).2). The titer of oxytetracycline was determined to be approximately 10 mg per liter of fermentation broth. Quantification was performed in triplicate by LC-MS analysis using a standard curve generated from commercial oxytetracycline. Negative controls of M. xanthus DK1622 cultures processed under identical conditions did not contain detectable levels of oxytetracycline.Open in a separate windowFIG. 2.LC-MS ion extraction analysis of the molecular ion [M+H]⁺ of standard and culture extracts. (A) Oxytetracycline standard. (B) M. xanthus ΔasgE Kan^r mutant containing the oxytetracycline biosynthetic pathway. (C) Wild-type M. xanthus DK1622.These data indicate that M. xanthus can heterologously express the oxytetracycline polyketide synthase biosynthetic pathway in S. rimosus. Several factors affect the successful heterologous production of polyketide synthase pathways, including codon usage, mRNA stability, functionality of regulatory elements, and the presence of all necessary starter and extender units (14). As codon usages between M. xanthus and the genus Streptomyces are very similar and myxobacteria are known to produce polyketide products requiring a wide diversity of starter and extender units, neither codon usage nor starter and extender unit availability was considered likely to affect the ability of M. xanthus to heterologously express streptomycete biosynthetic pathways. As Streptomyces strains do not appear to be effective at heterologous expression of myxobacterial biosynthetic pathways, we were concerned that Myxococcus and Streptomyces strains may possess substantially different regulatory elements. Our data indicate that the regulatory elements present in streptomycete-derived biosynthetic pathways are sufficient to enable expression of the biosynthetic genes in M. xanthus. Further work exploring the regulatory elements present in myxobacterial polyketide biosynthetic gene clusters is needed to evaluate this hypothesis.This study demonstrates that M. xanthus can heterologously express streptomycete-derived polyketide biosynthetic pathways in addition to myxobacterial polyketide biosynthetic pathways. The observed titer of 10 mg/liter of culture broth is comparable to titers reported for the heterologous expression of myxobacterial polyketide biosynthetic pathways in myxobacteria (11) and streptomycete-derived polyketide biosynthetic pathways in Streptomyces (14, 23) and is sufficient for characterization of the polyketide product. Pseudomonas putida, which has a more favorable growth profile, has been shown to be a good host for heterologous expression of myxobacterial polyketide biosynthetic pathways, with product titers in the range of 0.6 to 40 mg/liter of culture broth (14, 21, 23). The observed breadth of polyketide pathways accessible and the titers of the polyketide products produced make M. xanthus an attractive potential candidate for a “universal” host for facilitating heterologous expression of polyketide biosynthetic pathways derived from environmental samples of metagenomic DNA.     

RedEx: a method for seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters

Biosynthesis reprograming is an important way to diversify chemical structures. The large repetitive DNA sequences existing in polyketide synthase genes make seamless DNA manipulation of the polyketide biosynthetic gene clusters extremely challenging. In this study, to replace the ethyl group attached to the C-21 of the macrolide insecticide spinosad with a butenyl group by refactoring the 79-kb gene cluster, we developed a RedEx method by combining Redαβ mediated linear-circular homologous recombination, ccdB counterselection and exonuclease mediated in vitro annealing to insert an exogenous extension module in the polyketide synthase gene without any extra sequence. RedEx was also applied for seamless deletion of the rhamnose 3′-O-methyltransferase gene in the spinosad gene cluster to produce rhamnosyl-3′-desmethyl derivatives. The advantages of RedEx in seamless mutagenesis will facilitate rational design of complex DNA sequences for diverse purposes.     

Operon for Biosynthesis of Lipstatin,the Beta-Lactone Inhibitor of Human Pancreatic Lipase

Lipstatin, isolated from Streptomyces toxytricini as a potent and selective inhibitor of human pancreatic lipase, is a precursor for tetrahydrolipstatin (also known as orlistat, Xenical, and Alli), the only FDA-approved antiobesity medication for long-term use. Lipstatin features a 2-hexyl-3,5-dihydroxy-7,10-hexadecadienoic-β-lactone structure with an N-formyl-l-leucine group attached as an ester to the 5-hydroxy group. It has been suggested that the α-branched 3,5-dihydroxy fatty acid β-lactone moiety of lipstatin in S. toxytricini is derived from Claisen condensation between two fatty acid substrates, which are derived from incomplete oxidative degradation of linoleic acid based on feeding experiments. In this study, we identified a six-gene operon (lst) that was essential for the biosynthesis of lipstatin by large-deletion, complementation, and single-gene knockout experiments. lstA, lstB, and lstC, which encode two β-ketoacyl–acyl carrier protein synthase III homologues and an acyl coenzyme A (acyl-CoA) synthetase homologue, were indicated to be responsible for the generation of the α-branched 3,5-dihydroxy fatty acid backbone. Subsequently, the nonribosomal peptide synthetase (NRPS) gene lstE and the putative formyltransferase gene lstF were involved in decoration of the α-branched 3,5-dihydroxy fatty acid chain with an N-formylated leucine residue. Finally, the 3β-hydroxysteroid dehydrogenase-homologous gene lstD might be responsible for the reduction of the β-keto group of the biosynthetic intermediate, thereby facilitating the formation of the unique β-lactone ring.     

Construction of new vectors for high-level expression in actinomycetes

A new integrative vector (pCJR24) was constructed for use in the erythromycin producer Saccharopolyspora erythraea and in other actinomycetes. It includes the pathway-specific activator gene actII–ORF4 from the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. The actI promoter and the associated ribosome binding site are located upstream of an NdeI site (5′-CATATG-3′) which encompasses the actI start codon allowing protein(s) to be produced at high levels in response to nutritional signals if these signals are faithfully mediated by the ActII–ORF4 activator. Several polyketide synthase genes were cloned in pCJR24 and overexpressed in S. erythraea after integration of the vector into the chromosome by homologous recombination, indicating the possibility that the S. coelicolor promoter/activator functions appropriately in S. erythraea. pCJR24-mediated recombination was also used to place the entire gene set for the erythromycin-producing polyketide synthase under the control of the actI promoter. The resulting strain produced copious quantities of erythromycins and precursor macrolides when compared with wild-type S. erythraea. The use of this system provides the means for rational strain improvement of antibiotic-producing actinomycetes.     

Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis

Modular polyketide synthases (PKSs) of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS) domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT), ketoreductase (KR), and dehydratase (DH)–KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.     

Aspergillus oryzae CsyB Catalyzes the Condensation of Two β-Ketoacyl-CoAs to Form 3-Acetyl-4-hydroxy-6-alkyl-α-pyrone

The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C₉–C₁₇ in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs.     

A gene cluster involved in nogalamycin biosynthesis fromStreptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway

We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS.     

A Single Module Type I Polyketide Synthase Directs de Novo Macrolactone Biogenesis during Galbonolide Biosynthesis in Streptomyces galbus

Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-¹³C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed β-keto group modifications.     

Cloning and Structure-Function Analyses of Quinolone- and Acridone-producing Novel Type III Polyketide Synthases from Citrus microcarpa

Two novel type III polyketide synthases, quinolone synthase (QNS) and acridone synthase (ACS), were cloned from Citrus microcarpa (Rutaceae). The deduced amino acid sequence of C. microcarpa QNS is unique, and it shared only 56–60% identities with C. microcarpa ACS, Medicago sativa chalcone synthase (CHS), and the previously reported Aegle marmelos QNS. In contrast to the quinolone- and acridone-producing A. marmelos QNS, C. microcarpa QNS produces 4-hydroxy-N-methylquinolone as the “single product” by the one-step condensation of N-methylanthraniloyl-CoA and malonyl-CoA. However, C. microcarpa ACS shows broad substrate specificities and produces not only acridone and quinolone but also chalcone, benzophenone, and phloroglucinol from 4-coumaroyl-CoA, benzoyl-CoA, and hexanoyl-CoA, respectively. Furthermore, the x-ray crystal structures of C. microcarpa QNS and ACS, solved at 2.47- and 2.35-Å resolutions, respectively, revealed wide active site entrances in both enzymes. The wide active site entrances thus provide sufficient space to facilitate the binding of the bulky N-methylanthraniloyl-CoA within the catalytic centers. However, the active site cavity volume of C. microcarpa ACS (760 ų) is almost as large as that of M. sativa CHS (750 ų), and ACS produces acridone by employing an active site cavity and catalytic machinery similar to those of CHS. In contrast, the cavity of C. microcarpa QNS (290 ų) is significantly smaller, which makes this enzyme produce the diketide quinolone. These results as well as mutagenesis analyses provided the first structural bases for the anthranilate-derived production of the quinolone and acridone alkaloid by type III polyketide synthases.     

The Streptomyces peucetius dpsY and dnrX Genes Govern Early and Late Steps of Daunorubicin and Doxorubicin Biosynthesis

The Streptomyces peucetius dpsY and dnrX genes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions of dpsY and dnrX. Replacement of dpsY with a mutant form disrupted by insertion of the aphII neomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation of dnrX in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest that dnrX, analogous to the role of the S. peucetius dnrH gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria.     

Insights into the Biosynthesis of the Benzoquinone Ansamycins Geldanamycin and Herbimycin, Obtained by Gene Sequencing and Disruption

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.     

A Single Point Mutation in the Gene Encoding Gb3/CD77 Synthase Causes a Rare Inherited Polyagglutination Syndrome

Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR_int, and NOR2, in which Gal(α1–4), GalNAc(β1–3)Gal(α1–4), and Gal(α1–4)GalNAc(β1–3)Gal(α1–4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1–4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1–4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1–4)Gal and Gal(α1–4)GalNAc moieties.     

Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum

Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.