RESUMO
BACKGROUND: Alginate is an industrially important polysaccharide, currently produced commercially by harvesting of marine brown sea-weeds. The polymer is also synthesized as an exo-polysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter, and these organisms may represent an alternative alginate source in the future. The current work describes an attempt to rationally develop a biological system tuned for very high levels of alginate production, based on a fundamental understanding of the system through metabolic modeling supported by transcriptomics studies and carefully controlled fermentations. RESULTS: Alginate biosynthesis in Pseudomonas fluorescens was studied in a genomics perspective, using an alginate over-producing strain carrying a mutation in the anti-sigma factor gene mucA. Cells were cultivated in chemostats under nitrogen limitation on fructose or glycerol as carbon sources, and cell mass, growth rate, sugar uptake, alginate and CO(2) production were monitored. In addition a genome scale metabolic model was constructed and samples were collected for transcriptome analyses. The analyses show that polymer production operates in a close to optimal way with respect to stoichiometric utilization of the carbon source and that the cells increase the uptake of carbon source to compensate for the additional needs following from alginate synthesis. The transcriptome studies show that in the presence of the mucA mutation, the alg operon is upregulated together with genes involved in energy generation, genes on both sides of the succinate node of the TCA cycle and genes encoding ribosomal and other translation-related proteins. Strains expressing a functional MucA protein (no alginate production) synthesize cellular biomass in an inefficient way, apparently due to a cycle that involves oxidation of NADPH without ATP production. The results of this study indicate that the most efficient way of using a mucA mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation. CONCLUSIONS: The insights gained in this study should be very useful for a future efficient production of microbial alginates.
Assuntos
Proteínas de Bactérias/metabolismo , Reatores Biológicos , Biotecnologia/métodos , Modelos Biológicos , Pseudomonas fluorescens/metabolismo , Alginatos , Proteínas de Bactérias/genética , Células Cultivadas , Fermentação , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Ácido Glucurônico/biossíntese , Ácidos Hexurônicos , Análise em Microsséries , Mutação/genética , Análise de Componente PrincipalRESUMO
Polyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT(0) in the nystatin PKS loading module NysA with the propionate-specific AT(1) from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT(0) domain by site-directed mutagenesis were accomplished. However, none of the NysA mutants constructed was able to initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids were functional, providing for different levels of nystatin biosynthesis. An interplay between certain residues in AT(0) and an active site residue in the ketosynthase (KS)-like domain of NysA in initiation of nystatin biosynthesis was revealed. Some hybrids between the NysA and RimA loading modules carrying the NysA AT(0) domain were able to prime rimocidin PKS with both acetate and butyrate units upon complementation of a rimA-deficient mutant of the rimocidin/CE-108 producer Streptomyces diastaticus. Expression of the PimS0 loading module from the pimaricin producer in the same host, however, resulted in production of CE-108 only. Taken together, these data indicate relaxed substrate specificity of NysA AT(0) domain, which is counteracted by a strict specificity of the first extender module KS domain in the nystatin PKS of Streptomyces noursei.
Assuntos
Anti-Infecciosos/metabolismo , Macrolídeos/metabolismo , Polienos/metabolismo , Policetídeo Sintases/metabolismo , Streptomyces/enzimologia , Streptomyces/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Cromatografia Líquida de Alta Pressão , Teste de Complementação Genética , Humanos , Redes e Vias Metabólicas/genética , Modelos Moleculares , Estrutura Molecular , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Policetídeo Sintases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Streptomyces/química , Streptomyces/genéticaRESUMO
The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the beta-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.
Assuntos
Hexosaminas/biossíntese , Família Multigênica , Nistatina/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Animais , Western Blotting , Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Vetores Genéticos/genética , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Hemólise/efeitos dos fármacos , Hexosaminas/química , Hexosaminas/farmacologia , Cavalos , Espectrometria de Massas , Estrutura Molecular , Nistatina/química , Nistatina/farmacologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Transaminases/genética , Transaminases/metabolismoRESUMO
Although polyene macrolides are efficient antifungal agents with fungicidal mode of action, their use in medical practice is problematic due to their low solubility and significant human toxicity. In an attempt to address the solubility problem, we have obtained two analogues of nystatin with hydroxy groups at positions C31 and C33 through manipulation of the nystatin polyketide synthase in the producing organism Streptomyces noursei. Structures of the analogues were confirmed by nuclear magnetic resonance (NMR), and their solubility was found to be more than 2000 times higher than that of nystatin. However, both analogues were shown to have lost antifungal activity, implying that the integrity of the hydrophobic polyene region of the nystatin molecule is crucial for the fungicidal action. NMR data and computer modeling performed for the new analogues suggested conformational changes together with a significantly increased structural disorder, which may account for both increased solubility and the loss of activity.
Assuntos
Macrolídeos/química , Nistatina/análogos & derivados , Nistatina/química , Polienos/química , Antifúngicos/química , Antifúngicos/farmacologia , Engenharia Genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nistatina/biossíntese , Solubilidade , Streptomyces/enzimologia , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismo , Relação Estrutura-AtividadeRESUMO
Enoyl reductase (ER) domains in module 5 of nystatin and amphotericin polyketide synthase (PKS) are responsible for reduction of the C28-C29 unsaturated bond on the nascent polyketide chain during biosynthesis of both macrolides, resulting in production of tetraenes nystatin A(1) and amphotericin A, respectively. Data obtained in fermentations under glucose limitation conditions demonstrated that the efficiency of the ER5 domain can be influenced by carbon source availability in the amphotericin producer Streptomyces nodosus, but not in the nystatin producer Streptomyces noursei. Two S. noursei ER5 domain mutants were constructed, GG5073SP and S5016N, both producing the heptaene nystatin analogue S44HP with unsaturated C28-C29 bond. While the GG5073SP mutant, with altered ER5 NADPH binding site, produced S44HP exclusively, the S5016N mutant synthesized a mixture of nystatin and S44HP. Comparative studies on the S5016N S. noursei mutant and S. nodosus, both producing mixtures of tetraenes and heptaenes, revealed that the ratio between these two types of metabolites was significantly more affected by glucose limitation in S. nodosus. These data suggest that mutation S5016N in NysC "locks" the ER5 domain in a state of intermediate activity which, in contrast to the ER5 domain in the amphotericin PKS, is not significantly influenced by physiological conditions.
Assuntos
Antibacterianos/biossíntese , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Glucose/metabolismo , Macrolídeos/metabolismo , Mutação Puntual , Polienos/metabolismo , Streptomyces/enzimologia , Anfotericina B/análogos & derivados , Anfotericina B/biossíntese , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Mutagênese Sítio-Dirigida , Nistatina/biossíntese , Policetídeo Sintases/química , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Streptomyces/genética , Streptomyces/crescimento & desenvolvimentoRESUMO
The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Antibacterianos/biossíntese , Genes Bacterianos/fisiologia , Nistatina/biossíntese , Streptomyces/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/antagonistas & inibidores , Transporte Biológico , Família Multigênica , Streptomyces/genética , Vanadatos/farmacologiaRESUMO
The gram-positive bacterium Streptomyces noursei ATCC 11455 produces a complex mixture of polyene macrolides generally termed nystatins. Although the structures for nystatins A(1) and A(3) have been reported, the identities of other components of the nystatin complex remain obscure. Analyses of the culture extract from the S. noursei wild type revealed the presence of several nystatin-related compounds for which chemical structures could be suggested on the basis of their molecular weights, their UV spectra, and knowledge of the nystatin biosynthetic pathway. Nuclear magnetic resonance (NMR) studies with one of these polyene macrolides identified it as a nystatin analogue containing a mycarose moiety at C-35. A similar investigation was performed with the culture extract of the ERD44 mutant, which has a genetically altered polyketide synthase (PKS) NysC and which was previously shown to produce a heptaene nystatin analogue. The latter compound, tentatively named S44HP, and its derivative, which contains two deoxysugar moieties, were purified; and their structures were confirmed by NMR analysis. Nystatin analogues with an expanded macrolactone ring were also observed in the extract of the ERD44 mutant, suggesting that the altered PKS can "stutter" during the polyketide chain assembly. These data provide new insights into the biosynthesis of polyene macrolide antibiotics and the functionalities of PKSs and post-PKS modification enzymes.
Assuntos
Antibacterianos/biossíntese , Macrolídeos/metabolismo , Polienos/metabolismo , Policetídeo Sintases/biossíntese , Streptomyces/enzimologia , Antibacterianos/farmacologia , Candida albicans/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Macrolídeos/farmacologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Conformação Molecular , Peso Molecular , Nistatina/química , Nistatina/farmacologia , Polienos/farmacologia , Policetídeo Sintases/genética , Recombinação Genética , Streptomyces/genéticaRESUMO
Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.
Assuntos
Antibacterianos/biossíntese , Dioxigenases , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Nistatina/biossíntese , Streptomyces/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catecol 2,3-Dioxigenase , Deleção de Genes , Genes Bacterianos , Genes Reporter , Teste de Complementação Genética , Dados de Sequência Molecular , Família Multigênica , Oxigenases/genética , Oxigenases/metabolismo , Regiões Promotoras Genéticas , Alinhamento de Sequência , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismoRESUMO
The loading module for the nystatin polyketide synthase (PKS) in Streptomyces noursei is represented by the NysA protein composed of a ketosynthase (KS(S)), acyltransferase, dehydratase, and an acyl carrier protein. The absolute requirement of this protein for initiation of nystatin biosynthesis was demonstrated by the in-frame deletion of the nysA gene in S. noursei. The role of the NysA KS(S) domain, however, remained unclear, since no data on the significance of the "active site" serine (Ser-170) residue in the loading modules of type I PKSs were available. Site-specific mutagenesis of Ser-170 both in the wild-type NysA and in the hybrid loading module containing malonyl-specific acyltransferase domain from the extender module had no effect on nystatin biosynthesis. A second mutation (S413N) of the NysA KS(S) domain was discovered that completely abolished the ability of the hybrids to restore nystatin biosynthesis, presumably by affecting the ability of the resulting proteins to catalyze the required substrate decarboxylation. In contrast, NysA and its Ser-170 mutants bearing the same S413N mutation were able to restore nystatin production to significant levels, probably by using acetyl-CoA as a starter unit. Together, these data suggest that the KS(S) domain of NysA differs from the KS(Q) domains found in the loading modules of several PKS type I systems in that the active site residue is not significant for its activity.