Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 96
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Antibiot (Tokyo) ; 72(10): 769-774, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31341273

RESUMO

A novel sansanmycin analogue, sansanmycin Q (1), was identified by genome mining from the fermentation broth of Streptomyces sp. SS (CPCC 200442). In comparison with other sansanmycin compounds, sansanmycin Q has an extra glycine residue at the N-terminus of the pseudopeptide backbone. The additional glycine was proved to be assembled to sansanmycin A by SsaB, a tRNA-dependent aminoacyltransferase, based on the results of rescrutiny of sansanmycin biosynthetic gene cluster, and then overexpression and knockout of ssaB in the wild-type strain. The structure of sansanmycin Q was assigned by interpretation of NMR and mass spectral data. The results of the bioassay disclosed that sansanmycin Q exhibited more potency against Mycobacterium tuberculosis H37Rv and a rifampicin- and isoniazid-resistant strain than sansanmycin A.


Assuntos
Antituberculosos/metabolismo , Antituberculosos/farmacologia , Vias Biossintéticas/genética , Família Multigênica , Oligopeptídeos/biossíntese , Oligopeptídeos/farmacologia , Streptomyces/metabolismo , Uridina/análogos & derivados , Antituberculosos/química , Biologia Computacional , Fermentação , Genoma Bacteriano , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Oligopeptídeos/química , Streptomyces/crescimento & desenvolvimento , Uridina/biossíntese , Uridina/química , Uridina/farmacologia
2.
Metab Eng ; 49: 248-256, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30189293

RESUMO

Uridine is a kind of pyrimidine nucleoside that has been widely applied in the pharmaceutical industry. Although microbial fermentation is a promising method for industrial production of uridine, an efficient microbial cell factory is still lacking. In this study, we constructed a metabolically engineered Escherichia coli capable of high-yield uridine production. First, we developed a CRISPR/Cas9-mediated chromosomal integration strategy to integrate large DNA into the E. coli chromosome, and a 9.7 kb DNA fragment including eight genes in the pyrimidine operon of Bacillus subtilis F126 was integrated into the yghX locus of E. coli W3110. The resultant strain produced 3.3 g/L uridine and 4.5 g/L uracil in shake flask culture for 32 h. Subsequently, five genes involved in uridine catabolism were knocked out, and the uridine titer increased to 7.8 g/L. As carbamyl phosphate, aspartate, and 5'-phosphoribosyl pyrophosphate are important precursors for uridine synthesis, we further modified several metabolism-related genes and synergistically improved the supply of these precursors, leading to a 76.9% increase in uridine production. Finally, nupC and nupG encoding nucleoside transport proteins were deleted, and the extracellular uridine accumulation increased to 14.5 g/L. After 64 h of fed-batch fermentation, the final engineered strain UR6 produced 70.3 g/L uridine with a yield and productivity of 0.259 g/g glucose and 1.1 g/L/h, respectively. To the best of our knowledge, this is the highest uridine titer and productivity ever reported for the fermentative production of uridine.


Assuntos
Escherichia coli , Engenharia Metabólica , Microrganismos Geneticamente Modificados , Uridina/biossíntese , Bacillus subtilis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Loci Gênicos , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Óperon , Uridina/genética
3.
Appl Microbiol Biotechnol ; 102(20): 8753-8762, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30120523

RESUMO

In this study, a uridine and acetoin co-production pathway was designed and engineered in Bacillus subtilis for the first time. A positive correlation between acetoin and uridine production was observed and investigated. By disrupting acetoin reductase/2,3-butanediol dehydrogenasegenebdhA, the acetoin and uridine yield was increased while 2,3-butanediol formation was markedly reduced. Subsequent overexpression of the alsSD operon further improved acetoin yield and abolished acetate formation. After optimization of fermentation medium, key supplementation strategies of yeast extract and soybean meal hydrolysate were identified and applied to improve the co-production of uridine and acetoin. With a consumption of 290.33 g/L glycerol, the recombinant strain can accumulate 40.62 g/L uridine and 60.48 g/L acetoin during 48 h of fed-batch fermentation. The results indicate that simultaneous production of uridine and acetoin is an efficient strategy for balancing the carbon metabolism in engineered Bacillus subtilis. More importantly, co-production of value-added products is a possible way to improve the economics of uridine fermentation.


Assuntos
Acetoína/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Uridina/biossíntese , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fermentação , Engenharia Metabólica , Óperon
4.
Nucleic Acids Res ; 46(17): 9160-9169, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-29982645

RESUMO

Derivatives of 5-hydroxyuridine (ho5U), such as 5-methoxyuridine (mo5U) and 5-oxyacetyluridine (cmo5U), are ubiquitous modifications of the wobble position of bacterial tRNA that are believed to enhance translational fidelity by the ribosome. In gram-negative bacteria, the last step in the biosynthesis of cmo5U from ho5U involves the unique metabolite carboxy S-adenosylmethionine (Cx-SAM) and the carboxymethyl transferase CmoB. However, the equivalent position in the tRNA of Gram-positive bacteria is instead mo5U, where the methyl group is derived from SAM and installed by an unknown methyltransferase. By utilizing a cmoB-deficient strain of Escherichia coli as a host and assaying for the formation of mo5U in total RNA isolates with methyltransferases of unknown function from Bacillus subtilis, we found that this modification is installed by the enzyme TrmR (formerly known as YrrM). Furthermore, X-ray crystal structures of TrmR with and without the anticodon stemloop of tRNAAla have been determined, which provide insight into both sequence and structure specificity in the interactions of TrmR with tRNA.


Assuntos
Bacillus subtilis/enzimologia , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , RNA de Transferência/metabolismo , Uridina/análogos & derivados , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Metiltransferases/química , Metiltransferases/genética , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA de Transferência/química , S-Adenosilmetionina/metabolismo , Uridina/biossíntese , Uridina/metabolismo
5.
Nucleic Acids Res ; 46(4): 1565-1583, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29390138

RESUMO

Modified uridine containing taurine, 5-taurinomethyluridine (τm5U), is found at the anticodon first position of mitochondrial (mt-)transfer RNAs (tRNAs). Previously, we reported that τm5U is absent in mt-tRNAs with pathogenic mutations associated with mitochondrial diseases. However, biogenesis and physiological role of τm5U remained elusive. Here, we elucidated τm5U biogenesis by confirming that 5,10-methylene-tetrahydrofolate and taurine are metabolic substrates for τm5U formation catalyzed by MTO1 and GTPBP3. GTPBP3-knockout cells exhibited respiratory defects and reduced mitochondrial translation. Very little τm5U34 was detected in patient's cells with the GTPBP3 mutation, demonstrating that lack of τm5U results in pathological consequences. Taurine starvation resulted in downregulation of τm5U frequency in cultured cells and animal tissues (cat liver and flatfish). Strikingly, 5-carboxymethylaminomethyluridine (cmnm5U), in which the taurine moiety of τm5U is replaced with glycine, was detected in mt-tRNAs from taurine-depleted cells. These results indicate that tRNA modifications are dynamically regulated via sensing of intracellular metabolites under physiological condition.


Assuntos
RNA de Transferência/metabolismo , Taurina/deficiência , Uridina/análogos & derivados , Animais , Proteínas de Transporte/fisiologia , Gatos , Pré-Escolar , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/fisiologia , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , RNA de Transferência/química , Uridina/biossíntese
6.
Biotechnol Lett ; 40(1): 151-155, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29038923

RESUMO

OBJECTIVES: To construct a Bacillus subtilis strain for improved uridine production. RESULTS: The AAG2846-2848 fragment of the pyrAB gene, encoding carbamoylphosphate synthetase, was deleted in B. subtilis TD246 leading to a 245% increase of uridine production and the conversion from glucose to uridine increased by 10.5%. Overexpression of the pyr operon increased the production of uridine by a further 31% and the conversion rate of glucose to uridine was increased by 18%. In addition, the blocking of arginine synthesis or disabling of glutamate dehydrogenase significantly enhanced the uridine production. The highest-producing strain, B. subtilis TD297, accumulated 11 g uridine/l with a yield of 240 mg uridine/g glucose in shake-flask cultivation. CONCLUSION: This is the first report of engineered B. subtilis strains which can produce more than 11 g uridine/l, with a yield reaching 240 mg uridine/g glucose in shake-flask cultivation.


Assuntos
Bacillus subtilis/metabolismo , Engenharia Metabólica/métodos , Uridina/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Deleção de Genes , Expressão Gênica , Glucose/metabolismo , Redes e Vias Metabólicas/genética
7.
PLoS One ; 12(5): e0176545, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28472077

RESUMO

In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5'-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain.


Assuntos
Bacillus subtilis/metabolismo , Ensaios de Triagem em Larga Escala , Mutagênese , Uridina/biossíntese , Bacillus subtilis/genética , Sequência de Bases , Fermentação , Homologia de Sequência do Ácido Nucleico , Temperatura
8.
Science ; 355(6330)2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28302796

RESUMO

Uridine, a pyrimidine nucleoside present at high levels in the plasma of rodents and humans, is critical for RNA synthesis, glycogen deposition, and many other essential cellular processes. It also contributes to systemic metabolism, but the underlying mechanisms remain unclear. We found that plasma uridine levels are regulated by fasting and refeeding in mice, rats, and humans. Fasting increases plasma uridine levels, and this increase relies largely on adipocytes. In contrast, refeeding reduces plasma uridine levels through biliary clearance. Elevation of plasma uridine is required for the drop in body temperature that occurs during fasting. Further, feeding-induced clearance of plasma uridine improves glucose metabolism. We also present findings that implicate leptin signaling in uridine homeostasis and consequent metabolic control and thermoregulation. Our results indicate that plasma uridine governs energy homeostasis and thermoregulation in a mechanism involving adipocyte-dependent uridine biosynthesis and leptin signaling.


Assuntos
Adipócitos/metabolismo , Regulação da Temperatura Corporal , Metabolismo Energético , Jejum/metabolismo , Eliminação Hepatobiliar , Uridina/biossíntese , Uridina/sangue , Animais , Glicemia/metabolismo , Humanos , Leptina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
9.
Biomed Res Int ; 2017: 2520347, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29333435

RESUMO

To enhance nucleoside production in Hirsutella sinensis, the biosynthetic pathways of purine and pyrimidine nucleosides were constructed and verified. The differential expression analysis showed that purine nucleoside phosphorylase, inosine monophosphate dehydrogenase, and guanosine monophosphate synthase genes involved in purine nucleotide biosynthesis were significantly upregulated 16.56-fold, 8-fold, and 5.43-fold, respectively. Moreover, dihydroorotate dehydrogenase, uridine nucleosidase, uridine/cytidine monophosphate kinase, and inosine triphosphate pyrophosphatase genes participating in pyrimidine nucleoside biosynthesis were upregulated 4.53-fold, 10.63-fold, 4.26-fold, and 5.98-fold, respectively. To enhance the nucleoside production, precursors for synthesis of nucleosides were added based on the analysis of biosynthetic pathways. Uridine and cytidine contents, respectively, reached 5.04 mg/g and 3.54 mg/g when adding 2 mg/mL of ribose, resulting in an increase of 28.6% and 296% compared with the control, respectively. Meanwhile, uridine and cytidine contents, respectively, reached 10.83 mg/g 2.12 mg/g when adding 0.3 mg/mL of uracil, leading to an increase of 176.3% and 137.1%, respectively. This report indicated that fermentation regulation was an effective way to enhance the nucleoside production in H. sinensis based on biosynthetic pathway analysis.


Assuntos
Vias Biossintéticas/genética , Núcleosídeo-Fosfato Quinase/genética , Nucleosídeos/biossíntese , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Hypocreales , Núcleosídeo-Fosfato Quinase/metabolismo , Purinas/biossíntese , Pirimidinas/biossíntese , Uridina/biossíntese
10.
Wei Sheng Wu Xue Bao ; 56(1): 56-67, 2016 Jan 04.
Artigo em Chinês | MEDLINE | ID: mdl-27305780

RESUMO

OBJECTIVE: We studied several crucial factors influencing the uridine biosynthesis in Bacillus subtilis, including mutations of phosphoribosylpyrophosphate synthetase (PRPP synthetase) (prs) and carbamyl phosphate synthetase (pyrAA/pyrAB), and overexpression of heterologous 5'-nucleotidase (sdt1). METHODS: According to the inferred allosteric sites, we introduced point mutation into coding sequences of prs and pyrAB. The mutated prs gene was integratedly expressed in the xylR locus of the chromosome and the pyrAB gene was modified in-situ. The sdt1 gene was overexpressed in the saB locus of the chromosome. The effect of the genetic modification on uridine biosynthesis was characterized by the analysis of uridine, cytidine and uracil in the fermentation broth. RESULTS: The mutations of Asn120Ser, Leu135Ile, Glu52Gly or Val312Ala on PRPP synthase resulted in an increase of uridine production by 67% and 96%, respectively. The mutations of Ser948Phe, Thr977Ala and Lys993Ile on carbamyl phosphate synthase resulted in a 182% increase of uridine yield to 6.97 g/L. The overexpression of heterologous 5'-nucleotidase resulted in a 17% increase of uridine yield to 8.16 g/L. CONCLUSION: The activity and regulation mechanism of PRPP synthase and carbamyl phosphate synthase was an important factor to limit the excessive synthesis of uridine. Asn120Ser and Leu135Ile mutations of PRPP synthase and Ser948Phe, Thr977Ala and Lys993Ile mutations of carbamyl phosphate synthase will facilitate the biosynthesis of uridine. The additional Glu52Gly and Val312Ala mutations of PRPP synthase were beneficial for uridine biosynthesis. The reaction from UMP to uridine also limited the biosynthesis of uridine in B. subtilis.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Uridina/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Clonagem Molecular , Ribose-Fosfato Pirofosfoquinase/genética , Ribose-Fosfato Pirofosfoquinase/metabolismo
12.
Microb Cell Fact ; 15: 77, 2016 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-27154005

RESUMO

BACKGROUND: Sansanmycins are uridyl peptide antibiotics (UPAs), which are inhibitors of translocase I (MraY) and block the bacterial cell wall biosynthesis. They have good antibacterial activity against Pseudomonas aeruginosa and Mycobacterium tuberculosis strains. The biosynthetic gene cluster of sansanmycins has been characterized and the main biosynthetic pathway elucidated according to that of pacidamycins which were catalyzed by nonribosomal peptide synthetases (NRPSs). Sananmycin A is the major compound of Streptomyces sp. SS (wild type strain) and it bears a non-proteinogenic amino acid, meta-tyrosine (m-Tyr), at the N-terminus of tetrapeptide chain. RESULTS: ssaX deletion mutant SS/XKO was constructed by the λ-RED mediated PCR targeting method and confirmed by PCR and southern blot. The disruption of ssaX completely abolished the production of sansanmycin A. Complementation in vivo and in vitro could both recover the production of sansanmycin A, and the overexpression of SsaX apparently increased the production of sansanmycin A by 20%. Six new compounds were identified in the fermentation culture of ssaX deletion mutant. Some more novel sansanmycin analogues were obtained by mutasynthesis, and totally ten sansanmycin analogues, MX-1 to MX-10, were purified and identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). The bioassay of these sansanmycin analogues showed that sansanmycin MX-1, MX-2, MX-4, MX-6 and MX-7 exhibited comparable potency to sansanmycin A against M. tuberculosis H37Rv, as well as multi-drug-resistant (MDR) and extensive-drug-resistant (XDR) strains. Moreover, sansanmycin MX-2 and MX-4 displayed much better stability than sansanmycin A. CONCLUSIONS: We demonstrated that SsaX is responsible for the biosynthesis of m-Tyr in vivo by gene deletion and complementation. About twenty novel sansanmycin analogues were obtained by mutasynthesis in ssaX deletion mutant SS/XKO and ten of them were purified and structurally identified. Among them, MX-2 and MX-4 showed promising anti-MDR and anti-XDR tuberculosis activity and greater stability than sansanmycin A. These results indicated that ssaX deletion mutant SS/XKO was a suitable host to expand the diversity of the N-terminus of UPAs, with potential to yield more novel compounds with improved activity and/or other properties.


Assuntos
Antibacterianos/química , Proteínas de Bactérias/genética , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Peptídeo Sintases/genética , Streptomyces/metabolismo , Uridina/análogos & derivados , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Conformação Molecular , Família Multigênica , Mycobacterium tuberculosis/efeitos dos fármacos , Oligopeptídeos/biossíntese , Oligopeptídeos/farmacologia , Peptídeo Sintases/metabolismo , Plasmídeos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray , Streptomyces/genética , Uridina/biossíntese , Uridina/química , Uridina/genética , Uridina/farmacologia
14.
Appl Environ Microbiol ; 81(18): 6314-23, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26150462

RESUMO

The state-of-the-art procedure for gene insertions into Trichoderma reesei is a cotransformation of two plasmids, one bearing the gene of interest and the other a marker gene. This procedure yields up to 80% transformation efficiency, but both the number of integrated copies and the loci of insertion are unpredictable. This can lead to tremendous pleiotropic effects. This study describes the development of a novel transformation system for site-directed gene insertion based on auxotrophic markers. For this purpose, we tested the applicability of the genes asl1 (encoding an enzyme of the l-arginine biosynthesis pathway), the hah1 (encoding an enzyme of the l-lysine biosynthesis pathway), and the pyr4 (encoding an enzyme of the uridine biosynthesis pathway). The developed transformation system yields strains with an additional gene at a defined locus that are prototrophic and ostensibly isogenic compared to their parental strain. A positive transformation rate of 100% was achieved due to the developed split-marker system. Additionally, a double-auxotrophic strain that allows multiple genomic manipulations was constructed, which facilitates metabolic engineering purposes in T. reesei. By employing goxA of Aspergillus niger as a reporter system, the influence on the expression of an inserted gene caused by the orientation of the insertion and the transformation strategy used could be demonstrated. Both are important aspects to be considered during strain engineering.


Assuntos
Genoma Fúngico , Mutagênese Insercional/métodos , Transformação Genética , Trichoderma/genética , Arginina/biossíntese , Aspergillus niger/genética , Southern Blotting , Genes Fúngicos , Genes Reporter , Engenharia Genética/métodos , Lisina/biossíntese , Plasmídeos , Trichoderma/metabolismo , Trichoderma/ultraestrutura , Uridina/biossíntese
15.
J Biochem ; 158(6): 513-21, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26112661

RESUMO

Dihydrouridine (D) is formed by tRNA dihydrouridine synthases (Dus). In mesophiles, multiple Dus enzymes bring about D modifications at several positions in tRNA. The extreme-thermophilic eubacterium Thermus thermophilus, in contrast, has only one dus gene in its genome and only two D modifications (D20 and D20a) in tRNA have been identified. Until now, an in vitro assay system for eubacterial Dus has not been reported. In this study, therefore, we constructed an in vitro assay system using purified Dus. Recombinant T. thermophilus Dus lacking bound tRNA was successfully purified. The in vitro assay revealed that no other factors in living cells were required for D formation. A dus gene disruptant (Δdus) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The Δdus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNA(Phe) transcript at 60°C, demonstrating that other modifications in tRNA are not essential for Dus activity. However, a comparison of the formation of D in native tRNA(Phe) purified from the Δdus strain and tRNA(Phe) transcript revealed that other tRNA modifications are required for D formation at high temperatures.


Assuntos
Proteínas de Bactérias/química , Oxirredutases/química , RNA de Transferência/química , Thermus thermophilus/enzimologia , Uridina/análogos & derivados , Proteínas de Bactérias/isolamento & purificação , Ensaios Enzimáticos , Oxirredução , Oxirredutases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura , Uridina/biossíntese
16.
Microb Cell Fact ; 14: 54, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25890046

RESUMO

BACKGROUND: Cytidine and uridine are produced commercially by Bacillus subtilis. The production strains of cytidine and uridine were both derivatives from mutagenesis. However, the exact metabolic and genetic factors affecting the productivity remain unknown. Genetic engineering may be a promising approach to identify and confirm these factors. RESULTS: With the deletion of the cdd and hom genes, and the deregulation of the pyr operon in Bacillus subtilis168, the engineered strain produced 200.9 mg/L cytidine, 14.9 mg/L uridine and 960.1 mg/L uracil. Then, the overexpressed prs gene led to a dramatic increase of uridine by 25.9 times along with a modest increase of cytidine. Furthermore, the overexpressed pyrG gene improved the production of cytidine, uridine and uracil by 259.5%, 11.2% and 68.8%, respectively. Moreover, the overexpression of the pyrH gene increasesd the yield of cytidine by 40%, along with a modest augments of uridine and uracil. Lastly, the deletion of the nupC-pdp gene resulted in a doubled production of uridine up to 1684.6 mg/L, a 14.4% increase of cytidine to 1423 mg/L, and a 99% decrease of uracil to only 14.2 mg/L. CONCLUSIONS: The deregulation of the pyr operon and the overexpression of the prs, pyrG and pyrH genes all contribute to the accumulation of pyrimidine nucleoside compounds in the medium. Among these factors, the overexpression of the pyrG and pyrH genes can particularly facilitate the production of cytidine. Meanwhile, the deletion of the nupC-pdp gene can obviously reduce the production of uracil and simultaneously improve the production of uridine.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Citidina/biossíntese , Uridina/biossíntese , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Fermentação , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Homosserina Desidrogenase/genética , Homosserina Desidrogenase/metabolismo , Engenharia Metabólica/métodos , Mutagênese , Óperon/genética , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
J Biol Chem ; 290(22): 13710-24, 2015 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-25855790

RESUMO

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures.


Assuntos
Aminoglicosídeos/biossíntese , Aminoglicosídeos/genética , Antibacterianos/biossíntese , Farmacorresistência Bacteriana , Família Multigênica , Uridina/análogos & derivados , Uridina/química , Aminoglicosídeos/química , Antibacterianos/química , Sequência de Bases , Desenho de Fármacos , Escherichia coli/metabolismo , Heme/química , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosforilação , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Recombinantes/química , Streptomyces/metabolismo , Treonina/química , Transaldolase/metabolismo , Uridina/biossíntese , Uridina Monofosfato/química
18.
Appl Microbiol Biotechnol ; 98(15): 6621-32, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24769901

RESUMO

Adenosine triphosphate (ATP), the most important energy source for metabolic reactions and pathways, plays a vital role in control of metabolic flux. Considering the importance of ATP in regulation of the glycolytic pathway, the use of ATP-oriented manipulation is a rational and efficient route to regulate metabolic flux. In this paper, a series of efficient ATP-oriented regulation methods, such as changing ambient temperature and altering reduced nicotinamide adenine dinucleotide (NADH), was developed. To satisfy the different demand for ATP at different phases in directed biosynthesis of uridine-phosphoryl compounds, a multiphase ATP supply regulation strategy was also used to enhance to yield of target metabolites.


Assuntos
Trifosfato de Adenosina/metabolismo , Saccharomyces cerevisiae/metabolismo , Uridina/biossíntese , Glucose/metabolismo , Glicólise , NAD/metabolismo
19.
Microb Cell Fact ; 13(1): 59, 2014 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-24751325

RESUMO

BACKGROUND: Nikkomycins are competitive inhibitors of chitin synthase and inhibit the growth of filamentous fungi, insects, acarids and yeasts. The gene cluster responsible for biosynthesis of nikkomycins has been cloned and the biosynthetic pathway was elucidated at the genetic, enzymatic and regulatory levels. RESULTS: Streptomyces ansochromogenes ΔsanL was constructed by homologous recombination and the mutant strain was fed with benzoic acid, 4-hydroxybenzoic acid, nicotinic acid and isonicotinic acid. Two novel nikkomycin analogues were produced when cultures were supplemented with nicotinic acid. These two compounds were identified as nikkomycin Px and Pz by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Bioassays against Candida albicans and Alternaria longipes showed that nikkomycin Px and Pz exhibited comparatively strong inhibitory activity as nikkomycin X and Z produced by Streptomyces ansochromogenes 7100 (wild-type strain). Moreover, nikkomycin Px and Pz were found to be more stable than nikkomycin X and Z at different pH and temperature conditions. CONCLUSIONS: Two novel nikkomycin analogues (nikkomycin Px and Pz) were generated by mutasynthesis with the sanL inactivated mutant of Streptomyces ansochromogenes 7100. Although antifungal activities of these two compounds are similar to those of nikkomycin X and Z, their stabilities are much better than nikkomycin X and Z under different pHs and temperatures.


Assuntos
Aminoglicosídeos/biossíntese , Dipeptídeos/biossíntese , Nucleosídeos/biossíntese , Streptomyces/metabolismo , Uridina/análogos & derivados , Alternaria/efeitos dos fármacos , Aminoglicosídeos/química , Aminoglicosídeos/farmacologia , Candida albicans/efeitos dos fármacos , Dipeptídeos/isolamento & purificação , Dipeptídeos/farmacologia , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Molecular , Família Multigênica , Mutação , Niacina/farmacologia , Nucleosídeos/isolamento & purificação , Nucleosídeos/farmacologia , Streptomyces/efeitos dos fármacos , Espectrometria de Massas em Tandem , Temperatura , Transaminases/genética , Uridina/biossíntese , Uridina/isolamento & purificação , Uridina/farmacologia
20.
J Bacteriol ; 195(10): 2232-43, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23475969

RESUMO

Sansanmycins, produced by Streptomyces sp. strain SS, are uridyl peptide antibiotics with activities against Pseudomonas aeruginosa and multidrug-resistant Mycobacterium tuberculosis. In this work, the biosynthetic gene cluster of sansanmycins, comprised of 25 open reading frames (ORFs) showing considerable amino acid sequence identity to those of the pacidamycin and napsamycin gene cluster, was identified. SsaA, the archetype of a novel class of transcriptional regulators, was characterized in the sansanmycin gene cluster, with an N-terminal fork head-associated (FHA) domain and a C-terminal LuxR-type helix-turn-helix (HTH) motif. The disruption of ssaA abolished sansanmycin production, as well as the expression of the structural genes for sansanmycin biosynthesis, indicating that SsaA is a pivotal activator for sansanmycin biosynthesis. SsaA was proved to directly bind several putative promoter regions of biosynthetic genes, and comparison of sequences of the binding sites allowed the identification of a consensus SsaA binding sequence, GTMCTGACAN2TGTCAGKAC. The DNA binding activity of SsaA was inhibited by sansanmycins A and H in a concentration-dependent manner. Furthermore, sansanmycins A and H were found to directly interact with SsaA. These results indicated that SsaA strictly controls the production of sansanmycins at the transcriptional level in a feedback regulatory mechanism by sensing the accumulation of the end products. As the first characterized regulator of uridyl peptide antibiotic biosynthesis, the understanding of this autoregulatory process involved in sansanmycin biosynthesis will likely provide an effective strategy for rational improvements in the yields of these uridyl peptide antibiotics.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Oligopeptídeos/biossíntese , Streptomyces/metabolismo , Uridina/análogos & derivados , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Streptomyces/genética , Uridina/biossíntese
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA