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Nat Prod Bioprospect ; 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415420


Betulin (BE) has exceedingly become a potential natural product, providing multiple pharmacological and biological activities, including anti-cancer, anti-viral, and anti-inflammatory benefits. Previous research indicated that the solvatomorphism of BE can easily occur through crystallization with different organic solvents. This property of BE can directly affect its extraction, isolation, and preparation process. In this study, a system of thermogravimetry (TG)-differential thermal analysis (DTA) coupled with mass spectrometry (MS) with electron ionization (EI) and photoionization (PI) capability, equipped with the skimmer-type interface (i.e., skimmer-type interfaced TG-DTA-EI/PI-MS system), as a real-time and onsite analysis technique, was employed. Then, four solvatomorphs of BE, namely, with pyridine and water (A), sec-butanol (B), n,n-dimethylformamide (DMF) (C), and isopropanol (V), were analyzed for the first time. Finally, five kinds of the main volatile gaseous species, including H2O, pyridine, sec-butanol, DMF, and isopropanol, were identified clearly. Furthermore, the multi-step desolvation processes of the four solvatomorphs of BE were revealed by this system for the first time. This system showed great potential for the rapid and accurate analysis of various solvatomorphs of natural products.

Appl Environ Microbiol ; 82(6): 1898-1905, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26773081


Daptomycin is a potent cyclic lipopeptide antibiotic. It is widely used against various Gram-positive bacterial pathogens. Historically, a poor understanding of the transcriptional regulation of daptomycin biosynthesis has limited the options for targeted genetic engineering toward titer improvement. Here, we isolated a TetR family transcriptional regulator, DepR1, from the industrial producer Streptomyces roseosporus SW0702 using a biotinylated dptE promoter (dptEp) as a probe. The direct interaction between DepR1 and dptEp then was confirmed by electrophoretic mobility shift assays and DNase I footprinting assays. The deletion of depR1 led to a reduction in dptEp activity and the cessation of daptomycin production. The ΔdepR1 mutant produced less red pigment and failed to sporulate on R5 medium. This suggests that DepR1 plays a positive role in the control of morphological differentiation. Moreover, DepR1 was positively autoregulated by directly binding to its own promoter. This might account for the positive feedback regulation of daptomycin production. Based on these positive effects, genetic engineering by overexpression of depR1 raised daptomycin production and shortened the fermentation period both in flask and in fermentor.

Antibacterianos/biossíntese , Daptomicina/biossíntese , Regulação Bacteriana da Expressão Gênica , Streptomyces/genética , Streptomyces/metabolismo , Fatores de Transcrição/genética , Pegada de DNA , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Pigmentos Biológicos/biossíntese , Ligação Proteica , Esporos Bacterianos/crescimento & desenvolvimento , Streptomyces/crescimento & desenvolvimento , Transcrição Genética
Appl Microbiol Biotechnol ; 99(6): 2715-26, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25724582


The roles of many sigma factors are unclear in regulatory mechanism of secondary metabolism in Streptomyces. Here, we report the regulation network of a group 3 sigma factor, WhiGch, from a natamycin industrial strain Streptomyces chattanoogensis L10. WhiGch regulates the growth and morphological differentiation of S. chattanoogensis L10. The whiG ch deletion mutant decreased natamycin production by about 30 % and delayed natamycin production more than 24 h by delaying the growth. Overexpression of the whiG ch gene increased natamycin production in large scale production medium by about 26 %. WhiGch upregulated the transcription of natamycin biosynthetic gene cluster and inhibited the expression of migrastatin and jadomycin analog biosynthetic polyketide synthase genes. WhiGch positively regulated natamycin biosynthetic gene cluster by directly binding to the promoters of scnC and scnD, which were involved in natamycin biosynthesis, and these binding sites adjacent to translation start codon were determined. Thus, this paper further elucidates the high natamycin yield mechanisms of industrial strains and demonstrates that a valuable improvement in the yield of the target metabolites can be achieved through manipulating the transcription regulators.

Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Natamicina/biossíntese , Fator sigma/genética , Streptomyces/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Fragmentação do DNA , Fermentação , Deleção de Genes , Análise em Microsséries , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Família Multigênica , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Streptomyces/metabolismo
Microbiol Res ; 173: 25-33, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25801968


The polyene antibiotic natamycin is widely used as an antifungal agent in both human therapy and the food industry. Here we obtained four natamycin analogs with high titers, including two new compounds, by engineering of six post-polyketide synthase (PKS) tailoring enzyme encoding genes in a natamycin industrial producing strain, Streptomyces chattanoogensis L10. Precise analysis of S. chattanoogensis L10 culture identified natamycin and two natamycin analogs, 4,5-deepoxy-natamycin and 4,5-deepoxy-natamycinolide. The scnD deletion mutant of S. chattanoogensis L10 did not produce natamycin but increased the titer of 4,5-deepoxy-natamycin. Inactivation of each of scnK, scnC, and scnJ in S. chattanoogensis L10 abolished natamycin production and accumulated 4,5-deepoxy-natamycinolide. Deletion of scnG in S. chattanoogensis L10 resulted in production of two new compounds, 4,5-deepoxy-12-decarboxyl-12-methyl-natamycin and its dehydration product without natamycin production. Inactivation of the ScnG-associated ferredoxin ScnF resulted in impaired production of natamycin. Bioassay of these natamycin analogs showed that three natamycin analogs remained antifungal activities. We found that homologous glycosyltransferases genes including amphDI and nysDI can partly complement the ΔscnK mutant. Our results here also support that ScnG, ScnK, and ScnD catalyze carboxylation, glycosylation, and epoxidation in turn in the natamycin biosynthetic pathway. Thus this paper provided a method to generate natamycin analogs and shed light on the natamycin biosynthetic pathway.

Natamicina/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Natamicina/análogos & derivados , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Streptomyces/enzimologia
Clin Chem Lab Med ; 48(1): 109-14, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19943813


BACKGROUND: The free beta subunit of human chorionic gonadotropin (free beta-hCG) is an important serum marker for biochemical screening. Its weekly median value varies with ethnicity. Most of the fluorometers for lanthanide chelates are designed for the detection of signals from europium (Eu(3+)) chelates only. METHODS: We developed a two-site, one-step assay using two monoclonal antibodies (MAbs) against free beta subunit and beta subunit with Eu(3+) chelates as labels. Using the present assay, we evaluated 24,634 normal serum samples in Chinese pregnant women during 8-20 weeks of gestation. RESULTS: The detection limit using this assay was <0.05 ng/mL. The within-run and between-run imprecision was <6.0% and 7.0% using control material. Free beta-hCG concentrations measured using the current assay in 999 maternal serum samples correlated well with those obtained by samarium (Sm(3+))-labeled DELFIA free hCGbeta assay (r=0.987). The medians for 8-20 weeks for maternal serum free beta-hCG were higher in the women from mainland China compared to reports from other countries. CONCLUSIONS: The present assay is suitable for use in biochemical screening of women in mainland China. Our study on the median concentrations of free beta-hCG will help establish reference values that are specific for ethnic populations from the Chinese mainland. These will be useful for studying the importance of ethnic factors in biochemical screening.

Gonadotropina Coriônica Humana Subunidade beta/sangue , Fluorimunoensaio/métodos , China , Reações Cruzadas , Európio/química , Feminino , Idade Gestacional , Humanos , Limite de Detecção , Gravidez , Valores de Referência , Fatores de Tempo