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1.
Nature ; 622(7983): 603-610, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37699521

RESUMEN

Non-segmented negative-strand RNA viruses, including Ebola virus (EBOV), rabies virus, human respiratory syncytial virus and pneumoviruses, can cause respiratory infections, haemorrhagic fever and encephalitis in humans and animals, and are considered a substantial health and economic burden worldwide1. Replication and transcription of the viral genome are executed by the large (L) polymerase, which is a promising target for the development of antiviral drugs. Here, using the L polymerase of EBOV as a representative, we show that de novo replication of L polymerase is controlled by the specific 3' leader sequence of the EBOV genome in an enzymatic assay, and that formation of at least three base pairs can effectively drive the elongation process of RNA synthesis independent of the specific RNA sequence. We present the high-resolution structures of the EBOV L-VP35-RNA complex and show that the 3' leader RNA binds in the template entry channel with a distinctive stable bend conformation. Using mutagenesis assays, we confirm that the bend conformation of the RNA is required for the de novo replication activity and reveal the key residues of the L protein that stabilize the RNA conformation. These findings provide a new mechanistic understanding of RNA synthesis for polymerases of non-segmented negative-strand RNA viruses, and reveal important targets for the development of antiviral drugs.


Asunto(s)
Ebolavirus , ARN Viral , ARN Polimerasa Dependiente del ARN , Replicación Viral , Animales , Humanos , Antivirales/farmacología , Ebolavirus/enzimología , Ebolavirus/genética , Ebolavirus/crecimiento & desarrollo , Fiebre Hemorrágica Ebola/virología , ARN Viral/biosíntesis , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Genoma Viral , Conformación de Ácido Nucleico , Mutagénesis , Estabilidad del ARN
2.
Metab Eng ; 81: 210-226, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38142854

RESUMEN

Streptomyces has an extensive array of bioactive secondary metabolites (SMs). Nevertheless, devising a framework for the heterologous production of these SMs remains challenging. We here reprogrammed a versatile plug-and-play Streptomyces super-chassis and established a universal pipeline for production of diverse SMs via understanding of the inherent pleiotropic effects of ethanol shock on jadomycin production in Streptomyces venezuelae. We initially identified and characterized a set of multiplex targets (afsQ1, bldD, bldA, and miaA) that contribute to SM (jadomycin) production when subjected to ethanol shock. Subsequently, we developed an ethanol-induced orthogonal amplification system (EOAS), enabling dynamic and precise control over targets. Ultimately, we integrated these multiplex targets into functional units governed by the EOAS, generating a universal and plug-and-play Streptomyces super-chassis. In addition to achieving the unprecedented titer and yield of jadomycin B, we also evidenced the potential of this super-chassis for production of diverse heterologous SMs, including antibiotic oxytetracycline, anticancer drug doxorubicins, agricultural herbicide thaxtomin A, and plant growth regulator guvermectin, all with the yields of >10 mg/g glucose in a simple mineral medium. Given that the production of SMs all required complexed medium and the cognate yields were usually much lower, our achievement of using a universal super-chassis and engineering pipeline in a simple mineral medium is promising for convenient heterologous production of SMs.


Asunto(s)
Adenosina/análogos & derivados , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Antibacterianos , Etanol/metabolismo , Minerales/metabolismo , Minerales/farmacología
3.
Environ Monit Assess ; 195(10): 1221, 2023 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-37718385

RESUMEN

Heavy metal pollution has attracted increasing concern due to its high toxicity and persistence. A suitable extraction procedure for available heavy metals in soil is necessary for assessing the ecological risk. In this work, the single extraction methods aided by shaking and microwaves were investigated and analyzed for their ability to extract available heavy metals from soil samples, and a total of 42 soil samples were collected from suburbs of Zhengzhou city in China. The extraction efficiency of Cu, Zn, As, and Cd in the certified fluvo-aquic soil was compared using eight different types of solutions: CaCl2, CH3COONH4, NH4NO3, CH3COOH, Na2EDTA, DTPA, HNO3, and NH4H2PO4. Results indicated that the shaking-assisted method that utilized Na2EDTA as an extractant demonstrated satisfactory efficiency and was chosen for further optimization and that the optimal conditions were obtained using 0.05 M Na2EDTA at pH 7, soil-liquid ratio 1:20, and extraction duration 2 h, which gained the perfect extraction efficiency ranging from 85.8 to 109.5%. The proposed approach has been applied to extract available Cu, Zn, As, and Cd in soils of Zhengzhou suburbs, where the mean values varied from 0.129 to 6.881 mg/kg. The bioavailability of different heavy metals in the soil varies greatly, with Cd having the highest activity in the survey region. Significant (p < 0.01) positive relationships were observed between the available state and the total amount of all the heavy metals. The assessment of health risks associated with heavy metals indicated that there was no risk for chronic non-carcinogenic effects. Even though the total amount of metal elements in suburban soil of Zhengzhou is 1.6% with high carcinogenic risk, the risk of available elements is still within the acceptable range, which verified that the risk grade obtained by the total amount is higher than the actual risk.


Asunto(s)
Cadmio , Metales Pesados , Ácido Edético , Monitoreo del Ambiente , China , Medición de Riesgo , Suelo
4.
Proc Natl Acad Sci U S A ; 115(11): 2818-2823, 2018 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-29483275

RESUMEN

Seven-carbon-chain-containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses-containing antibiotics. To date, very little is known about the biosynthesis of these compounds. Here we sequenced the genome of the septacidin producer and identified the septacidin gene cluster by heterologous expression. After determining the boundaries of the septacidin gene cluster, we studied septacidin biosynthesis by in vivo and in vitro experiments and discovered that SepB, SepL, and SepC can convert d-sedoheptulose-7-phosphate (S-7-P) to ADP-l-glycero-ß-d-manno-heptose, exemplifying the involvement of ADP-sugar in microbial natural product biosynthesis. Interestingly, septacidin, a secondary metabolite from a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with the gram-negative bacterium LPS. In addition, two acyltransferase-encoding genes sepD and sepH, were proposed to be involved in septacidin side-chain formation according to the intermediates accumulated in their mutants. In hygromycin B biosynthesis, an isomerase HygP can recognize S-7-P and convert it to ADP-d-glycero-ß-d-altro-heptose together with GmhA and HldE, two enzymes from the Escherichia coli LPS heptose biosynthetic pathway, suggesting that the d-heptopyranose moiety of hygromycin B is also derived from S-7-P. Unlike the other S-7-P isomerases, HygP catalyzes consecutive isomerizations and controls the stereochemistry of both C2 and C3 positions.


Asunto(s)
Escherichia coli/metabolismo , Higromicina B/biosíntesis , Fosfatos de Azúcar/metabolismo , Vías Biosintéticas , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Heptosas/metabolismo , Higromicina B/química , Nucleósidos de Purina/biosíntesis , Nucleósidos de Purina/química , Fosfatos de Azúcar/química
5.
Appl Microbiol Biotechnol ; 103(20): 8459-8472, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31422450

RESUMEN

Streptomyces avermitilis is well known as the producer of anthelmintic agent avermectins, which are widely used in agriculture, veterinary medicine, and human medicine. aveI encodes a TetR-family regulator, which is the homolog of AtrA. It was reported that deletion of aveI caused enhanced avermectin production. In this study, we investigated the regulatory function of the AveI in S. avermitilis. By binding to the 15-nt palindromic sequence in the promoter regions, AveI directly regulates at least 35 genes. AveI represses avermectin production by directly regulating the transcription of the cluster-situated regulator gene aveR and structural genes aveA1, aveA3, and aveD. AveI represses oligomycin production by repressing the CSR gene olmRII and structural genes olmC. AveI activates melanin biosynthesis by activating the expression of melC1C2 operon. AveI activates morphological differentiation by activating the expression of ssgR and ssgD genes, repressing the expression of wblI gene. Besides, AveI regulates many genes involved in primary metabolism, including substrates transport, the metabolism of amino acids, lipids, and carbohydrates. Therefore, AveI functions as a global regulator in S. avermitilis, controls not only secondary metabolism and morphological differentiation, but also primary metabolism.


Asunto(s)
Productos Biológicos/metabolismo , Regulación Bacteriana de la Expresión Génica , Ivermectina/análogos & derivados , Melaninas/metabolismo , Oligomicinas/metabolismo , Streptomyces/metabolismo , Factores de Transcripción/metabolismo , Ivermectina/metabolismo , Streptomyces/citología , Streptomyces/genética , Factores de Transcripción/genética
6.
Proc Natl Acad Sci U S A ; 112(39): 12181-6, 2015 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-26374838

RESUMEN

There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.


Asunto(s)
Productos Biológicos/aislamiento & purificación , Descubrimiento de Drogas/métodos , Citometría de Flujo/métodos , Proteínas Fluorescentes Verdes/metabolismo , Elementos Reguladores de la Transcripción/genética , Streptomyces/química , Biología Sintética/métodos , Sitios de Unión/genética , Productos Biológicos/análisis , Descubrimiento de Drogas/tendencias , Microscopía Confocal , Familia de Multigenes/genética , Regiones Promotoras Genéticas/genética , Propidio , Biología Sintética/tendencias
7.
Appl Microbiol Biotechnol ; 101(9): 3811-3820, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28352998

RESUMEN

Since the lacZα-based blue/white screening system was introduced to molecular biology, several different visual reporter systems were developed and used for various purposes in Escherichia coli. A common limit to the existent visual reporter systems is that an extracellular chromogenic substrate has to be added for the visible pigment production. In this study, we developed a new blue/white screening system based on a non-ribosomal peptide synthetase encoded by idgS from Streptomyces and a phosphopantetheinyl transferase encoded by sfp from Bacillus. When IdgS is activated from an apo-form to a holo-form via a posttranslational modification catalyzed by Sfp, it can synthesize a blue pigment indigoidine using L-glutamine, the amino acid abundant in cells, as a substrate. The new blue/white screening system contains a recipient E. coli strain with an optimized idgS gene cassette and a cloning vector harboring an sfp gene with an in-frame insertion of a multiple cloning site close to its N-terminal. We demonstrated that the IdgS/Sfp-based blue/white screening system is a powerful alternative to the lacZα-based screening system, which does not require any external substrate addition.


Asunto(s)
Clonación Molecular , Escherichia coli/genética , Pruebas Genéticas/métodos , Vectores Genéticos , Genética Microbiana/métodos , Biología Molecular/métodos , Pigmentos Biológicos/análisis , Bacillus/enzimología , Bacillus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Color , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Streptomyces/enzimología , Streptomyces/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo
8.
J Gen Virol ; 97(8): 1797-1806, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27217257

RESUMEN

Influenza A viruses have the potential to cause pandemics due to the introduction of novel subtypes against which human hosts have little or no preexisting immunity. Such viruses may result from reassortment between human and animal influenza viruses. Recently, new influenza-like viruses were identified in bats, raising the concern for a new reservoir of potentially harmful influenza viruses that could form reassortants with categorized human influenza A viruses. However, until now, it has not been possible to generate a recombinant reassortant virus containing a single functional gene or domain from H17N10 that could propagate. Here, we demonstrate that a recombinant A/Puerto Rico/8/1934 (H1N1) virus with NS1 gene from H17N10 influenza-like virus can be successfully rescued. We used luciferase reporter assays and quantitative reverse transcriptase PCR to show that the NS1 protein from H17N10 inhibited Sendai-virus (SeV)-induced activation of IFN-ß expression with an efficiency similar to NS1 from an H5N1 strain. Moreover, the crystal structure of the NS1 (H17N10) RNA-binding domain is also similar to that of other NS1s. These results demonstrate that H17N10 influenza-like virus indeed contains functional genes that are compatible with categorized influenza A viruses. Although the chance of this particular event occurring in nature seems negligible, further research is needed to address the possibility of the natural formation of reassortants.


Asunto(s)
Quirópteros/virología , Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/veterinaria , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Línea Celular , Cristalografía por Rayos X , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Modelos Moleculares , Infecciones por Orthomyxoviridae/virología , Conformación Proteica , Virus Reordenados/genética , Virus Reordenados/fisiología , Genética Inversa , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética
9.
Metab Eng ; 33: 41-51, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26506462

RESUMEN

Because high-throughput screening tools are typically unavailable when using the pathway-engineering approach, we developed a new strategy, named intermediate sensor-assisted push-pull strategy, which enables sequential pathway optimization by incorporating a biosensor targeting a key pathway intermediate. As proof of concept, we constructed an L-Trp biosensor and used it to optimize the deoxyviolacein biosynthetic pathway, which we divided into two modules with L-Trp being the product of the upstream and the substrate of the downstream module for deoxyviolacein synthesis. Using the biosensor and fluorescence-activated cell sorting, the activities of the two modules were sequentially and independently optimized in Escherichia coli to achieve the desired phenotypes. By this means, we increased the deoxyviolacein titer 4.4-fold (1.92 g/L), which represents the greatest deoxyviolacein production reported. This work suggests that a biosynthetic pathway can be enhanced to produce a value-added secondary metabolite(s) without available end-product screening method by using a central metabolic junction molecule biosensor(s).


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Indoles/metabolismo , Ingeniería Metabólica/métodos , Análisis de Flujos Metabólicos/métodos , Proteínas de Escherichia coli/genética , Mejoramiento Genético/métodos , Indoles/aislamiento & purificación , Transducción de Señal/fisiología , Triptófano/genética , Triptófano/metabolismo
10.
Synth Syst Biotechnol ; 9(1): 69-77, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38273864

RESUMEN

Streptomyces has enormous potential to produce novel natural products (NPs) as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters (BGCs). However, the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery. Here, we developed an easy-to-use, highly flexible DNA assembly toolkit for gene cluster engineering. The DNA assembly toolkit is compatible with various DNA assembling approaches including Biobrick, Golden Gate, CATCH, yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly. This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters. To demonstrate the utility of this toolkit, we quantified a library of modular regulatory parts, and engineered a gene cluster (act) using characterized promoters that led to increased production. Overall, this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.

11.
World J Microbiol Biotechnol ; 29(10): 1869-75, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23579768

RESUMEN

The S-adenosylmethionine synthetase gene (metK) from Streptomyces avermitilis was cloned into multi-copy vector pIJ653 and integrative vector pSET152 yielding two metK expression plasmids pYJ02 and pYJ03, respectively. When wild-type strain ATCC31267 was transformed with these two plasmids, avermectin production was increased about 2.0-fold and 5.5-fold, respectively. The introduction of integrative expression plasmid pYJ03 into the engineered strain GB-165, which produces only avermectin B, promoted the production of avermectin approximately 2.0-fold. However, introduction of pYJ02 did not influence avermectin accumulation in GB-165. Moreover, transformation of the avermectin-overproducing industry strain 76-05 with these two plasmids did not stimulate avermectin production. These results showed that there were different effects of metK expression levels on avermectin production in various S. avermitilis strains. Additionally, the transcript levels of metK, aveR (the avermectin pathway-specific regulatory gene) and aveA1 (one avermectin biosynthesis gene) meet the expectation of fermentation levels of avermectin in wild-type strain and its recombinant strains. The gene expression levels of metK, aveR and aveA1 in GB-165 and 76-05 were much higher then those in wild-type strain, which probably limited the increasement of avermectin by overexpression of metK.


Asunto(s)
Expresión Génica , Ivermectina/análogos & derivados , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Clonación Molecular , Vectores Genéticos , Ivermectina/metabolismo , Ingeniería Metabólica , Plásmidos
12.
Life (Basel) ; 12(5)2022 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-35629358

RESUMEN

Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized Streptomyces hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the txtED operon, txtABH operon and txtC gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 µg/mL in heterologous Streptomyces coelicolor M1154. To further optimize the biosynthetic pathway, we used constraint-based combinatorial design to build 27 refactored gene clusters by varying the promoter strength of every operon, and the highest titer of thaxtomin A production reached 504.6 µg/mL. Taken altogether, this work puts forward a multiplexed promoter engineering strategy to engineer secondary metabolism gene clusters for efficiently improving fermentation titers.

13.
Synth Syst Biotechnol ; 7(3): 972-981, 2022 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-35756964

RESUMEN

The application of the valuable natural product thaxtomin A, a potent bioherbicide from the potato scab pathogenic Streptomyces strains, has been greatly hindered by the low yields from its native producers. Here, we developed an orthogonal transcription system, leveraging extra-cytoplasmic function (ECF) sigma (σ) factor 17 (ECF17) and its cognate promoter Pecf17, to express the thaxtomin gene cluster and improve the production of thaxtomin A. The minimal Pecf17 promoter was determined, and a Pecf17 promoter library with a wide range of strengths was constructed. Furthermore, a cumate inducible system was developed for precise temporal control of the ECF17 transcription system in S. venezuelae ISP5230. Theoretically, the switchable ECF17 transcription system could reduce the unwanted influences from host and alleviate the burdens introduced by overexpression of heterologous genes. The yield of thaxtomin A was significantly improved to 202.1 ± 15.3  µ g/mL using the switchable ECF17 transcription system for heterologous expression of the thaxtomin gene cluster in S. venezuelae ISP5230. Besides, the applicability of this transcription system was also tested in Streptomyces albus J1074, and the titer of thaxtomin A was raised to as high as 239.3 ± 30.6 µg/mL. Therefore, the inducible ECF17 transcription system could serve as a complement of the generally used transcription systems based on strong native constitutive promoters and housekeeping σ factors for the heterologous expression of valuable products in diverse Streptomyces hosts.

14.
Life (Basel) ; 11(11)2021 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-34833131

RESUMEN

CRISPR-based enzymes have offered a unique capability to the design of genetic switches, with advantages in designability, modularity and orthogonality. CRISPR-based genetic switches operate on multiple levels of life, including transcription and translation. In both prokaryotic and eukaryotic cells, deactivated CRISPR endonuclease and endoribonuclease have served in genetic switches for activating or repressing gene expression, at both transcriptional and translational levels. With these genetic switches, more complex circuits have been assembled to achieve sophisticated functions including inducible switches, non-linear response and logical biocomputation. As more CRISPR enzymes continue to be excavated, CRISPR-based genetic switches will be used in a much wider range of applications.

15.
Viruses ; 13(8)2021 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-34452298

RESUMEN

Influenza A viruses are serious zoonotic pathogens that continuously cause pandemics in several animal hosts, including birds, pigs, and humans. Indole derivatives containing an indole core framework have been extensively studied and developed to prevent and/or treat viral infection. This study evaluated the anti-influenza activity of several indole derivatives, including 3-indoleacetonitrile, indole-3-carboxaldehyde, 3-carboxyindole, and gramine, in A549 and MDCK cells. Among these compounds, 3-indoleacetonitrile exerts profound antiviral activity against a broad spectrum of influenza A viruses, as tested in A549 cells. Importantly, in a mouse model, 3-indoleacetonitrile with a non-toxic concentration of 20 mg/kg effectively reduced the mortality and weight loss, diminished lung virus titers, and alleviated lung lesions of mice lethally challenged with A/duck/Hubei/WH18/2015 H5N6 and A/Puerto Rico/8/1934 H1N1 influenza A viruses. The antiviral properties enable the potential use of 3-indoleacetonitrile for the treatment of IAV infection.


Asunto(s)
Antivirales/farmacología , Indoles/farmacología , Indoles/uso terapéutico , Virus de la Influenza A/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Células A549 , Animales , Antivirales/uso terapéutico , Antivirales/toxicidad , Perros , Femenino , Humanos , Indoles/toxicidad , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/fisiología , Virus de la Influenza A/fisiología , Pulmón/patología , Pulmón/virología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Sulfuros/farmacología , Carga Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos
16.
Can J Microbiol ; 55(12): 1355-63, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20029527

RESUMEN

The avermectin analogue doramectin (CHC-B1), which is produced in mutants that have an altered biosynthesis pathway of avermectin, is one of the most effective agricultural pesticides and antiparasitics. We report here the construction of a bkdF olmA double-deletion mutant lacking one of the branched-chain alpha-keto acid dehydrogenase encoding genes (bkdF) and the oligomycin PKS encoding gene cluster (olmA) in Streptomyces avermitilis 76-05. We then characterized the production of various antibiotics in cultures of the deletion mutant. In a fermentation medium supplemented with cyclohexanecarboxylic acid, this double mutant produced doramectin and its analogues but no oligomycin. The mutant proved to be genetically stable, without any antibiotic resistance markers inserted into its chromosome, and could potentially become an industrial doramectin-producing strain after further improvement.


Asunto(s)
Antihelmínticos/metabolismo , Insecticidas/metabolismo , Ivermectina/análogos & derivados , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Streptomyces/metabolismo , Fermentación , Eliminación de Gen , Ingeniería Genética , Microbiología Industrial , Ivermectina/metabolismo , Complejos Multienzimáticos/genética , Familia de Multigenes , Mutación , Sintasas Poliquetidas/genética , Streptomyces/genética
17.
Nat Commun ; 10(1): 3665, 2019 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-31413323

RESUMEN

Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity. Due to the challenge of cultivation and genetic intractability, assessing the capability of their biosynthetic gene clusters (BGCs) for secondary metabolite production requires an efficient heterologous expression system. However, this kind of host system is still unavailable. Here, we use the facultative anaerobe Streptococcus mutans UA159 as a heterologous host for the expression of BGCs from anaerobic bacteria. A natural competence based large DNA fragment cloning (NabLC) technique was developed, which can move DNA fragments up to 40-kb directly and integrate a 73.7-kb BGC to the genome of S. mutans UA159 via three rounds of NabLC cloning. Using this system, we identify an anti-infiltration compound, mutanocyclin, from undefined BGCs from human oral bacteria. We anticipate this host system will be useful for heterologous expression of BGCs from anaerobic bacteria.


Asunto(s)
Bacterias Anaerobias/genética , Vías Biosintéticas/genética , Clonación Molecular/métodos , Familia de Multigenes/genética , Streptococcus mutans/genética , Humanos , Microbiota/genética , Boca , Péptidos , Policétidos , Terpenos
18.
ACS Synth Biol ; 6(2): 211-216, 2017 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-27718551

RESUMEN

We developed an in vitro DNA detection system using a pair of dCas9 proteins linked to split halves of luciferase. Luminescence was induced upon colocalization of the reporter pair to a ∼44 bp target sequence defined by sgRNAs. We used the system to detect Mycobacterium tuberculosis DNA with high specificity and sensitivity. The reprogrammability of dCas9 was further leveraged in an array design that accesses sequence information across the entire genome.


Asunto(s)
Secuencia de Bases/genética , Endonucleasas/genética , Ácidos Nucleicos/genética , Sistemas CRISPR-Cas/genética , ADN Bacteriano/genética , Genoma Bacteriano/genética , Luciferasas/genética , Luminiscencia , Mycobacterium tuberculosis/genética , Sensibilidad y Especificidad
19.
ACS Synth Biol ; 5(11): 1308-1317, 2016 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-27133230

RESUMEN

As a product of a multistep enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here, we reported a semirational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon cloned from the native producer Ralstonia entropha (R. entropha). Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high-throughput screening, and detailed in-depth analysis. Applying this approach, strains accumulating 0%-92% PHB contents in cell dry weight (CDW) were achieved. PHB with various weight-average molecular weights (Mw) of 2.7-6.8 × 106 were also efficiently produced in relatively high contents. These results suggest that the semirational approach combining library design, construction, and proper screening is an efficient way to optimize PHB and other multienzyme pathways.


Asunto(s)
Clonación Molecular , Escherichia coli/genética , Genes Bacterianos , Secuenciación de Nucleótidos de Alto Rendimiento , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Fragmentación del ADN , Escherichia coli/metabolismo , Peso Molecular , Operón , Plásmidos/genética , Plásmidos/metabolismo
20.
J Biol Eng ; 9: 23, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26702298

RESUMEN

BACKGROUND: Imbalances in gene expression of a metabolic pathway can result in less-yield of the desired products. Several targets were intensively investigated to balance the gene expression, such as promoter, ribosome binding site (RBS), the order of genes, as well as the species of the enzymes. However, the capability of simultaneous manipulation of multiple targets still needs to be explored. RESULTS: We reported a new DNA assembling method to vary all the above types of regulatory targets simultaneously, named oligo-linker mediated assembly (OLMA) method, which can incorporate up to 8 targets in a single assembly step. Two experimental cases were used to demonstrate the capability of the method: (1) assembly of multiple pieces of lacZ expression cassette; (2) optimization of four enzymes in lycopene biosynthetic pathway. Our results indicated that the OLMA method not only exploited larger combinatorial space, but also reduced the inefficient mutants. CONCLUSIONS: The unique feature of oligo-linker mediated assembly (OLMA) method is inclusion of a set of chemically synthetic double-stranded DNA oligo library, which can be designed as promoters and RBSs, or designed with different overhang to bridge the genes in different orders. The inclusion of the oligos resulted in a PCR-free and zipcode-free DNA assembly reaction for OLMA.

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