RESUMEN
This study aimed to obtain reliable high Vip3A production from Bacillus thuringiensis (Bt) by modifying Vip3A to acquire higher thermostability in a suitable host. Bt117 is a great host for Vip3A production due to protein production consistency, low protease activity in culture media, and large amounts of mostly full-length protein, but it produces Vip3A with lower thermostability (Vip3Aa35). The C-terminal region of Bt117 Vip3A was replaced with that of a Vip3A with higher thermostability (Vip3Aa64 from Bt294) to generate the recombinant Bt117-Vip3Aa64-C. Like the parental strain Bt117, this strain expressed mostly full-length protein and exhibited low protease activity and similar protein expression profiles in culture media but retained greater larvicidal activity upon 37 °C storage like Bt294 Vip3Aa64. Importantly, every culture batch of Bt117-Vip3Aa64-C yielded over 200 mg/l Vip3A, which is a notable improvement over the original Vip3Aa64-producing strain Bt294 where 45% of culture batches failed to produce Vip3A at the same level. Successfully, we combined the superior qualities of two Bt strains, Bt294, which produces thermostable Vip3A but at low and inconsistent levels, and Bt117, which produces Vip3A with low thermostability but at consistently high levels. Protein engineering of Vip3A in Bt117 ultimately yielded an improved strain producing a thermostable Vip3A with reliably high protein production.
Asunto(s)
Bacillus thuringiensis , Proteínas Bacterianas , Ingeniería de Proteínas , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Larva/microbiología , Estabilidad ProteicaRESUMEN
A vegetative insecticidal protein, Vip3A, is highly active against lepidopteran pests, which are the most important pests in most tropical countries. An important aspect of the successful commercial production of this bacterial insecticide is the development of bacterial culture media that maximize the titres of this protein and cost reduction. This study aimed to investigate and optimize Vip3A production by Bacillus thuringiensis Bt294 using statistical methods and 3-step sequential approaches. The experimental design showed that the production of Vip3A was maximized to 300 mg/L when the bacterium was cultivated in medium composed of 5.05 g/L glycerol, 49.17 g/L soytone, 30.05 g/L casein hydrolysate, 1.99 g/L CaCl2.2H2O, 7.5 mg/L CuSO4, 15 mg/L MnSO4.H2O, 9.4 g/L K2HPO4, 2.2 g/L KH2PO4, 0.2 g/L MgSO4.7H2O, 5 g/L yeast extract, 2.5 mg/L NiCl2.6H2O and 3 mL/L vitamin solution. B. thuringiensis Bt294 Vip3A toxin was highly toxic to Spodoptera exigua with LC50 values of 187.1 ng/cm2 at 7 days. This result demonstrated that a high titre of Vip3A produced by B. thuringiensis Bt294 will be useful as a biological control agent. This optimization will allow production to be scaled up for commercial production in the future.
RESUMEN
Insecticidal protein Vip3A secreted from B. thuringiensis is a potential biocontrol agent for control of lepidopteran pests. Under laboratory conditions, high albeit variable Vip3A production from the local isolate Bt294 was only obtained from a much enriched TB culture medium. Proteomic analysis and strain improvement were therefore performed to improve Vip3A production. Studies indicated that the buffer capacity, carbon source, and nitrogen source are critical to efficiently produce Vip3A. Medium with lower amounts of peptone and yeast extract (compared to TB), with an additional carbon source and phosphate buffer (LB*G medium) was found to give reasonable yields of Vip3A. Proteomic analysis revealed higher expression of proteins involved in glutamate and histidine biosynthesis in cells cultured in TB compared to LB about 58 and 33 times, respectively. Experiments confirmed that glutamate supplementation could increase Vip3A production. In addition, promoter substitution with that of cry3A increased Vip3A yields by about 20-30%. Overall, very high yields of Vip3A could be obtained by culturing Bt294 (Pcry3A-vip3Aa64) in LB*G medium with glutamate supplementation.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Agentes de Control Biológico/metabolismo , Insecticidas/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/crecimiento & desarrollo , Toxinas de Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Endotoxinas/genética , Proteínas Hemolisinas/genética , Regiones Promotoras Genéticas , ProteómicaRESUMEN
BACKGROUND: Vip3Aa is a vegetative insecticidal protein produced by Bacillus thuringiensis. The protein is produced as an 88-kDa protoxin that could be processed by insect gut proteases into a 22-kDa N-terminal and a 66-kDa C-terminal fragments. The C-terminal part could bind to a specific receptor while the N-terminal part is required for toxicity and structural stability. OBJECTIVE: To demonstrate the antagonistic effect of truncated fragments on the insecticidal activity of the full-length Vip3Aa. METHODS: The full-length protein (Vip3Aa), a 66-kDa C-terminal fragment (Vip3Aa-D199) and a predicted carbohydrate binding module (CBM) were produced in Escherichia coli. Purified proteins were mixed at different ratios and fed to Spodoptera litura and Spodoptera exigua larvae. Mortality was recorded and compared between larvae fed with individual toxin and mixtures of the full-length and truncated toxins. RESULTS: Production level of the Vip3Aa-D199 was significantly decreased comparing to that of the full-length protein. Vip3Aa-D199 and CBM fragment were not toxic to insect larvae whereas Vip3Aa showed high toxicity with LC50 about 200 ng/cm2. Feeding the larvae with mixtures of the Vip3Aa and Vip3Aa-D199 at different ratios revealed antagonistic effect of the Vip3Aa-D199 on the toxicity of Vip3Aa. Results showed that the lethal time (LT 50 and LT 95) of larvae fed the mixture toxins was longer than those fed the Vip3Aa alone. In addition, a CBM fragment could inhibit toxicity of the full-length Vip3Aa. CONCLUSION: Our results demonstrated that the Vip3Aa-D199 and a CBM fragment could complete for the membrane binding thus rendering activity of the full-length Vip3Aa.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/toxicidad , Interacciones Huésped-Patógeno/efectos de los fármacos , Insecticidas/toxicidad , Larva/efectos de los fármacos , Spodoptera/efectos de los fármacos , Animales , Proteínas Bacterianas/metabolismo , Insecticidas/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Control Biológico de Vectores , Spodoptera/crecimiento & desarrollo , Spodoptera/metabolismoRESUMEN
Vip3Aa (vegetative insecticidal protein) secreted by Bacillus thuringiensis (Bt) is highly toxic to lepidopteran insects. The Bt isolate M190 produces Vip3Aa35 at high concentrations, and Vip3Aa35 was found to be very effective against Spodoptera exigua. Unfortunately, the use of Vip3Aa35 in pest control is limited by its short shelf life when stored at high temperatures, retaining activity for only 1 month at 37 °C. To find a more stable alternative, we screened 500 isolates of Bt collected from various locations in Thailand and discovered Bt isolate 294 which produced large amounts of Vip3Aa64 that exhibited high toxicity against S. exigua but could be stored at 37 °C for up to 3 months. Vip3Aa35 and Vip3Aa64 have only nine amino acid differences between them, with six of those residues being located at the C terminus. Vip3Aa35 and Vip3Aa64 chimeras revealed that the C-terminal sequence is important for the retained larvicidal activity observed with Vip3Aa64. Various single amino acid substitutions were created to identify the key amino acids responsible for this stability. A single residue, Tyr776, was found to be solely responsible, with the Vip3Aa35:N776Y acquiring thermostability similar to Vip3Aa64 while the Vip3Aa64:Y776N exhibited Vip3Aa35-like thermostability.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Agentes de Control Biológico/farmacología , Spodoptera/microbiología , Sustitución de Aminoácidos/genética , Animales , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Clonación MolecularRESUMEN
Cyt2Aa2 is a mosquito larvicidal and cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. The toxin becomes inactive when isoleucine at position 150 was replaced by alanine. To investigate the functional role of this position, Ile150 was substituted with Leu, Phe, Glu and Lys. All mutant proteins were produced at high level, solubilized in carbonate buffer and yielded protease activated product similar to those of the wild type. Intrinsic fluorescence spectra analysis suggested that these mutants retain similar folding to the wild type. However, mosquito larvicidal and hemolytic activities dramatically decreased for the I150K and were completely abolished for I150A and I150F mutants. Membrane binding and oligomerization assays demonstrated that only I150E and I150L could bind and form oligomers on lipid membrane similar to that of the wild type. Our results suggest that amino acid at position 150 plays an important role during membrane binding and oligomerization of Cyt2Aa2 toxin.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Biopolímeros/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Isoleucina/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Secuencia de Bases , Biopolímeros/química , Calorimetría , Cartilla de ADN , Endotoxinas/química , Proteínas Hemolisinas/química , Isoleucina/química , Unión ProteicaRESUMEN
The binary toxin produced from Bacillus sphaericus is highly toxic against larvae of Culex and Anopheles mosquitoes. The two major components of the binary toxin are 42-kDa BinA and 51-kDa BinB, which are produced as crystalline inclusions during sporulation. Currently, there is no detailed knowledge of the molecular mechanism of the binary toxin, mainly due to the lack of structural information. Herein, we describe an expression protocol with modified conditions allowing production of soluble, biologically active BinA and BinB for further structural analysis. The binA and binB genes from B. sphaericus 2297 strain were independently cloned and fused with a polyhistidine tag at their N-termini. Both (His)(6)-tagged BinA and (His)(6)-tagged BinB were expressed as soluble forms at low temperature. Highly pure proteins were obtained after two-step purification by Ni-NTA affinity and size exclusion chromatography. In vitro activation by trypsin digestion generated a resistant fragment, of 40kDa for BinA, and of 45kDa for BinB, and an oligomeric complex of BinA and BinB in solution was observed after proteolytic activation. Their functional and structural properties were confirmed by a biological assay and far-UV circular dichroism, respectively. The mixture of BinA and BinB, either as a protoxin or as a trypsin-activated form, exhibited high mosquito-larvicidal activity against Culex quinquefasciatus larvae with LC(50) of about 10ng/ml, while no toxicity was observed from the single binary toxin component. Results from far-UV circular dichroism of BinA and BinB suggest the presence of mainly ß-structure. The expression and purification protocols reported here will be useful for the production of the active and homogeneous binary toxin to allow further detailed structural investigation.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Toxinas Bacterianas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Bacillus , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/química , Toxinas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/farmacología , Cromatografía en Gel , Culex/efectos de los fármacos , Insecticidas/química , Insecticidas/aislamiento & purificación , Insecticidas/farmacología , Larva/efectos de los fármacos , Dosificación Letal Mediana , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , SolubilidadRESUMEN
Mtx1 and Mtx2 are mosquitocidal toxins produced by some strains of Bacillus sphaericus during vegetative phase of growth. Mtx1 from B. sphaericus 2297 shows higher toxicity against Culex quinquefasciatus larvae than to Aedes aegypti larvae whereas Mtx2 from B. sphaericus 2297 shows lower toxicity against C. quinquefasciatus than to A. aegypti larvae. To test synergism of these toxins against A. aegypti larvae, mtx1 and mtx2 genes were cloned into a single plasmid and expressed in Escherichia coli. Cells producing both Mtx1 and Mtx2 toxins exhibited high synergistic activity against A. aegypti larvae approximately 10 times compared to cells expressing only a single toxin. Co-expression of both toxins offers an alternative to improve efficacy of recombinant bacterial insecticides. There is a high possibility to develop these toxins to be used as an environmentally friendly mosquito control agent.
Asunto(s)
Aedes/efectos de los fármacos , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Insecticidas/toxicidad , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Toxinas Bacterianas/aislamiento & purificación , Sinergismo Farmacológico , Escherichia coli/genética , Larva/efectos de los fármacos , Dosificación Letal MedianaRESUMEN
Cyt2Aa2 produced by Bacillus thuringiensis subsp. darmstadiensis exhibits in vitro cytolytic activity against broad range of cells but shows specific in vivo toxicity against larvae of Dipteran insects. To investigate the role of amino acids in alphaA and alphaC of this toxin, 3 single-point mutants (A61C, S108C and V109A) were generated. All 3 mutant proteins were highly produced as inclusion bodies that could be solubilized and activated by proteinase K similar to that of the wild type. Hemolytic activity of A61C and S108C mutants was significantly reduced whereas the V109A mutant showed comparable hemolytic activity to the wild type. Interestingly, the A61C mutant exhibited high larvicidal activity to both Aedes aegypti and Culex quinquefasciatus. S108C and V109A mutants showed low activity against C. quinquefasciatus but relatively high toxicity to A. aegypti. These results demonstrated for the first time that amino acids in alphaA and alphaC are involved in the selectivity of the Cyt toxin to the targeted organism.
Asunto(s)
Sustitución de Aminoácidos , Bacillus thuringiensis/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Culicidae/fisiología , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Hemólisis/fisiología , Control de Mosquitos , Subunidades de Proteína/metabolismo , Secuencia de Aminoácidos , Animales , Bacillus thuringiensis/efectos de los fármacos , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Culicidae/efectos de los fármacos , Endotoxinas/química , Eritrocitos/efectos de los fármacos , Proteínas Hemolisinas/química , Hemólisis/efectos de los fármacos , Larva/efectos de los fármacos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Ovinos , Solubilidad/efectos de los fármacos , Especificidad de la EspecieRESUMEN
Fungal type I polyketide (PK) compounds are highly valuable for medical treatment and extremely diverse in structure, partly because of the enzymatic activities of reducing domains in polyketide synthases (PKSs). We have cloned several PKS genes from the fungus Xylaria sp. BCC 1067, which produces two polyketides: depudecin (reduced PK) and 19,20-epoxycytochalasin Q (PK-nonribosomal peptide (NRP) hybrid). Two new degenerate primer sets, KA-series and XKS, were designed to amplify reducing PKS and PKS-NRP synthetase hybrid genes, respectively. Five putative PKS genes were amplified in Xylaria using KA-series primers and two more with the XKS primers. All seven are predicted to encode proteins homologous to highly reduced (HR)-type PKSs. Previously designed primers in LC-, KS-, and MT-series identified four additional PKS gene fragments. Selected PKS fragments were used as probes to identify PKS genes from the genomic library of this fungus. Full-length sequences for five PKS genes were obtained: pks12, pks3, pksKA1, pksMT, and pksX1. They are structurally diverse with 1-9 putative introns and products ranging from 2162 to 3654 amino acids in length. The finding of 11 distinct PKS genes solely by means of PCR cloning supports that PKS genes are highly diverse in fungi. It also indicates that our KA-series primers can serve as powerful tools to reveal the genetic potential of fungi in production of multiple types of HR PKs, which the conventional compound screening could underestimate.
Asunto(s)
Ascomicetos/genética , Variación Genética , Sintasas Poliquetidas/genética , Polimorfismo Genético , Ascomicetos/enzimología , Cartilla de ADN , ADN de Hongos/química , ADN de Hongos/genética , Proteínas Fúngicas/genética , Intrones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
A novel secondary metabolite, pughiinin A, together with pycnidione, mevalonolactone, and 7-hydroxy-2-methylchromanone, was isolated from the seed fungus Kionochaeta pughii BCC 3878. The chemical structure was established by spectroscopic methods and by single crystal X-ray crystallography. Pughiinin A and pycnidione exhibited in vitro antiplasmodial activity against Plasmodium falciparum (K1 strain). Pycnidione also showed anti-cancer activity against KB and BC cell lines with the IC 50 values of 2.0 and 1.6 microg/mL, respectively.