Short-Term Evaluation in Growing Rats of Diet Containing Bacillus thuringiensis Cry1Ia12 Entomotoxin: Nutritional Responses and Some Safety Aspects

The Cry1Ia12 entomotoxin from a Brazilian Bacillus thuringiensis strain is currently being expressed in cotton cultivars to confer resistance to insect-pests. The present study aimed to assess the effects of a diet containing Cry1Ia12 protein on growing rats. A test diet containing egg white and Cry1Ia12 (0.1% of total protein) as a protein source was offered to rats for ten days. In addition, an acute toxicity bioassay was performed in rats with a single oral dose of the entomotoxin (12 mg/animal). No adverse effects were observed in the animals receiving the test diet when compared to those receiving a control diet (egg white). The analysed parameters included relative dry weight of internal organs, duodenum histology, blood biochemistry, and nutritional parameters. The results of the acute toxicity test showed no mortality or behaviour alteration. Thus, Cry1Ia12 toxin at the tested concentration does not cause deleterious effects on growing rats when incorporated in the diet for 10 days

Novel homologous lactate transporter improves l‑lactic acid production from glycerol in recombinant strains of Pichia pastoris

Background: Crude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous l-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for l-lactic acid production. Results: Batch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g. Conclusions: We showed that P. pastoris has a great potential as a fermentative organism for producing l-lactic acid using glycerol as the carbon source at limited oxygenation conditions (below 0.05 % DO in the bioreactor). The best strain had both the LDHb and the homologous lactate transporter encoding genes expressed, and reached a titer 1.5 times higher than the strain with the S. cerevisiae transporter. Finally, it was also shown that increased lactic acid production was concomitant to reduction of acetic acid formation by half

Plant Ureases and Related Peptides: Understanding Their Entomotoxic Properties

Recently, ureases were included in the arsenal of plant defense proteins, alongside many other proteins with biotechnological potential such as insecticides. Isoforms of Canavalia ensiformis urease (canatoxin—CNTX and jack bean urease—JBURE-I) are toxic to insects of different orders. This toxicity is due in part to the release of a 10 kDa peptide from the native protein, by cathepsin-like enzymes present in the insect digestive tract. The entomotoxic peptide, Jaburetox-2Ec, exhibits potent insecticidal activity against several insects, including many resistant to the native ureases. JBURE-I and Jaburetox-2Ec cause major alterations of post-feeding physiological processes in insects, which contribute to, or can be the cause of, their entomotoxic effect. An overview of the current knowledge on plant urease processing and mechanisms of action in insects is presented in this review

Ureases de Canavalia ensiformis e peptídeo inseticida derivado

Urease, uma enzima encontrada em diversas espécies de plantas, catalisa a hidrólise de uréia, formando amônia e dióxido de carbono. Esta enzima é encontrada também em bactérias, fungos e alguns invertebrados e apresenta três domínios: alfa, beta e gama. Em Canavalia ensiformis, foram descritas mais de uma isoforma de urease: a urease clássica JBU (Jack bean urease - cDNA e proteína), o cDNA jbure-II e a proteína canatoxina. A sequência jbure-II, obtida previamente, codificava uma proteína hipotética incompleta nos domínios alfa e gama (correspondentes as regiões terminais 5´ e 3´ do cDNA). Neste trabalho, demonstramos a clonagem de um cDNA denominado jbure-IIB, que codifica uma proteína predita com os domínios completos. Análises filogenéticas e modelagem molecular da proteína predita foram realizadas. A estrutura proposta para a proteína hipotética JBURE-IIB possui uma forma similar às das ureases bacterianas, exceto pela presença de duas regiões de ligação, que conectam os três domínios das ureases, ausentes nas ureases bacterianas. Todos os resíduos críticos para a atividade ureásica foram detectados. Em seguida, o cDNA completo de jbure-IIB foi obtido através de sobreposição dos fragmentos clonados (5’, interno e 3’), utilizando uma técnica de “PCR overlap” modificada. O cDNA foi clonado no vetor pET101, a proteína heteróloga foi produzida em Escherichia coli e sua presença confirmada por análises de Western blot. A atividade da urease recombinante foi observada em placas das colônias transformadas induzidas, na presença de uréia e níquel. A proteína canatoxina apresenta atividade inseticida contra diferentes espécies de insetos. Sua toxicidade depende da liberação de um peptídeo interno de 10 kDa (pepcanatox) pelas catepsinas do sistema digestivo dos insetos suscetíveis. Baseado na seqüência N-terminal e tamanho de pepcanatox, projetamos oligonucleotideos para amplificar um fragmento de 270 pb, usando o cDNA jbure-II como molde. Este fragmento, denominado jaburetox-2 (“Jack bean urease toxin 2”) foi clonado em vetor pET101 e expresso em E. coli. O peptídeo recombinante foi denominado jaburetox-2Ec (“Jack bean urease toxin 2” expresso em células de E. coli, contendo uma metionina inicial e acrescido, na região Cxiii terminal, do epitopo V-5 e seis resíduos de Histidina). Sua atividade inseticida foi testada contra ninfas de Dysdercus peruvianus e larvas de Spodoptera frugiperda, obtendo-se 100% de mortalidade. Em seguida, cultivou-se a bactéria recombinante em biorreatores para obtenção de grandes quantidades de peptídeo, que foi purificado e testado contra Rhodnius prolixus (4º instars) e Triatoma infestans (5º instars e adultos). A injeção de jaburetox-2Ec (1 μg/mg de peso vivo do inseto) resultou em 100% de mortalidade. Em contraste, altas doses do peptídeo foram inócuas quando injetadas ou ingeridas por ratos neonatos e camundongos. Corroborando com estes resultados, a modelagem molecular “ab initio” de jaburetox-2Ec revelou um motivo de “grampo beta” consistente com atividade inseticida, possivelmente baseada em neurotoxicidade ou alteração de permeabilidade celular. Adicionalmente, plantas de tabaco foram transformadas com o vetor pCAMBIA contendo o fragmento de cDNA jaburetox-2 (acrescido de um códon de iniciação e um códon de terminação da tradução) e as plantas transformadas, PCR positivas, foram testadas contra S. frugiperda. Realizou-se alimentação direta com as folhas de tabaco e observaram-se diferentes níveis de mortalidade (50-100%), provocadas por diferentes plantas, após 15-30 dias. O conjunto de dados desta tese demonstra o potencial uso de jaburetox-2 como um transgene para construção de plantas resistentes a insetos e contribui para elucidação do mecanismo de ação da atividade inseticida de ureases, bem como seu possível papel na defesa da planta.Urease, an ubiquitous enzyme in plants, catalyzes the hydrolysis of urea to form ammonia and carbon dioxide. Urease is also found in bacteria, fungi and some invertebrates. In Canavalia ensiformis there are more than one isoform of urease: the classic JBU (the major isoform), JBURE-II and the protein Canatoxin. In a previous study, a partial sequence of jbure-II was obtained that putatively codify for an enzyme incomplete at the alfa and gamma domains. In this work, we report the cloning of a cDNA named jbure-IIB, encoding a complete urease protein with the expected 90 kDa size. Phylogenetic studies of the urease sequence and the molecular modeling of the putative protein are also presented. Its modeled structure has an overall shape similar to that of related bacterial ureases except for the presence of two linking regions that connect the three domains of ureases. All critical residues for urease activity are present. The complete cDNA was obtained by overlapping the three parts of cloned cDNA (5’, middle part and 3’) using a modified PCR overlap technique. The cDNA was cloned into PET101 vector and the heterologous protein was produced in Escherichia coli cells and confirmed by Western blot analyses. The activity of the recombinant urease was observed in a urea segregation agar containing urea and nickel. Canatoxin displays insecticidal activity against different insect species. The entomotoxicity relies on an internal 10 kDa peptide (pepcanatox), released by hydrolysis of Canatoxin by cathepsins in the digestive system of susceptible insects. Here, based on the N-terminal sequence of pepcanatox, we designed primers to amplify by PCR a 270-bp fragment corresponding to pepcanatox using jbure-II cDNA as a template. This amplicon named jaburetox-2 (“jack bean urease toxin 2”) was cloned into pET101 vector and expressed in E. coli. The recombinant peptide was named jaburetox-2Ec and its insecticidal effect was demonstrated against Dysdercus peruvianus and Spodoptera frugiperda larvaes, in which it induced 100% mortality. Bacterial cultivation in bioreactors was carried out with lactose as inducer to obtain large amounts of recombinant peptide. It was tested against Rhodnius prolixus (4th instars) and Triatoma infestans (5th instars and xv adults) by injection and 1 μg/mg insect body weight resulted in 100% mortality. In contrast, high doses of jaburetox-2Ec were innocuous when injected or ingested by mice and neonate rats. Modeling of jaburetox-2Ec, and comparison with other peptide structures, revealed a prominent β-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or alteration of cell permeability. Finally, tobacco plants were transformed with pCAMBIA vector containing the jaburetox-2 fragment (with a start and a stop codon) and the transformed plants were tested against S. frugiperda. Mortality varying from 50-100% of the insects feeding on different transformed plants was observed after 15-30 days. Our results showed the potential use of jaburetox-2 as transgene to engineering insect resistance into plant and contribute to elucidation of mechanisms of action of urease insecticidal activity, as well as its possible role in plant defense

Presença de soyatoxina, uma proteína tóxica de soja (Glycine max) em amostras de SBTI comercial

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