Platforms for Production of Protein-Based Vaccines: From Classical to Next-Generation Strategies

To date, vaccination has become one of the most effective strategies to control and reduce infectious diseases, preventing millions of deaths worldwide. The earliest vaccines were developed as live-attenuated or inactivated pathogens, and, although they still represent the most extended human vaccine types, they also face some issues, such as the potential to revert to a pathogenic form of live-attenuated formulations or the weaker immune response associated with inactivated vaccines. Advances in genetic engineering have enabled improvements in vaccine design and strategies, such as recombinant subunit vaccines, have emerged, expanding the number of diseases that can be prevented. Moreover, antigen display systems such as VLPs or those designed by nanotechnology have improved the efficacy of subunit vaccines. Platforms for the production of recombinant vaccines have also evolved from the first hosts, Escherichia coli and Saccharomyces cerevisiae, to insect or mammalian cells. Traditional bacterial and yeast systems have been improved by engineering and new systems based on plants or insect larvae have emerged as alternative, low-cost platforms. Vaccine development is still time-consuming and costly, and alternative systems that can offer cost-effective and faster processes are demanding to address infectious diseases that still do not have a treatment and to face possible future pandemics

Main Variables Affecting a Chemical-Enzymatic Method to Obtain Protein and Amino Acids from Resistant Microalgae

he development of microalgae uses requires further investigation in cell disruption alternatives to reduce the costs associated to this processing stage. This study aimed to evaluate the main variables affecting an extraction method to obtain protein and amino acids from microalgae. The method was based on a sequential alkaline-enzymatic process, with separate extractions and noncontrolled pH, and was applied to fresh biomass of a resistant species. The processed microalgae were composed of a consortium with Nannochloropsis sp. as predominant species. After the optimization of the pH of the alkaline reaction, the effect of the time of the alkaline reaction (30-120min), the time (30-120min) and temperature (40-60 degrees C) of the enzymatic reaction, and the biomass concentration (50-150mgml(-1)), on the extraction yields of protein and free amino nitrogen (FAN) and on the final concentration of protein in the extract, was studied using a response surface methodology. Even though all the variables and some interactions among them had a significant effect, the biomass concentration was the most important factor affecting the overall process. The results showed relevant information about the different options in order to maximize not only the response variables individually but also different combinations of them. Assays with optimized values reached maximum yields of 80.3% and 1.07% of protein (% of total protein) and FAN (% of total biomass), respectively, and a protein concentration in the extract of 15.2mgml(-1). The study provided the essential information of an alternative approach to obtain protein and amino acids from fresh biomass of resistant microalgae with a high yield, also opening perspectives for further research in particular aspects

Metabolic engineering for the optimization of hydrogen production in Escherichia coli: A review

El hidrógeno es una fuente potencial de energía sostenible y podría convertirse en una alternativa a la combustión de combustibles fósiles, contribuyendo así a reducir las emisiones de gases de efecto invernadero. La producción biológica de hidrógeno, en lugar de su síntesis química, es una posibilidad prometedora, ya que este proceso requiere menos energía y es más sostenible y respetuoso con el medio ambiente. Se han utilizado varios microorganismos con este fin, pero Escherichia coli es uno de los más utilizados en este campo. La literatura en esta área ha aumentado exponencialmente en los últimos 10 años y se han reportado varias estrategias en un esfuerzo por mejorar la producción de hidrógeno. En este trabajo, el estado del arte de la biosíntesis de hidrógeno por E. coli y Metab. Eng. para mejorar la producción de hidrógeno. Este trabajo incluye una discusión sobre los complejos hidrogenasa responsables de la síntesis de hidrógeno en este microorganismo y las vías centrales del metabolismo del carbono relacionadas con este proceso. Las principales estrategias Metab. Eng. aplicadas, como la expresión génica heteróloga, la evolución adaptativa y la ingeniería metabólica y proteica. Por otra parte, también se han considerado las condiciones de cultivo, incluido el uso de fuentes de carbono como el glicerol, la glucosa o los residuos orgánicos. También se comparan los rendimientos y productividades de las cepas de ingeniería más relevantes de las que se tiene constancia que utilizan diversas fuentes de carbono

Versatile method to obtain protein- and/or amino acid-enriched extracts from fresh biomass of recalcitrant microalgae without mechanical pretreatment

The aim of the study was to develop a sequential alkaline-enzymatic method to obtain protein- and/or amino acid-enriched extracts from fresh biomass of recalcitrant microalgae without any supplementary pretreatment. The effects of the initial biomass concentration, the use of freeze-dried or fresh biomass, enzyme dosage, processing procedure (two-step and single-step, with and without pH control) and species were studied. The method was evaluated with a consortium of microalgae isolated from a landfill leachate and was tested on other recognized recalcitrant microalgae such as Chlorella vulgaris, Nannochloropsis gaditana and Scenedesmus obliquus. The approach includes alternative pathways, provides high extraction yields of proteinaceous material and produces protein- and/or hydrolyzed peptide-enriched extracts with different amino acid compositions (e.g., a pathway without pH control achieves a yield of 81% of total protein and a concentration of 29 mg mL−1 of proteinaceous material). The versatility in processing procedures and the range of products obtained, along with applicability to different microalgal species, make this method an interesting option for algae biomass treatment. Furthermore, the high yield and simplicity from a technological point of view, gives it a great potential for process development and encourages further research for a wide variety of applications, such as feed, biostimulants, culture media, bulk chemicals, and biogas

Estudio de la temperatura del suelo en Puerto Rico.

Soil temperature measurements from a climate monitoring network in Puerto Rico were evaluated and the difference between mean summer and mean winter soil temperature, known as isotivity value, was calculated. Air and soil temperature was collected from five weather stations of the USDA-Natural Resources Conservation Service from sea level to 1,019 m above sea level and from different soil moisture regimes. Isotivity values ranged from 1.2 to 3.9° C with an average of 2.6° C. The 750-m elevation was identified as the limit between the isohyperthermic and isothermic soil temperature regimes in the perudic soil moisture regime in Puerto Rico. The greatest differences between mean annual soil temperature and mean annual air temperature were observed at Guánica, Combate and Guilarte (2.1 ° C) stations. The smallest differences were observed at Maricao (0.8° C) and Isabela (1.8° C) stations. The study also indicated that the mean annual soil temperature in Puerto Rico can be estimated by adding 1.8° C to the mean annual air temperature or by the equation y = -0.007x + 28.0° C. The equation indicates that 97 percent of the time the behavior of the mean annual soil temperature is a function of elevation. According to the updated soil temperature regime boundaries, eight soil series were established in the Soil Survey of San Germán Area. In an area under the isothermic soil temperature regime, four soil series were classified as Oxisols (Haploperox), two soil series as Inceptisols (Eutrudepts) and two soil series as Mollisols (Argiudolls). This is the first field recognition of the Haploperox soil great group in the United States and its territories.Se evaluaron mediciones de temperatura del suelo de la red de monitoreo de clima en Puerto Rico y se calculó la diferencia entre la temperatura promedio del suelo de verano y de invierno, conocida como valor de isotividad. Se tomaron datos de temperatura del aire y del suelo de cinco estaciones climatológicas del Servicio de Conservación de Recursos Naturales del Departamento de Agricultura de los Estados Unidos desde el nivel del mar hasta 1,019 m de elevación sobre el nivel del mar y a diferentes regímenes de humedad del suelo. Los valores de isotividad oscilaron entre 1.2 y 3.9° C con una media de 2.6° C. El límite entre el régimen de temperatura de suelo isohipertérmico e isotérmico en el régimen de humedad perúdico en Puerto Rico fue identificado a una elevación de 750 m. En las estaciones de Guánica, Combate y Guilarte se observaron las mayores diferencias entre la media anual de la temperatura del suelo y la media anual de la temperatura del aire (2.1° C). En las estaciones de Maricao (0.8° C) e Isabela (1.8° C) se observaron las menores diferencias. El estudio también indica que la media anual de la temperatura del suelo en Puerto Rico puede ser estimada añadiéndole 1.8° C a la media anual de la temperatura del aire o por la ecuación y = -0.007x + 28.0° C. La ecuación indica que el 97 por ciento del tiempo el comportamiento de la media anual de la temperatura del suelo es una función de la elevación. De acuerdo con la actualización de los límites de los regímenes de temperatura de suelo, se establecieron ocho series de suelo en el Catastro de Suelos del Área de San Germán. Cuatro series de suelo clasificaron como Oxisoles (Haploperox), dos series de suelos como Inceptisoles (Eutrudepts) y dos series de suelos como Molisoles (Argiudolls) en el área bajo régimen de temperatura isotérmico. Este es el primer reconocimiento en el campo del gran grupo de suelos Haploperox en los Estados Unidos de América y sus territorios

Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2′-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties

Benzohydroxamic acids, such as 4-hydroxy-(2H)- 1,4-benzoxazin-3(4H)-one (D-DIBOA), exhibit interesting herbicidal, fungicidal and bactericidal properties. Recently, the chemical synthesis of D-DIBOA has been simplified to only two steps. In a previous paper, we demonstrated that the second step could be replaced by a biotransformation using Escherichia coli to reduce the nitro group of the precursor, ethyl 2-(2′-nitrophenoxy)acetate and obtain D-DIBOA. The NfsA and NfsB nitroreductases and the NemA xenobiotic reductase of E. coli have the capacity to reduce one or two nitro groups from a wide variety of nitroaromatic compounds, which are similar to the precursor. By this reason, we hypothesised that these three enzymes could be involved in this biotransformation. We have analysed the biotransformation yield (BY) of mutant strains in which one, two or three of these genes were knocked out, showing that only in the double nfsA/nfsB and in the triple nfsA/nfsB/nemA mutants, the BY was 0%. These results suggested that NfsA and NfsB are responsible for the biotransformation in the tested conditions. To confirm this, the nfsA and nfsB open reading frames were cloned into the pBAD expression vector and transformed into the nfsA and nfsB single mutants, respectively. In both cases, the biotransformation capacity of the strains was recovered (6.09±0.06% as in the wild-type strain) and incremented considerably when NfsA and NfsB were overexpressed (40.33%±9.42% and 59.68%±2.0% respectively)

Nitric oxide synthase-dependent immune response against gram negative bacteria in a crustacean, Litopenaeus vannamei

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In previous studies, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide in vitro e in vivo. Hyperimmune serum was obtained from rabbits immunized with a P. argus eNOS fragment of 31 kDa produced in Escherichia coli, which specifically detected the recombinant polypeptide and the endogenous NOS from lobster hemocytes by western blotting and immunofluorescence. In the present work, we demonstrate that the hyperimmune serum obtained against P. argus NOS also recognizes Litopenaeus vannamei NOS in hemocytes by western blotting and immunofluorescence. Our data also show that while the hemolymph of L. vannamei has a strong antibacterial activity against the Gram negative bacteria Aeromonas hydrophila, the administration of the anti NOS serum reduce the natural bacterial clearance. These results strongly suggest that NOS is required for the shrimp immune defense toward Gram negative bacteria. Therefore, the monitoring of induction of NOS could be an important tool for testing immunity in shrimp farming

Overexpression of the nitroreductase NfsB in an E. coli strain as a whole-cell biocatalyst for the production of chlorinated analogues of the natural herbicide DIBOA

Los ácidos benzohidroxámicos, como el DIBOA (2,4-dihidroxi-2 H)-1,4-benzoxazin-3(4 H)-ona), son productos vegetales que presentan interesantes propiedades herbicidas, fungicidas y bactericidas. Una alternativa viable a su purificación a partir de fuentes naturales es la síntesis de compuestos análogos como el D-DIBOA (2-deoxi-DIBOA) y sus derivados clorados. Su síntesis química se ha simplificado en dos pasos. Sin embargo, el segundo paso es una reacción exotérmica e implica la liberación de hidrógeno, lo que hace que esta metodología sea cara y difícil de ampliar. En el presente estudio se estudia la posibilidad de producir los derivados clorados de las benzoxazinonas mediante un proceso biocatalítico in vivo que utiliza la capacidad de la cepa de E. coli nfsB-/pBAD-NfsB modificada para catalizar la síntesis de 6-Cl- D-DIBOA y 8-Cl- D-DIBOA a partir de sus respectivos precursores (PCs). Los resultados muestran que esta cepa es capaz de crecer en medios que contienen estos compuestos y producir el producto de biotransformación con unos rendimientos del 59,3% y el 46,7%. respectivamente. Además, la cepa es capaz de procesarel precursor (PC) no purificados del primer paso químico para obtener rendimientos similares a los obtenidos a partir del PC purificado. Se estudió la cinética de la reacción in in vitro con la nitrorreductasa NfsB recombinante purificada para caracterizar la catálisis y evaluar los efectos que varios componentes del precursor no purificado tiene durante el proceso de síntesis. Los resultados revelaron que la cinética es la de una enzima alostérica. El efecto inhibitorio del sustrato en el primer paso de la síntesis química, se encuentra también en algunas etapas previas a la purificación del precursor

Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in Escherichia coli

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production

A genetically engineered Escherichia coli strain overexpressing the nitroreductase NfsB is capable of producing the herbicide D‑DIBOA with 100% molar yield

Background The use of chemical herbicides has helped to improve agricultural production, although its intensive use has led to environmental damages. Plant allelochemicals are interesting alternatives due to their diversity and degradability in the environment. However, the main drawback of this option is their low natural production, which could be overcome by its chemical synthesis. In the case of the allelochemical DIBOA ((2,4-dihydroxy-2H)-1,4-benzoxazin-3(4H)-one), the synthesis of the analogous compound D-DIBOA (2-deoxy-DIBOA) has been achieved in two steps. However, the scale up of this synthesis is hindered by the second step, which uses an expensive catalyst and is an exothermic reaction, with hydrogen release and a relatively low molar yield (70%). We have previously explored the “Green Chemistry” alternative of using E. coli strains overexpressing the nitroreductase NfsB as a whole-cell-biocatalyst to replace this second step, although the molar yield in this case was lower than that of the chemical synthesis. Results In this work, we engineered an E. coli strain capable of carrying out this reaction with 100% molar yield and reaching a D-DIBOA concentration up to 379% respect to the highest biotransformation yield previously reported. This was achieved by a screening of 34 E. coli mutant strains in order to improve D-DIBOA production that led to the construction of the ΔlapAΔfliQ double mutant as an optimum genetic background for overexpression of the NfsB enzyme and D-DIBOA synthesis. Also, the use of a defined medium instead of a complex one, the optimization of the culture conditions and the development of processes with several substrate loads allowed obtaining maxima yields and concentrations. Conclusions The high yields and concentrations of D-DIBOA reached by the microbial-cell-factory approach developed in this work will facilitate its application to industrial scale. Also, the use of an optimized defined medium with only an organic molecule (glucose as carbon and energy source) in its composition will also facilitate the downstream processes