Comparative analysis of the Squamosa Promoter Binding-Like (SPL) gene family in Nicotiana benthamiana and Nicotiana tabacum.

SQUAMOSA PROMOTER BINDING-LIKE (SPL) proteins constitute a large family of transcription factors known to play key roles in growth and developmental processes, including juvenile-to-adult and vegetative-to-reproductive phase transitions. This makes SPLs interesting targets for precision breeding in plants of the Nicotiana genus used as e.g. recombinant biofactories. We report the identification of 49 SPL genes in Nicotiana tabacum cv. K326 and 43 SPL genes in Nicotiana benthamiana LAB strain, which were classified into eight phylogenetic groups according to the SPL classification in Arabidopsis. Exon-intron gene structure and DNA-binding domains were highly conserved between homeologues and orthologues. Thirty of the NbSPL genes and 33 of the NtSPL genes were found to be possible targets of microRNA 156. The expression of SPL genes in leaves was analysed by RNA-seq at three different stages, revealing that genes not under miR156 control were in general constitutively expressed at high levels, whereas miR156-regulated genes showed lower expression, often developmentally regulated. We selected the N. benthamiana SPL13_1a gene as target for a CRISPR/Cas9 knock-out experiment. We show here that a full knock-out in this single gene leads to a significant delay in flowering time, a trait that could be exploited to increase biomass for recombinant protein production.

The apocarotenoid production in microbial biofactories: An overview.

Carotenoids are a vast group of natural pigments that come in a variety of colors ranging from red to orange. Apocarotenoids are derived from these carotenoids, which are hormones, pigments, retinoids, and volatiles employed in the textiles, cosmetics, pharmaceutical, and food industries. Due to the high commercial value and poor natural host abundance, they are significantly undersupplied. Microbes like Saccharomyces cerevisiae and Escherichia coli act as heterologous hosts for apocarotenoid production. This article briefly reviews categories of apocarotenoids, their biosynthetic pathway commencing from the MVA and MEP, its significance, the tool enzymes for apocarotenoid biosynthesis like CCDs, their biotechnological production in microbial factories, and future perspectives.

Production of monoclonal antibodies for therapeutic purposes: A review.

Monoclonal antibodies (mAbs) have been used in the development of immunotherapies that target a variety of diseases, such as cancer, autoimmune diseases, and even viral infections; they play a key role in immunization and are expected after vaccination. However, some conditions do not promote the development of neutralizing antibodies. Production and use of mAbs, generated in biofactories, represent vast potential as aids in immunological responses when the organism cannot produce them on their own, these convey unique specificity by recognizing and targeting specific antigen. Antibodies can be defined as heterotetrametric glycoproteins of symmetric nature, and they participate as effector proteins in humoral responses. Additionally, there are different types of mAbs (murine, chimeric, humanized, human, mAbs as Antibody-drug conjugates and bispecific mAbs) discussed in the present work. When these molecules are produced in vitro as mAbs, several common techniques, such as hybridomas or phage display are used. There are several preferred cell lines that function as biofactories, for the production of mAbs, the selection of which rely on the variation of adaptability, productivity and both phenotypic and genotypic shifts. After the cell expression systems and culture techniques are used, there are diverse specialized downstream processes to achieve desired yield and isolation as well as product quality and characterization. Novel perspectives regarding these protocols represent a potential improvement for mAbs high-scale production.

Hypes, hopes, and the way forward for microalgal biotechnology.

The urge for food security and sustainability has advanced the field of microalgal biotechnology. Microalgae are microorganisms able to grow using (sun)light, fertilizers, sugars, CO2, and seawater. They have high potential as a feedstock for food, feed, energy, and chemicals. Microalgae grow faster and have higher areal productivity than plant crops, without competing for agricultural land and with 100% efficiency uptake of fertilizers. In comparison with bacterial, fungal, and yeast single-cell protein production, based on hydrogen or sugar, microalgae show higher land-use efficiency. New insights are provided regarding the potential of microalgae replacing soy protein, fish oil, and palm oil and being used as cell factories in modern industrial biotechnology to produce designer feed, recombinant proteins, biopharmaceuticals, and vaccines.

Plastid Transformation: New Challenges in the Circular Economy Era.

In a circular economy era the transition towards renewable and sustainable materials is very urgent. The development of bio-based solutions, that can ensure technological circularity in many priority areas (e.g., agriculture, biotechnology, ecology, green industry, etc.), is very strategic. The agricultural and fishing industry wastes represent important feedstocks that require the development of sustainable and environmentally-friendly industrial processes to produce and recover biofuels, chemicals and bioactive molecules. In this context, the replacement, in industrial processes, of chemicals with enzyme-based catalysts assures great benefits to humans and the environment. In this review, we describe the potentiality of the plastid transformation technology as a sustainable and cheap platform for the production of recombinant industrial enzymes, summarize the current knowledge on the technology, and display examples of cellulolytic enzymes already produced. Further, we illustrate several types of bacterial auxiliary and chitinases/chitin deacetylases enzymes with high biotechnological value that could be manufactured by plastid transformation.

Optimization of Vectors and Targeting Strategies Including GoldenBraid and Genome Editing Tools: GoldenBraid Assembly of Multiplex CRISPR /Cas12a Guide RNAs for Gene Editing in Nicotiana benthamiana.

New breeding techniques, especially CRISPR/Cas, could facilitate the expansion and diversification of molecular farming crops by speeding up the introduction of new traits that improve their value as biofactories. One of the main advantages of CRISPR/Cas is its ability to target multiple loci simultaneously, a key feature known as multiplexing. This characteristic is especially relevant for polyploid species, as it is the case of Nicotiana benthamiana and other species of the same genus widely used in molecular farming. Here, we describe in detail the making of a multiplex DNA construct for genome editing in N. benthamiana using the GoldenBraid modular cloning platform. In this case, the procedure is adapted for the requirements of LbCas12a (Lachnospiraceae bacterium Cas12a), a nuclease whose cloning strategy differs from that of the more often used SpCas9 (Streptococcus pyogenes Cas9) enzyme. LbCas12a-mediated edition has several advantages, as its high editing efficiency, described for different plant species, and its T/A-rich PAM sequence, which expands the range of genomic loci that can be targeted by site-specific nucleases. The protocol also includes recommendations for the selection of protospacer sequences and indications for the analysis of editing results.

Compendium on Food Crop Plants as a Platform for Pharmaceutical Protein Production.

Tremendous advances in crop biotechnology related to the availability of molecular tools and methods developed for transformation and regeneration of specific plant species have been observed. As a consequence, the interest in plant molecular farming aimed at producing the desired therapeutic proteins has significantly increased. Since the middle of the 1980s, recombinant pharmaceuticals have transformed the treatment of many serious diseases and nowadays are used in all branches of medicine. The available systems of the synthesis include wild-type or modified mammalian cells, plants or plant cell cultures, insects, yeast, fungi, or bacteria. Undeniable benefits such as well-characterised breeding conditions, safety, and relatively low costs of production make plants an attractive yet competitive platform for biopharmaceutical production. Some of the vegetable plants that have edible tubers, fruits, leaves, or seeds may be desirable as inexpensive bioreactors because these organs can provide edible vaccines and thus omit the purification step of the final product. Some crucial facts in the development of plant-made pharmaceuticals are presented here in brief. Although crop systems do not require more strictly dedicated optimization of methodologies at any stages of the of biopharmaceutical production process, here we recall the complete framework of such a project, along with theoretical background. Thus, a brief review of the advantages and disadvantages of different systems, the principles for the selection of cis elements for the expression cassettes, and available methods of plant transformation, through to the protein recovery and purification stage, are all presented here. We also outline the achievements in the production of biopharmaceuticals in economically important crop plants and provide examples of their clinical trials and commercialization.

Plant-Based Systems for Vaccine Production.

Plant systems have been used as biofactories to produce recombinant proteins since 1983. The huge amount of data, collected so far in this framework, suggests that plants display several key advantages over existing traditional platforms when they are intended for therapeutic uses, including safety, scalability, and the speed in obtaining the final product.Here, we describe a method that could be applied for the expression and production of a candidate subunit vaccine in Nicotiana benthamiana plants by transient expression, defining all the protocols starting from plant cultivation to target recombinant protein purification.

The C-Terminal Half of SARS-CoV-2 Nucleocapsid Protein, Industrially Produced in Plants, Is Valid as Antigen in COVID-19 Serological Tests.

BACKGROUND: The fight against the current coronavirus disease 2019 (COVID-19) pandemic has created a huge demand of biotechnological, pharmaceutical, research and sanitary materials at unprecedented scales. One of the most urgent demands affects the diagnostic tests. The growing need for rapid and accurate laboratory diagnostic tests requires the development of biotechnological processes aimed at producing reagents able to cope with this demand in a scalable, cost-effective manner, with rapid turnaround times. This is particularly applicable to the antigens employed in serological tests. Recombinant protein expression using plants as biofactories is particularly suitable for mass production of protein antigens useful in serological diagnosis, with a neat advantage in economic terms. METHODS: We expressed a large portion of the nucleoprotein (N) derived from SARS-CoV-2 in Nicotiana benthamiana plants. After purification, the recombinant N protein obtained was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to SARS-CoV-2 in human sera. To validate the ELISA, a panel of 416 sera from exposed personnel at essential services in Madrid City Council were tested, and the results compared to those obtained by another ELISA, already validated, used as reference. Furthermore, a subset of samples for which RT-PCR results were available were used to confirm sensitivity and specificity of the test. RESULTS: The performance of the N protein expressed in plants as antigen in serologic test for SARS-CoV-2 antibody detection was shown to be highly satisfactory, with calculated diagnostic sensitivity of 96.41% (95% CI: 93.05-98.44) and diagnostic specificity of 96.37 (95% CI: 93.05-98.44) as compared to the reference ELISA, with a kappa (K) value of 0.928 (95% CI:0.892-0.964). Furthermore, the ELISA developed with plant-derived N antigen detected SARS-CoV-2 antibodies in 84 out of 93 sera from individuals showing RT-PCR positive results (86/93 for the reference ELISA). CONCLUSION: This study demonstrates that the N protein part derived from SARS-CoV-2 expressed in plants performs as a perfectly valid antigen for use in COVID-19 diagnosis. Furthermore, our results support the use of this plant platform for expression of recombinant proteins as reagents for COVID-19 diagnosis. This platform stands out as a convenient and advantageous production system, fit-for-purpose to cope with the current demand of this type of biologicals in a cost-effective manner, making diagnostic kits more affordable.

Pilot Production of SARS-CoV-2 Related Proteins in Plants: A Proof of Concept for Rapid Repurposing of Indoor Farms Into Biomanufacturing Facilities.

The current CoVid-19 crisis is revealing the strengths and the weaknesses of the world's capacity to respond to a global health crisis. A critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. On the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions. In this work, we explore how, in a context of rapid exchange of scientific information, plant biofactories can serve as a rapid and easily adaptable solution for local manufacturing of bioreagents, more specifically recombinant antibodies. For this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against SARS-CoV-2 in Nicotiana benthamiana. For the design of the antibodies, we took advantage, among other data sources, of the DNA sequence information made rapidly available by other groups in preprint publications. mAbs were engineered as single-chain fragments fused to a human gamma Fc and transiently expressed using a viral vector. In parallel, we also produced the recombinant SARS-CoV-2 N protein and the receptor binding domain (RBD) of the Spike protein in planta and used them to test the binding specificity of the recombinant mAbs. Finally, for two of the antibodies, we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. Our results indicate that gram amounts of anti-SARS-CoV-2 antibodies could be easily produced in little more than 6 weeks in repurposed greenhouses with little infrastructure requirements using N. benthamiana as production platform. Similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid therapeutic responses.

Aplicaciones biotecnológicas de los microorganismos / Biotechnological applications of microorganisms

Resumen La biodiversidad de los microorganismos así como la naturaleza única y las capacidades biosintéticas en condiciones ambientales específicas hacen que los microorganismos sean los probables candidatos para resolver problemas de escases de alimentos, contro de plagas, biodegradación de los xenobióticos, descomposición de la basura, las pilas de desechos producidas, entre otros. Los microorganismos ofrecen un gran potencial para la exploración de moléculas y procesos, y el conocimiento de las especies no convencionales, especialmente dentro del grupo Archaea, ha estimulado la investigación molecular de genes de interés. Estos nuevos genes pueden incorporarse mediante tecnología recombinante en especies biológicamente conocidas, como E. coli y S. cerevisiae, para la síntesis a gran escala de productos. La microbiología tecnológica tiene grandes potenciales para explorar y obstáculos por superar. Por lo tanto, solo la investigación en esta área resulta prometedora para científicos en todo el mundo. En la presente revisión se presentan las aplicaciones más significativas de los microorganismos en la industria de alimentos, la agricultura, compuestos químicos, combustibles, farmacología y materiales.

Abstract The biodiversity of microorganisms as well as the unique nature and biosynthetic capabilities in specific environmental conditions make microorganisms the likely candidates to solve problems of food shortages, pest control, biodegradation of xenobiotics, decomposition of garbage, batteries of produced waste, among others. Microorganisms offer great potential for the exploration of molecules and processes, and knowledge of non-conventional species, especially within the Archaea group, has stimulated the molecular investigation of genes of interest. These new genes can be incorporated by recombinant technology into biologically known species, such as E. coli and S. cerevisiae, for the large-scale synthesis of products. Technological microbiology has great potentials to explore and obstacles to overcome. Therefore, only research in this area is promising for scientists around the world. In this review we present the most significant applications of microorganisms in the food industry, agriculture, chemical compounds, fuels, pharmacology and materials.

Genomic methylation in plant cell cultures: A barrier to the development of commercial long-term biofactories.

Plant cell biofactories offer great advantages for the production of plant compounds of interest, although certain limitations still need to be overcome before their maximum potential is reached. One obstacle is the gradual loss of secondary metabolite production during in vitro culture maintenance, which is an important impediment in the development of large-scale production systems. The relationship between in vitro maintenance and epigenetic changes has been demonstrated in several plant species; in particular, methylation levels have been found to increase in in vitro cultures over time. Higher DNA methylation levels have been correlated with a low yield of secondary metabolites in in vitro plant cell cultures. The longer the period of subculturing, the more methylated cytosines were found throughout the genome, and secondary metabolism decreased significantly. This review summarizes different studies on epigenetic changes during the maintenance of in vitro cell cultures and the insights they provide on the mechanisms involved. It concludes by looking at the perspectives for new approaches designed to avoid declines in metabolite production.

Biotechnological Production of Pharmaceuticals and Biopharmaceuticals in Plant Cell and Organ Cultures.

BACKGROUND: Plant biofactories are biotechnological platforms based on plant cell and organ cultures used for the production of pharmaceuticals and biopharmaceuticals, although to date only a few of these systems have successfully been implemented at an industrial level. Metabolic engineering is possibly the most straightforward strategy to boost pharmaceutical production in plant biofactories, but social opposition to the use of GMOs means empirical approaches are still being used. Plant secondary metabolism involves thousands of different enzymes, some of which catalyze specific reactions, giving one product from a particular substrate, whereas others can yield multiple products from the same substrate. This trait opens plant cell biofactories to new applications, in which the natural metabolic machinery of plants can be harnessed for the bioconversion of phytochemicals or even the production of new bioactive compounds. Synthetic biological pipelines involving the bioconversion of natural substrates into products with a high market value may be established by the heterologous expression of target metabolic genes in model plants. OBJECTIVE: To summarize the state of the art of plant biofactories and their applications for the pipeline production of cosme-, pharma- and biopharmaceuticals. RESULTS: In order to demonstrate the great potential of plant biofactories for multiple applications in the biotechnological production of pharmaceuticals and biopharmaceuticals, this review broadly covers the following: plant biofactories based on cell and hairy root cultures; secondary metabolite production; biotransformation reactions; metabolic engineering tools applied in plant biofactories; and biopharmaceutical production.

Zearalenone Uptake and Biotransformation in Micropropagated Triticum durum Desf. Plants: A Xenobolomic Approach.

A model was set up to elucidate the uptake, translocation, and metabolic fate of zearalenone (ZEN) in durum wheat. After treatment with ZEN, roots and shoots were profiled with LC-HRMS. A comprehensive description of in planta ZEN biotransformation and a biotechnological evaluation of the model were obtained. Up to 200 µg ZEN were removed by each plantlet after 14 days. Most ZEN and its masked forms were retained in roots, while minimal amounts were detected in leaves. Sixty-two chromatographic peaks were obtained, resulting in 7 putative phase I and 18 putative phase II metabolites. ZEN16Glc and ZEN14Glc were most abundant in roots, sulfo-conjugates and zearalenol derivatives were unable to gain systemic distribution, while distinct isomers of malonyl conjugates were found in leaves and roots. This study underlines the potential ZEN occurrence in plants without an ongoing Fusarium infection. Micropropagation may represent a tool to investigate the interplay between mycotoxins and wheat.

Technological Microbiology: Development and Applications.

Over thousands of years, modernization could be predicted for the use of microorganisms in the production of foods and beverages. However, the current accelerated pace of new food production is due to the rapid incorporation of biotechnological techniques that allow the rapid identification of new molecules and microorganisms or even the genetic improvement of known species. At no other time in history have microorganisms been so present in areas such as agriculture and medicine, except as recognized villains. Currently, however, beneficial microorganisms such as plant growth promoters and phytopathogen controllers are required by various agricultural crops, and many species are being used as biofactories of important pharmacological molecules. The use of biofactories does not end there: microorganisms have been explored for the synthesis of diverse chemicals, fuel molecules, and industrial polymers, and strains environmentally important due to their biodecomposing or biosorption capacity have gained interest in research laboratories and in industrial activities. We call this new microbiology Technological Microbiology, and we believe that complex techniques, such as heterologous expression and metabolic engineering, can be increasingly incorporated into this applied science, allowing the generation of new and improved products and services.

Expression and characterization of the gD protein of HSV-2 fused to the tetramerization domain of the transcription factor p53.

The highly immunogenic glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) is a very important element for entry of this virus into host cells. These characteristics have made this protein a very interesting HSV-2 subunit vaccine candidate. Despite efforts to prevent genital herpes using gD-based subunit vaccines, to date, clinical trials using this antigen have failed. Therefore, using a small animal model, we sought to determine if a tetramerized truncated form of gD subunit vaccine, produced by recombinant baculovirus infected insect larvae, would elicit better protection against genital herpes than a monomeric gD-2 subunit vaccine. Three out of 5 mice immunized with the tetramerized antigen produced in a baculovirus expression vector system, survived a lethal challenge with a wild type HSV-2 strain (for more than 3 weeks after challenge). In contrast, all the mice (5) immunized with the truncated protein, produced by the same methodology, died within 2 weeks after challenge. These results suggest that multimerization (increasing the structural complexity) of the truncated gD antigen might be more likely protective than the monomer form. Also the use of an alternative cost-efficient eukaryotic expression system is described.