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This manuscript describes a unique protocol for the rapid transformation of Medicago truncatula A17 cell suspension cultures mediated by Agrobacterium tumefaciens. Medicago cells were collected on day 7 of the growth curve, which corresponded to the beginning of the exponential phase. They were then co-cultured with Agrobacterium for 3 days before being spread onto a petri dish with appropriate antibiotic selection. The Receptor Binding Domain of the Spike protein of SARS-CoV-2 was used as a model to develop this protocol. The presence of the transgene was assessed using PCR, and the integrity of the product was evaluated by SDS-PAGE and Western-blotting.
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The COVID-19 pandemic, caused by the worldwide spread of SARS-CoV-2, has prompted the scientific community to rapidly develop efficient and specific diagnostics and therapeutics. A number of avenues have been explored, including the manufacture of COVID-related proteins to be used as reagents for diagnostics or treatment. The production of RBD and Spike proteins was previously achieved in eukaryotic cells, mainly mammalian cell cultures, while the production in microbial systems has been unsuccessful until now. Here we report the effective production of SARS-CoV-2 proteins in two plant model systems. We established transgenic tobacco BY-2 and Medicago truncatula A17 cell suspension cultures stably producing the full-length Spike and RBD recombinant proteins. For both proteins, various glycoforms were obtained, with higher yields in Medicago cultures than BY-2. This work highlights that RBD and Spike can be secreted into the culture medium, which will impact subsequent purification and downstream processing costs. Analysis of the culture media indicated the presence of the high molecular weight Spike protein of SARS-CoV-2. Although the production yields still need improvement to compete with mammalian systems, this is the first report showing that plant cell suspension cultures are able to produce the high molecular weight Spike protein. This finding strengthens the potential of plant cell cultures as production platforms for large complex proteins.
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Phaeodactylum tricornutum is a model diatom with numerous potential applications in the industry, including the production of high-value carotenoid pigments such as fucoxanthin. This compound is a potent antioxidant currently extracted mainly from brown macroalgae. Fucoxanthin exhibits several biological properties with well-known beneficial effects in the treatment and prevention of lifestyle-related diseases. P. tricornutum offers a valuable alternative to macroalgae for fucoxanthin production as it has a specific productivity that is 10-fold higher as compared with macroalgae. However, production processes still need to be optimised to become a cost-effective alternative. In this work, we investigated the optimal supplementation of nitrate in a cultivation medium that is currently used for P. tricornutum and how this nitrate concentration affects cell growth and fucoxanthin production. It has previously been shown that the addition of sodium nitrate increases productivity, but optimal conditions were not accurately determined. In this report, we observed that the continuous increase in nitrate concentration did not lead to an increase in biomass and fucoxanthin content, but there was rather a window of optimal values of nitrate that led to maximum growth and pigment production. These results are discussed considering both the scale up for industrial production and the profitability of the process, as well as the implications in the cell's metabolism and effects in fucoxanthin production.
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Carotenoids are a class of pigments with a biological role in light capture and antioxidant activities. High value ketocarotenoids, such as astaxanthin and canthaxanthin, are highly appealing for applications in human nutraceutical, cosmetic, and animal feed industries due to their color- and health-related properties. In this review, recent advances in metabolic engineering and synthetic biology towards the production of ketocarotenoids, in particular the red-orange canthaxanthin, are highlighted. Also reviewed and discussed are the properties of canthaxanthin, its natural producers, and various strategies for its chemical synthesis. We review the de novo synthesis of canthaxanthin and the functional ß-carotene ketolase enzyme across organisms, supported by a protein-sequence-based phylogenetic analysis. Various possible modifications of the carotenoid biosynthesis pathway and the present sustainable cost-effective alternative platforms for ketocarotenoids biosynthesis are also discussed.
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Histone deacetylases are involved in chromatin remodelling and thus play a vital role in the epigenetic regulation of gene expression. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as transcription. Novel HDAC inhibitors were designed and synthesised to promote higher levels of recombinant protein production in tobacco cell cultures. The effect of these chemical enhancers on the epigenetic profiles in plant cells has been evaluated by molecular docking, in vitro and in vivo studies. The addition of these novel enhancers led to an increase in histone H3 acetylation levels that promoted an increase in the accumulation levels of the recombinant protein in cell culture. These results can pave the way for the application of these enhancers to improve the production of high value products in plant cell based systems.
