Bernardo Bañuelos-Hernández

Over-expression of Dof-type transcription factor increases lipid production in Chlamydomonas reinhardtii

The high demand for less polluting, newer, and cheaper fuel resources has increased the search of the most innovative options for the production of the so-called biofuels. Chlamydomonas reinhardtii is a photosynthetic unicellular algae with multiple biotechnological advantages such as easy handling in the laboratory, a simple scale-up to industrial levels, as well as a feasible genetic modification at nuclear and chloroplast levels. Besides, its fatty acids can be used to produce biofuels. Previous studies in plants have found that the over expression of DOF-type transcription factor genes increases the synthesis and the accumulation of total lipids in seeds. In this context, the over-expression of a DOF-type transcription factor in C. reinhardtii was applied as approach to increase the amount of lipids. The results indicate higher amounts (around 2-fold) of total lipids, which are mainly fatty acids, in the genetically C. reinhardtii modified strains when compared with the non-genetically modified strain. In order to elucidate the possible function of the introduced Dof-type transcription factor, we performed a transcription profile of 8 genes involved in fatty acid biosynthesis and 6 genes involved in glycerolipid biosynthesis, by quantitative real time (qRT-PCR). Differential expression profile was observed, which can explain the increase in lipid accumulation. However, these strains did not show notable changes in the fatty acid profile. This work represents an early effort in generating a strategy to increase fatty acids production in C. reinhardtii and their use in biofuel synthesis.

Corn-based vaccines: current status and prospects

AbstractMain conclusionCorn is an attractive host for vaccine production and oral delivery. The present review provides the current outlook and perspectives for this field. Among seed-crops, corn represents a key source of biomass for food, fuel production, and other applications. Since the beginning of the development of plant-based vaccines, corn was explored for the production and delivery of vaccines. About a dozen of pathogens have been studied under this technology with distinct degrees of development. A vaccine prototype against enterotoxigenic Escherichia coli was evaluated in a phase I clinical trial and several candidates targeting bacterial and viral diseases are under preclinical evaluation. The present review provides an updated outlook on this topic highlighting the employed expression strategies; perspectives for the field are also provided.

Oxidative Stress Modifies the Levels and Phosphorylation State of Tau Protein in Human Fibroblasts

Since the tau protein is closely involved in the physiopathology of Alzheimers disease (AD), studying its behavior in cellular models might lead to new insights on understanding this devastating disease at molecular levels. In the present study, primary cultures of human fibroblasts were established and used to determine the expression and localization of the tau protein in distinct phosphorylation states in both untransfected and tau gene-transfected cells subjected to oxidative stress. Higher immunopositivity to phospho-tau was observed in cell nuclei in response to oxidative stress, while the levels of total tau in the cytosol remained unchanged. These findings were observed in both untransfected cells and those transfected with the tau gene. The present work represents a useful model for studying the physiopathology of AD at the cellular level in terms of tau protein implications.

Algevir: An Expression System for Microalgae Based on Viral Vectors

The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus) and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin), which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture), which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins.

Viral Vector-Based Expression Strategies

The need for high recombinant protein yields for the formulation of plant-derived vaccines has led to the development of innovative expression strategies. The efficient capacity of viruses to replicate themselves is a highly desirable characteristic with implications on achieving this goal. Plant viral vectors have been positioned as one of the most efficient approaches in terms of protein yield following strategies of full or deconstructed virus. Expression strategies based on the use of RNA and DNA plant virus are described in this chapter. Plant virus vectors based on Potexvirus, Tobamovirus, Comovirus, and Geminivirus are prominently used because of their high recombinant protein yields. The strategy of using virus-like particles has resulted in highly effective immunogens due to their complex structure, offering the production of chimeric particles bearing unrelated epitopes. Other viral elements that represent new developments are the internal ribosome entry sites (IRES) and the 2A peptide of foot-and-mouth disease virus, which may allow for the co-expression of distinct antigens from a single expression cassette, thus having implications on the formulation of improved bivalent or adjuvanted vaccines. Promising vaccination approaches using viral vector-based platforms are also presented, including vaccines against influenza, human immunodeficiency virus, human papillomavirus, and Norwalk virus.

Production of Biopharmaceuticals in Microalgae

Abstract Current production systems for recombinant proteins are composed of bacteria, yeasts, mammalian, or plant cells that have been genetically modified to produce several biopharmaceuticals (BFs) currently available in the market, such as hormones, monoclonal antibodies, vaccines, and others. Each system possesses its own features in terms of cost, safety, biosynthetic capacity, and downstream processing. Given the generalized high cost of BFs, emerging platforms are still desirable to address optimum features including low cost to increase product accessibility, especially in low-income countries. Besides serving as a valuable model in biological sciences, Chamydomonas reinhardtii is a microalga that has been successfully used during the last decade in the biotechnology area to establish alga-based platforms for BF production at low costs. This context augurs the exploitation of additional algae species, such as Phaeodactylum tricornutum, Dunaliella salina, and Nannochloropsis spp., for BFs production as a key trend in the field. In this chapter, the potential advantages of using marine microalgae species are analyzed.

Recombinant Hemagglutinin of Avian Influenza Virus H5 Expressed in the Chloroplast of Chlamydomonas reinhardtii and Evaluation of Its Immunogenicity in Chickens

SUMMARY Globally, avian influenza (AI) is a serious problem in poultry farming. Despite vaccination, the prevalence of AI in México highlights the need for new approaches to control AI and to reduce the economic losses associated with its occurrence in susceptible birds. Recombinant proteins from avian influenza virus (AIV) have been expressed in different organisms, such as plants. The present study investigated the feasibility of designing and expressing the HA protein of AIV in the transplastomic microalga Chlamydomonas reinhardtii as a novel approach for AIV control and taking advantage of culture conditions, its reproductive range, and safe use in consideration of the generally regarded as safe food ingredient regulatory classification. The results showed that the HA protein of AIV in C. reinhardtii presents antigenic activity by western blot test and through its application in chickens, demonstrating its feasibility as a recombinant antigen against AIV.

Expression of a Zika virus antigen in microalgae: Towards mucosal vaccine development

Zika virus (ZIKV) infection has extended rapidly all over the world in the last decades affecting humans of all ages, inducing severe illness such as the autoimmune Guillain-Barré syndrome as well as fetal neurodevelopmental defects. Despite the epidemiological importance of ZIKV, today there are no commercially available drugs or vaccines to combat or prevent this infection. Microalgae are attractive hosts to produce and deliver vaccines, with some candidates under preclinical evaluation. Herein, algae-based expression was assessed for the production of a new vaccine candidate against ZIKV called ZK. The Algevir technology was applied to express an antigenic protein called ZK comprising the B subunit of the heat labile Escherichia coli enterotoxin along with 3 epitopes from the ZIKV envelope glycoprotein. Efficient expression of the ZK antigen was achieved in Schizochytrium sp. with yields of up to 365 μg g-1 microalgae fresh weight. Upon oral administration in mice, the microalgae-made ZK protein elicited significant humoral responses at a higher magnitude to those induced upon subcutaneous immunization. The algae-made ZK vaccine represents a promising candidate to formulate attractive vaccines against ZIKV.

Prospects on the Use of Schizochytrium sp. to Develop Oral Vaccines

Although oral subunit vaccines are highly relevant in the fight against widespread diseases, their high cost, safety and proper immunogenicity are attributes that have yet to be addressed in many cases and thus these limitations should be considered in the development of new oral vaccines. Prominent examples of new platforms proposed to address these limitations are plant cells and microalgae. Schizochytrium sp. constitutes an attractive expression host for vaccine production because of its high biosynthetic capacity, fast growth in low cost culture media, and the availability of processes for industrial scale production. In addition, whole Schizochytrium sp. cells may serve as delivery vectors; especially for oral vaccines since Schizochytrium sp. is safe for oral consumption, produces immunomodulatory compounds, and may provide bioencapsulation to the antigen, thus increasing its bioavailability. Remarkably, Schizochytrium sp. was recently used for the production of a highly immunoprotective influenza vaccine. Moreover, an efficient method for transient expression of antigens based on viral vectors and Schizochytrium sp. as host has been recently developed. In this review, the potential of Schizochytrium sp. in vaccinology is placed in perspective, with emphasis on its use as an attractive oral vaccination vehicle.