Current Status and Perspective on the Use of Viral-Based Vectors in Eukaryotic Microalgae

During the last two decades, microalgae have attracted increasing interest, both commercially and scientifically. Commercial potential involves utilizing valuable natural compounds, including carotenoids, polysaccharides, and polyunsaturated fatty acids, which are widely applicable in food, biofuel, and pharmaceutical industries. Conversely, scientific potential focuses on bioreactors for producing recombinant proteins and developing viable technologies to significantly increase the yield and harvest periods. Here, viral-based vectors and transient expression strategies have significantly contributed to improving plant biotechnology. We present an updated outlook covering microalgal biotechnology for pharmaceutical application, transformation techniques for generating recombinant proteins, and genetic engineering tactics for viral-based vector construction. Challenges in industrial application are also discussed.


Introduction
Microalgae are unicellular microorganisms found in marine and freshwater ecosystems over a wide range, from very small (a few micron) to large (a few hundreds of microns). They can rapidly produce biomass from solar energy, CO 2 , and nutrients, such as nitrogen, sulfur, and phosphorous. Simple maintenance and cultivation in artificial environments offer a profitable platform to produce and extract bioactive compounds compared with other bioresources. Here, microalgae produce various metabolites with applications in pharmaceutical, cosmetic, bioenergy, and food/feed industries [1,2]. Various microalgae-derived products for food and feed have already been commercialized by different companies worldwide, including A4F-Algae 4 Future (Portugal), Blue Biotech (Germany), DIC Lifetec (Japan), E.I.D Parry (India), Ocean Nutrition (Canada), Phycom (Netherlands), Chlorella Co. (Taiwan), and Solazyme, Inc. (San Francisco), all of which used their bioactive compounds as colorants, additives, or supplements [3].
Biopharmaceuticals are complex molecules of biological origin used to diagnose, prevent, treat, and cure diseases or conditions in human beings and animals. According to their biological structure, biopharmaceuticals can be classified into amino acids, nucleic acids, and vaccines. In biopharmaceutical terms, these molecules are specifically produced under biotechnological processes based on genetically engineered organisms used as an expression host [4]. The main organisms used here are bacteria, yeast, mammalian cells, and insect cells, with each system having their own advantages as well as limitations [5][6][7][8].
Recently, microalgae have attracted increasing scientific interest due to their versatile growth and functional metabolic properties, as well as their biopharmaceutical production. Microalgae possess distinct attributes that have attracted the attention of biotechnologists, who have developed advanced genetic and molecular tools to leverage microalgae as green bioreactors to produce biopharmaceuticals. These attributes include their ability to grow and culture under heterotrophic, autotrophic, and mixotrophic conditions, the capacity to realize post-translational modifications and proper protein maturation, and the distinction of some microalgae species as "Generally Recognized as Safe" by the Food and Drug Administration (FDA). This status is conferred to any substance, chemical, or a whole organism that is safe for human consumption, owing to the absence of pathogens, microorganism, or related endotoxins. Mostly heterotrophic microalgae are FDA-approved for biotechnological applications due to their large-scale growing capacity and high cell density compared with other organisms [9,10] To increase the yield and accelerate time to obtain and improve biopharmaceutical quality, microalgae biotechnology uses various expression methods and genetic and molecular biology strategies. These methods include stable nuclear and chloroplast expression and, in recent years, transient expression using viral-based vectors that allow high protein accumulation in a short period of time. However, the method using Agrobacterium tumefaciens transformation makes oral formulations of algal biomass unusable due to residual bacteria. Conversely, viral vectors for this purpose are limited and are mainly designed using elements derived from plant viruses. We present an updated outlook covering microalgal biotechnology for pharmaceutical applications, transformation techniques for obtaining recombinant proteins, and genetic engineering tactics for viral-based vectors construction ( Figure 1). Finally, we discuss the potential challenges in industrial application. (C). Method to transfer plasmid DNA using electroporation. (D). Introduction to target gene through the Agrobacterium-mediated method. (E). Stepwise protocol for the transfer of genes. (F). Traditional algae transformation method (glass beads). (G). Transformation methods (direct or Agrobacterium mediated) and integration into algae cell. (H). Stages of development for large-scale production of valuable biopharmaceuticals.

Genetic Engineering Transformation Methods for Biopharmaceutical Production of Microalgae
During the last 20 years, 40 different microalgae species genetic engineering methods have been developed [11,12]. Chlamydomonas reinhardtii, Dunaliella salina, Volvox carteri, Haematococcus pluvialis, and Phaeodactylum tricornutum are widely used microalgae for transforming foreign transgene expression studies, as well as biopharmaceutical protein production [13][14][15][16][17]. Here, microalgae genomes, such as nuclear, chloroplast, and mitochondrial transformation protocols, have been explored. In microalgae, four traditional methods are widely used to deliver foreign genes into microalgal genomes, including agitation with glass beads [18], particle bombardment [19], electroporation [20], and Agrobacterium-mediated transformation [21][22][23][24][25]. Of these methods, glass beads and Agrobacterium do not require any specialized apparatus, are less labor-intensive, and are relatively fast [18,26]. Additionally, bacterial conjugation as well as natural and liposome-mediated transformation also have been employed, each of these exhibiting their own advantages and drawbacks. The most notable disadvantages presented for some methods include the need for optimizing the transformation conditions, the low efficiency, and the high cost of the equipment or interface used [27].
Among these techniques, the particle gun method is the most efficient for direct DNA delivery into cells. Generally, the gene gun method shows high transformation efficiency; however, this method is costly. Both particle bombardment and electroporation can be applied to transfer not only endogenous DNA but also proteins into microalgae cells. The most important application introduced Cas9 protein-gRNA ribonucleoproteins (RNPs) into microalgae, namely, into C. reinhardtii, P. tricornutum, and Tetraselmis sp. cells, for DNA-free genome editing [80][81][82][83].
In addition to the aforementioned methods used to introduce foreign DNA into microalgae cells, other transformation methods are also available. Hawkins and Nakamura (1999) showed Chlorella sp. protoplast cells and plasmids can be generated by mixing with polyethylene glycol and dimethyl sulfoxide for human growth hormone gene transformation [84]. Similarly, Liu et al. (2013) described novel, simple, reliable, and cost-effective transformation of C. ellipsoidea protoplast cells by mixing foreign DNA with PNC solution (NaCl, CaCl 2 , and 40% PEG 4000) [71]. Other methods include stable nuclear transformation systems for Pleurochrysis carterae using polyethylene glycol (PEG)-mediated transfer of hygromycin B-resistance genes [85]. Recent reports present genetic transformation of microalgae by bacterial conjugation [86,87] and gene injection [88]. In addition to these techniques, other emerging methods, such as cell-penetrating peptides, nanoparticles, metal-organic frameworks, and liposomes, have not yet been demonstrated in microalgae [12,[89][90][91].

Microalgae Nuclear and Chloroplast-Based Expression
Microalgae contain nuclear, mitochondrial, and chloroplast genomes, each of which have their own transcription, translation, and post-translation properties [92]. Nuclear expression in microalgae offers numerous benefits, such as targeting recombinant protein expression in specific organelles, protein glycosylation, post-translational modification, and secretion [93]. In nuclear-based expression, the position of an exogenous gene into a microalgal genome occurs as a random insertion and usually transgenic cells are selected via phenotypic variation or antibiotic resistance. Generally, this approach results in low yields. Although the reasons for this phenomenon are not completely understood, possible explanations could be attributed to the RNA-silencing process, transcript instability, positional effects of transgenes, and an inaccessible chromatin structure [94].
Using chloroplasts to express foreign genes has become a promising alternative to the nuclear genome. Microalgae chloroplasts serve as the main cell factory for synthesizing several metabolic pathway enzymes and appropriate transformation objects for producing isoprenoids, carbohydrates, lipid, carotenoids, pigments, fatty acids, and proteins [95,96]. Further, this organelle lacks a gene-silencing mechanism and may be used to protect proteins from degradation and involve some post-translation modifications, such as phosphorylation. These multiple functions in a single cell organelle are the most important traits for its heterologous gene expression in microalgae [97,98]. For delivery, the foreign gene must pass through several membranes, which represent a greater challenge. The preferred method to achieve this goal is particle bombardment. In particular, C. reinhardtii has been descried in numerous transformation studies for producing foreign proteins due to the chloroplast genome being fully sequenced and offering a unique advantage in the transformation system [99]. Further, various transformation methods have been reported for C. reinhardtii chloroplasts, among which are the marker-free chloroplast transformation system [100] and glass bead agitation using cell wall-deficient cells [28][29][30]. Finally, a chloroplast transformation system based on electroporation has also been developed for Phaeodactylum tricornutum [101].

Algal Biotechnology in Pharmaceutical Applications
In biochemistry, metabolites are defined as small molecules of <1.5 kilodaltons (KDa) that act as intermediates or end products in cellular metabolism and are classified as pri-mary and secondary. Primary metabolites are directly involved in growth, development, and reproduction, whereas secondary are not implicated in these processes but offer an important ecological function and are typically linked to specific environmental conditions or developmental stages [102]. In microalgae, diverse bioactive metabolites have been studied for their antifungal, anticancer, antibacterial, and immunosuppressive properties [103][104][105][106][107].
Conversely, using single-cell engineering microalgae as a green factory to produce biopharmaceuticals includes recombinant expression of numerous antigenic proteins that act as human and animal vaccine candidates against viral or bacterial diseases and parasitic infections. Among these candidates, expression of viral epitopes from Zika virus [115], avian influenza [116], human papillomavirus [117], hepatitis B [41], and human immunodeficiency virus (HIV) [118], as well bacterial proteins from Staphylococcus aureus [76] and Histophilus somni [119], are well studied. Regarding parasitic infections, proteins from Plasmodium falciparum that cause malaria are also expressed in microalgae [120,121]. Furthermore, microalgae are also used to produce monoclonal antibodies, hormones, cytokines, growth factors, immunotoxins, and proteins to prevent non-communicable diseases [122][123][124][125]. A detailed recompilation of biopharmaceuticals produced in microalgae are summarized in Table 2. In addition, these recombinant microalgae cells can be utilized as an effective oral drug delivery platform formulated as pills, tablets, or freeze-dried cells [9]. A study by Kwon et al. (2019) demonstrated that the green fluorescent protein (GFP) expressed in chloroplasts of C. reinhardtii remained intact after biomass lyophilization [126].

Viral-Based Expression Vectors for Recombinant Protein, Vaccine, and Biopharmaceutical Production
Currently, biotechnology and genetic engineering is harnessing numerous viruses or their component parts to produce heterologous proteins for human and animal use. Given the expression of epitopes from influenza A virus can be fused with the hepatitis B core antigen in Nicotiana benthamiana plants, generation of virus-like particles (VLPs) in insect cells for the human papilloma virus as a vaccine-delivery vehicle for genetic material can generate an immune response in the human body, as recently developed for a COVID-19 vaccine [160][161][162]. Furthermore, polymerases and reverse-transcriptases from viral origins, in addition to elements such as transcriptional promoters, terminators, silencing suppressors, and internal ribosomal entry sites, form part of a molecular toolbox for genetic engineers, biologists, and biotechnologists.
The common approach for generating viral-based expression vectors involves inserting a determinate viral genome sequence into an expression vector downstream of a cell-typespecific promoter. The coding sequence of a heterologous gene is then inserted into the viral genome sequence as part of a viral polyprotein or downstream to a subgenomic promoter. The construct is then transferred to host cells for transcription and subsequent translation processes by host molecular machinery [163]. During the last decade, the design, generation, and use of viral-based expression vectors for producing heterologous proteins have gained increasing scientific interest, mainly in the plant biotechnology field. To achieve this goal, expression strategies have focused on RNA and DNA plant viruses, of which tobamovirus, comovirus, potexvirus, and geminivirus are the most exploited genera.
Developing and applying this approach has followed an interesting path. First, by creating first-generation expression vectors or full virus strategies based on expression of the gene of interest (GO), this approach has also produced its own viral genes and subsequent translation as an individual antigenic or fusion protein on the C-terminal of the capsid protein (CP). Using these vectors, several immunogens have been produced, reaching up to 10% of the total soluble protein (TSP) in Nicotiana benthamiana plants. However, stability is negatively related to insert size, hence the proteins larger than 30 KDa are poorly expressed in a chimeric CP form and epitopes should be 25 amino acids at maximum length [164][165][166]. These drawbacks slowed the development of second-generation viral vectors, whereby using a full virus was replaced with a deconstructed virus genome containing essential elements for replication and non-viral sequence integration to accomplish other functions, such as replicon formation using T-DNA delivered via A. tumefaciens. Using Agrobacterium for DNA delivery offers considerable advantages given the efficient transfer capacity by infiltration of plant leaves. Plants species using this approach include spinach, sunflower, red beetroot, and N. benthamiana, presenting maximum yields up to 50% of TSP in a 4-5 day period where the size of the GO can be up to 2 Kb and proteins of 80 KDa can be produced [167][168][169].
Special attention should be directed toward DNA virus-based vectors, specifically those applying elements from geminivirus, a twinned icosahedral virus with a single-strand DNA (ssDNA) arranged in one (monopartite) or two components (bipartite) encoding proteins essential for the replication process, pathogenicity, suppression of plant gene silencing, and intercellular and long-distance movement of the virus [170,171]. In general, these vectors are based on a transient expression system, the advantages of which include rapid product expression, high production rate, flexibility, and scalability. A geminivirus engineered for biopharmaceuticals is Bean Yellow Dwarf Virus (BeYDV), which has been modified to leverage its Rep protein under independent promoter control. With this strategy, diverse BeYDV-based expression vectors have been engineered and an assortment of antigens and monoclonal antibodies have been generated [172][173][174]. For microalgae, the geminiviral vector pBYR2e was used for expression of the receptor-binding domain (RBD) from SARS-CoV-2 and fibroblast growth factor (bFGF) in two freshwater microalgal species. Yields reached up to 1.61 µg/g and 1.14 µg/g for RBD when expressed in C. reinhardtii and C. vulgaris, respectively [35]. Conversely, Berndt et al. (2021) reported expression of RBD-fused GFP in C. reinhardtii. Interestingly, the protein targeted three different cellular localizations: (i) in the endoplasmic reticulum-Golgi pathway; (ii) secreted out of the cell into the culture media; and (iii) directed to the chloroplasts. In the latter, although under higher expression, the protein appeared to be truncated by~5 kDa at the amine end, whereas the end targeted to the ER was produced with the expected size and correct amino acid sequence. For obtaining proteins, the transgene was placed into the pBR9 and pOpt vectors; in particular, the pBR9 vector containing the sh ble zeocin resistance selection marker with a food and mouth disease virus (FMDV) 2A self-cleaving sequence placed between the coding sequences, resulted in accumulation of two separate proteins [175].
Another geminivirus-based vector, named Algevir, has been developed with diverse antigenic proteins and epitopes expressed in the marine microalgae Schizochytrium sp., which was engineered using the Rep protein and origin of replication (Ori) from the bego-movirus Ageratum enation to produce and replicate circular DNA carrying the GO and AlcR gene, as well as the AlcA promoter from Aspergillus nidulans to obtain ethanol-induced expression. This innovative system has produced viral and bacterial proteins at a maximum level production of 1.25 mg/g fresh biomass for GP1 from Zaire ebolavirus [79]. Table 3 shows the viral-based vectors used for biopharmaceutical production in microalgae. However, yields produced in microalgae with a nuclear approach and using viral-based vectors do not fully outcompete those produced in chloroplasts whereby targets allow production of 3.28 mg/L of culture medium [176]. The strategy based on protein production in this organelle requires a long time and construction of detailed vectors containing specific sequences for integration by homologous recombination. Here, optimizing viral-based vectors is needed to increase the protein yield and improve stability, which requires transient expression as a primary approach given that some transgene products may become toxic for host cells, leading to very low yields under stably transformed lines. Alternatively, microalgae viruses can be naturally used to drive gene expression at different infection stages and viral elements can be explored throughout the design process of novel viral-based vectors or when improving current models. Updating the functions of viral genes and the genome composition is an important requirement for executing a rational design in which regulatory elements, such as promoters, terminators, or replication proteins, help reach strong GO expression. Finally, exploring the possibility of directly purifying recombinant proteins using elements from lytic viruses presents an alternative approach [177].

Design of a Viral-Based Vector for Microalgae Use
In the virosphere, many species are capable of infecting microalgae. In addition to triggering high mortality rates, such species can reprogram host metabolism, including photosynthesis and important cycling processes, such as central carbon metabolism, phosphorus, nitrogen, and sulfur [178].
Recently, a list of 10 isolated and characterized viruses was published by Sandaa et al. (2022) [180]. These viruses can infect marine haptophytes species. Here, a rational design of a microalgal-specific viral vector to achieve higher protein yields, using viral elements that naturally infect microalgae, could be a promising strategy.
In a study published by Kadono et al. (2015) [181], a set of five potential promoter regions located upstream of the replication-associated protein (VP3) or structural protein (VP2), coding genes for three marine diatom-infecting viruses (DIVs), were evaluated and compared in the Pennales diatom Phaeodactylum tricornutum as a heterologous host ( Table 4). The gene-encoding fucoxanthin chlorophyll a/c-binding protein (fcp) was used as an endogenous promoter and eGFP as a protein reporter. In addition, the extrinsic promoter, such as Cauliflower mosaic virus 35S (CaMV35S), cytomegalovirus (CMV), and nopaline synthase gene (nos) promoter, were also used. The results show the novel promoter ClP1 mediated significantly higher transcription and translation rates according to mRNA transcripts and flow cytometry analysis, respectively. Further, the abundance of eGFP mRNA transcripts in the stationary phase were higher than those found in the log phase under both low and standard nutrient culture conditions. In addition to DIVs, other viruses can help explore their genetic elements and design a novel viral-based vector, particularly those with ssDNA or dsRNA genomes. Among them, viral species infecting the most commonly studied microalgal, such as the genus Chlorella, may offer a useful genetic toolbox. For example, the Paramecium bursaria Chlorella virus 1 (PBCV-1), a large dsDNA virus (>300 kb) infecting the green microalgae Chlorella variabilis NC64A, is now a model system for studying DNA virus/algal interactions, which has also been tested for biomass saccharification with subsequent bioethanol production and proteins involved in cell wall degradation [182][183][184][185]. Another virus fully sequenced that infects the Chlorella genus with potential biotechnology application are those that exclusively multiply in Syngen 2-3 or SAG 3.83 cells, which could lead to specific protein expression in microalgae strains. The prototype viruses are only Syngen viruses-NE5 (OSy-NE5) and Acanthocystis turfacea chlorella virus (ATCV-1) [186,187].

Challenges and Perspectives
In recent years, the current pandemic has pushed progress of several biomedical technologies, e.g., RNA vaccines and adenovirus-based vaccines. Based on these advances, what are the key insights from the field of algae-based biopharmaceuticals? Biopharmaceuticals using algae are considered a promising alternative for improving global health. Algae offer low production costs and some species are already used at industrial levels in the food industry and thus are considered safe for use as delivery vehicles, especially oral formulations. However, although the proof of concept for using algae to produce and even deliver biopharmaceutical has been reported by several groups, a number of challenges remain to be addressed in this field, including improving recombinant protein yield productivity.
Another critical path that deserves research attention in developing algae-made biopharmaceuticals is related to regulation. Defining the main guidelines for specific regulations applied to this type of biological agent is a major priority task. Performing clinical trials requires translating prototypes generated in academic labs to facilities with good laboratory practices that can approve and perform clinical trials. Moreover, implementing GMP-compliant processes in cooperation with pharmaceutical companies is urgently needed.
The current pandemic has increased support from several countries to invest in biomedical research and strengthen the developmental path for drugs and biologics. For example, several developing and emerging countries are increasing funding for research on innovative platforms for biopharmaceuticals production, including Thailand and México. We consider that the innovative green platforms required to produce biopharmaceuticals are a promising niche that could be accelerated by such initiatives. However, this should be a mid-term goal considering that conventional production systems with wellestablished regulatory frameworks will be the priority for such countries to provide rapid solutions for immediate needs. As biopharmaceuticals are inherently more complex than conventional chemical drugs, they demand a more complicated manufacturing process with varying quality and demands for extensive processes and product understanding. In addition, downstream processing represents another bottleneck. For algae, eliminating large amounts of lipids present in total extracts should be studied and the impact of differential glycosylation compared with mammalian glycosylation is another aspect that deserves attention.
Although the good manufacturing practice (GMP) standards of various regulatory authorities and international organizations are very similar and appropriate in addressing the manufacturing challenges, introducing innovative platforms always presents challenges. This challenge is exacerbated in developing or emerging countries that require affordable biopharmaceuticals. For instance, a recent study by Rahalkar et al. (2021) revealed that, in several emerging countries, the lack of standardized biosimilar development criteria and regulatory convergence across agencies led to challenges in multi-country biosimilar development, limiting our ability to introduce new, cheaper biosimilars into the market [188]. Unfortunately, for biopharmaceuticals produced in algae, this remains an ongoing challenge.
Although using viral vectors improves efficiency in expression systems, using Agrobacterium presents the need for complex purification steps to eliminate bacterial endotoxins. Therefore, expanding stable transformation systems to express viral replicons under an inducible approach is a possible solution to this limitation. Avoiding antibiotic-resistant markers is another challenge when designing vectors. Alternative markers, such as nutrientselective markers, are accruing more interest. Another possibility is developing oral formulations subjected to less strict regulations. It is clear that this field is still in its infancy; thus, exploring new constructs optimized for model species, especially C. reinhardtii, are required. Special emphasis on developing vectors based on new algae viruses is crucial.

Concluding Remarks
Although using viral-based expression systems in algae is still new, this technology has immense potential to revolutionize the algae-based biopharmaceuticals field by offering higher yields and shorter production times compared with chloroplast and nuclear stable transformation methods. The following decade will be critical, as technology will benefit from refreshed interest when supporting biomedical research in response to the COVID-19 pandemic. Research and development goals should be focused not only on generating prototypes in academic labs but also on critical regulatory issues to ensure the success of new products that enter the market and ultimately benefit human health, especially in developing and emerging countries. On February 2022, Medicago, a Canadian company, and GlaxoSmithKline (GSK) announced approval by the Health agency in Canada of COVIFENZ ® , a COVID-19 vaccine produced in plants. This is a milestone, as it is the first vaccine produced using a green platform approved for human use. Will algae-based products reach the same success? The following decade will be crucial in addressing this goal.