Recombinant Protein Expression and Its Biotechnological Applications in Chlorella spp.

: Recombinant protein expression is a fundamental aspect of both synthetic biology and biotechnology as well as a field unto itself. Microalgae, with their eukaryotic cellular machinery, high lipid content, cost-effective cultivation conditions, safety profile for human consumption, and environmentally friendly attributes, are a promising system for protein expression or metabolic engineering for sustainable chemical production. Amongst the incredible diversity of microalgae species, Chlorella spp. are heavily studied due to their high growth efficiency, potential for low-cost cultivation, and well-characterized scale-up process for large-scale cultivation. This review aims to comprehensively examine the ongoing advancements in the bioengineering of Chlorella spp. for recombinant protein production and its biotechnological applications. This includes genetic elements such as promoters, terminators, reporters and markers, enhancers, and tags successfully used in Chlorella spp.


Introduction
Recombinant protein expression is a foundational tool for molecular cell biology as well as for numerous biotechnologies, such as metabolic engineering and recombinant protein production (therapeutic proteins, vaccines, industrial enzymes, etc.).However, the focus of recombinant protein production is to achieve high titers, yields, and productivities in order to reduce the cost of protein production, because although recombinant protein production is now a well-established practice, large-scale production of proteins, particularly for low-cost applications, still remains economically challenging [1].
Recombinant protein production begins with the integration of recombinant DNA, often from a heterologous source, into the genome of the host organism under the control of protein expression elements.A DNA vector composed of various genetic elements such as the gene of interest, promoters, terminators, reporters, markers, and enhancers is transformed into the host cell using a variety of different transformation methods.Integration into the host cell genome can also be achieved using a variety of mechanisms such as CRISPR-mediated genome editing or random integration, and is largely influenced by the host cell type (bacterial, plant, yeast, cell culture systems) and species.The most used platforms include bacteria (Escherichia coli), yeast (Saccharomyces cerevisiae and Pichia pastoris), mammalian cells (CHO and HEK-293 cells), and baculovirus/insect cells (Sf9, Sf21) [2].However, proteins with complex post-translational modifications cannot be produced in the highly productive, low-cost bacteria or yeast systems, and therefore, costly cell culture systems are typically employed.Due to their costs, cell cultures systems are typically only used to produce high-value products like biologics.Thus, there remains a gap in the available production systems for the production eukaryotic proteins for low-cost applications like industrial enzymes.
Microalgae are microorganisms widely distributed throughout natural environments.Due to the low cost of their cultivation (low energy inputs and simple inorganic media), they have garnered interest as a low-cost platform to produce recombinant proteins.Prominent microalgae species that have been studied for recombinant protein production include numerous Chlorella spp.(vulgaris, ellipsoidea, and sorokiniana), Chlamydomonas reinhardtii [3], Phaeodactylum tricornutum [4], and Haematococcus pluvialis [5].Among them, Chlorella spp., unicellular and non-motile green microalgae [6], stand out due to their robust growth [7], their ability to grow well under both phototrophic and heterotrophic conditions [8], and their simple scale-up in either photobioreactors or outdoor ponds [9,10].When scaling protein production, high growth rates are critical for achieving high titers and productivities, while a low-cost medium is critical for the economical production of industrial enzymes, for example.Furthermore, the ability of many Chlorella spp. to grow as quickly as yeast on organic carbon sources like glucose greatly accelerates the genome editing process compared to using obligate phototrophs like P. tricornutum, although it should be noted that some strains have now been engineered to enable heterotrophy [11,12].As eukaryotic species, Chlorella spp.can produce proteins with complex post-translational modifications [13] while possessing smaller haploid genomes compared to mammalian cells (CHO), plants (Nicotiana tabacum) used for protein expression, and even other microalgae (Table 1).A number of Chlorella species are also considered Generally Regarded As Safe (GRAS status), which indicates that these species could be potential hosts for oral drug delivery, for example [14].However, recombinant protein production in Chlorella spp.remains challenging due to the difficulties in transforming species possessing cell walls and the lack of a robust genetic toolbox for engineering these species.E. coli, which is used extensively for recombinant protein production, has one of the most extensive toolkits for controlling protein expression, which allows users to tune the expression of a protein quickly in order to achieve the highest yields possible with relatively low effort.In order to advance the use of Chlorella spp. as a host for recombinant protein expression, a robust toolkit is also needed, along with a greater understanding of the best cultivation conditions for recombinant protein expression.This review aims to comprehensively assess the current progress in recombinant protein expression in Chlorella spp.and identify gaps that need to be addressed before Chlorella spp.can be effectively used for protein production.
As reviewed in the following sections of this paper, numerous heterologous and endogenous proteins have been expressed in various Chlorella species since the early 2000s (Figure 1), with a steady increase in the number of studies since the early 2010s, which coincides with several modern biotechnology advancements (development of CRISPR-Cas9 genome editing tools and advancements in next generation sequencing) that have reduced some of the challenges associated with microalgae genome engineering, such as a lack of genomic data.Three species have been used more extensively: C. vulgaris (26 reports), C. ellipsoidea (10 studies), and C. sorokiniana (6 studies).

Transformation Methods
There are numerous studies demonstrating that Agrobacterium-mediated transformation (AMT, Figure 2-Left) [24][25][26], electroporation (EP) [27-29], polyethylene glycol (PEG)-mediated transformation [30][31][32], and microparticle bombardment (MPB) [33][34][35] are all effective methods for the introduction of recombinant DNA into multiple Chlorella spp.(Figure 2-center).However, as PEG-mediated transformation is often performed after protoplasting, this method can be more tedious than direct EP, which is often performed with the cell wall intact.MPB requires more complex infrastructure and also results in 3.7-fold less transformants than EP in C. zofingiensis [36].AMT, while much lengthier than EP, potentially has the capacity to transfer up to 150 kbp of genetic information, which is much more than that typically transferred using electroporation [37].However, both the AMT and EP methods employed thus far have resulted in random integration of the recombinant DNA, and gene silencing, location-specific effects, and non-specific mutations may occur as a result [38,39].Therefore, more studies to increase the number of transformants in order to allow for more robust screening for healthy, stable strains would be welcome.It should be noted that a CRISPR-Cas9 system for the knockout (KO) of nitrate reductase (NR) and adenine phosphoribosyltransferase (APT) for site-directed deletions was recently demonstrated [40], and an AMT/CRISPR-Cas9 system was used to express Cas9 in C. vulgaris [41], but as of yet, no genes have been knocked in using this system.However, development of a site-directed tool for gene insertion is highly desirable to perform more reproducible studies and reduce the amount of colony screening needed.
Linearization of plasmid vectors has been observed to impact the number of colonies appearing on selection media using MPB for chloroplast transformation in C. vulgaris [29], but a more throughout examination of transformation parameters on the gene editing efficiency in Chlorella species would be a useful exercise, as it is currently difficult to tell which factors, such as using circular or linear DNA, are the most critical.Magnetic nanoparticle mediated transformation [42] and the use of HIV-TAT as a cell-penetrating peptide for transformation [43] have both recently been shown to be effective transformation methods as well.
The majority of studies use plasmids directly for the introduction of recombinant DNA; however, a commonly used plant gene expression tool, geminiviral expression vector delivered via AMT, was successfully demonstrated in C. vulgaris and C. reinhardtii for the expression of SARS-CoV-2 receptor-binding domain (RBD) and basic fibroblast

Transformation Methods
There are numerous studies demonstrating that Agrobacterium-mediated transformation (AMT, Figure 2-Left) [24][25][26], electroporation (EP) [27-29], polyethylene glycol (PEG)-mediated transformation [30][31][32], and microparticle bombardment (MPB) [33][34][35] are all effective methods for the introduction of recombinant DNA into multiple Chlorella spp.(Figure 2-center).However, as PEG-mediated transformation is often performed after protoplasting, this method can be more tedious than direct EP, which is often performed with the cell wall intact.MPB requires more complex infrastructure and also results in 3.7-fold less transformants than EP in C. zofingiensis [36].AMT, while much lengthier than EP, potentially has the capacity to transfer up to 150 kbp of genetic information, which is much more than that typically transferred using electroporation [37].However, both the AMT and EP methods employed thus far have resulted in random integration of the recombinant DNA, and gene silencing, location-specific effects, and non-specific mutations may occur as a result [38,39].Therefore, more studies to increase the number of transformants in order to allow for more robust screening for healthy, stable strains would be welcome.It should be noted that a CRISPR-Cas9 system for the knockout (KO) of nitrate reductase (NR) and adenine phosphoribosyltransferase (APT) for site-directed deletions was recently demonstrated [40], and an AMT/CRISPR-Cas9 system was used to express Cas9 in C. vulgaris [41], but as of yet, no genes have been knocked in using this system.However, development of a site-directed tool for gene insertion is highly desirable to perform more reproducible studies and reduce the amount of colony screening needed.
Linearization of plasmid vectors has been observed to impact the number of colonies appearing on selection media using MPB for chloroplast transformation in C. vulgaris [29], but a more throughout examination of transformation parameters on the gene editing efficiency in Chlorella species would be a useful exercise, as it is currently difficult to tell which factors, such as using circular or linear DNA, are the most critical.Magnetic nanoparticle mediated transformation [42] and the use of HIV-TAT as a cell-penetrating peptide for transformation [43] have both recently been shown to be effective transformation methods as well.
The majority of studies use plasmids directly for the introduction of recombinant DNA; however, a commonly used plant gene expression tool, geminiviral expression vector delivered via AMT, was successfully demonstrated in C. vulgaris and C. reinhardtii for the expression of SARS-CoV-2 receptor-binding domain (RBD) and basic fibroblast growth factor (bFGF).The result was transient expression of these proteins with yields in the range of 0.1-1.6 µg/g [24].
organisms can metabolize ammonia than nitrate, culture contamination may be more common in ammonia-containing media.
Only two reports of chloroplast transformation in Chlorella have been published, both for C. vulgaris [29,33].Both of these reports used homologous recombination (HR) with the chloroplast 16S-trn1 and the trnA-23S region of the chromosome to achieve integration.

Selectable Markers
For stable integration of the DNA into the genome, antibiotics have most often been employed (Table 2).The most prevalent selection marker in Chlorella sp. is Geneticin (G418) [30,48,49].The resistance mechanism is conferred by the nptII gene, which encodes a neomycin phosphotransferase II enzyme (EC 2.7.1.95)responsible for the inactivation of Selectable markers used for the selection of transformants can have negative effects on the host cell and are costly during larger-scale cultivation, but to date, no marker removal systems have been demonstrated in Chlorella spp.Two systems for markerless transformation have been reported: transformation using SV40 Large T antigen (TAg) [44] and a transformation system that depends on the KO ofNR using homologous recombination [28, [45][46][47].In this system, a double or triple cross-over event is used to disrupt the NR gene, which can be selected for by supplementing KClO 3 in the media (Figure 2-right).In the presence of an active NR, chlorate is reduced to the highly toxic chlorite, which prevents the growth of the wild-type cells.Since ammonium is generally a cheaper source of nitrogen than nitrate, loss of NR is not critical during scale-up, although as more organisms can metabolize ammonia than nitrate, culture contamination may be more common in ammonia-containing media.
Only two reports of chloroplast transformation in Chlorella have been published, both for C. vulgaris [29,33].Both of these reports used homologous recombination (HR) with the chloroplast 16S-trn1 and the trnA-23S region of the chromosome to achieve integration.

Selectable Markers
For stable integration of the DNA into the genome, antibiotics have most often been employed (Table 2).The most prevalent selection marker in Chlorella sp. is Geneticin (G418) [30,48,49].The resistance mechanism is conferred by the nptII gene, which encodes a neomycin phosphotransferase II enzyme (EC 2.7.1.95)responsible for the inactivation of aminoglycoside antibiotics including neomycin, kanamycin, geneticin, and paromomycin [50].Geneticin has been used extensively for various species of Chlorella, with concentrations typically ranging from 15-500 µg/mL, but typically, 30 µg/mL is used for agar plate selection, while kanamycin and paromomycin have only been used a handful of times.Geneticin has broad host range, as it binds and inhibits protein synthesis in both prokaryotic 16S rRNA of the 30S ribosomal subunit and eukaryotic 18S rRNA [51,52], while kanamycin targets the bacterial 16S rRNA of the 30S ribosomal subunit [53].As Chlorella sp. can grow both phototrophically and heterotrophically, media formulation must be carefully considered when using selectable markers that inhibit eukaryotic ribosomes in the ER or the prokaryotic ribosomes present in the chloroplast.Kanamycin selection may only work under phototrophic growth conditions, allowing escaped colonies to form when organic carbon is present, while the broad spectrum of geneticin may be less prone to this problem, resulting in a higher rate of positive transformants.How antibiotic effectiveness changes with the trophy of the culture has not yet been investigated; however, antibiotics have been used to purposefully inhibit photosynthesis to screen the microalgae Chrystotila roscoffensis for heterotrophy [54].For the second-most prevalent antibiotic, Hygromycin B [41,55,56], most frequently employed in plant studies using AMT, resistance is conferred by hygromycin phosphotransferase encoded by hpt and is an aminoglycoside-4 ′ -phosphotransferase that inhibits protein synthesis in both bacterial and eukaryotic ribosomes (EC 2.7.1.163)[57].While it has been used in a range from 15-500 µg/mL, typically, 50 µg/mL is used most frequently for plate selections.Phleomycin (Zeocin) resistance is encoded by ble is more commonly used in Chlamydomonas systems [58], but has also been used with C. vulgaris and C. ellipsoidae [59,60].Both chloramphenicol (cat) and spectinomycin (aadA) are typically used for bacterial systems because they target the 50S and 30S ribosomal subunits present in bacteria, respectively [61,62].Chloramphenicol has been shown to work in C. vulgaris [63] and spectinomycin in C. sorokiniana [64].Other bacterial-targeted antibiotics, spectinomycin and kanamycin, were demonstrated to be effective for the selection of chloroplast transformants in C. vulgaris [29,33].Finally, in addition to the antibiotics described above, the herbicide norflurazon was successfully used along with a mutant norflurazon-resistance phytoene desaturase (pds) for selection in C. zofingiensis [36], and the KO of NR for selection using KClO 3 containing media has been used to avoid the need for antibiotics altogether [45].
A final strategy that has been successfully employed has been the fusion of antibiotic resistance genes to the gene of interest using a 2A self-cleaving peptide tag between the two genes [59,65].The 2A self-cleaving peptide is derived from hand-foot-and-mouth disease, and separate polypeptides are generated by ribosome skipping caused by the 2A tag [66].This technique has the advantage of ensuring that both selection and gene of interests are expressed together, which may decrease the prevalence of escaped colonies or incomplete expression of the gene of interest.However, extensive study on the use of 2A tags in Chlorella spp.this has not yet been undertaken.

Promoters and Terminators
Promoters and terminators play a critical role in the effective expression of recombinant proteins because they regulate the initiation and termination of transcription (Figure 3).Most studies have focused on modifications to the nuclear genome, and the cauliflower mosaic virus 35S (CaMV35S) promoter is by far the most used promoter for protein expression from the nuclear genome of Chlorella spp.Its choice is obvious as one of the best-characterized constitutive promoters used in plant biotechnology; however, many variants of this promoter exist, including long, short, and dual variants, and authors sometimes do not directly report which variant was used.A Chlorella virus promoter was also tested in C. vulgaris and C. sorokiniana, but proved to be similar to that of CaMV35S [67].The ubiquitin 1 (Ubi) promoter derived from the maize plant has also been successfully used for numerous proteins.During transient expression of β-glucuronidase (GUS), GUS activity was 3.5-fold higher in the P ubi strains than the P CaMV35S strains [68].Other constitutive promoters used include actin 1 from rice and the nopaline synthase (NOS) promoter from A. tumefaciens.Endogenous constitutive promoters from Chlorella spp.have also been used, for example, phytoene desaturase (PDS) from C. zofigiensis [36] and ω-3 fatty acid desaturase (FAD) promoters from C. vulgaris [69].

Promoters and Terminators
Promoters and terminators play a critical role in the effective expression of recombinant proteins because they regulate the initiation and termination of transcription (Figure 3).Most studies have focused on modifications to the nuclear genome, and the cauliflower mosaic virus 35S (CaMV35S) promoter is by far the most used promoter for protein expression from the nuclear genome of Chlorella spp.Its choice is obvious as one of the bestcharacterized constitutive promoters used in plant biotechnology; however, many variants of this promoter exist, including long, short, and dual variants, and authors sometimes do not directly report which variant was used.A Chlorella virus promoter was also tested in C. vulgaris and C. sorokiniana, but proved to be similar to that of CaMV35S [67].The ubiquitin 1 (Ubi) promoter derived from the maize plant has also been successfully used for numerous proteins.During transient expression of β-glucuronidase (GUS), GUS activity was 3.5-fold higher in the Pubi strains than the PCaMV35S strains [68].Other constitutive promoters used include actin 1 from rice and the nopaline synthase (NOS) promoter from A. tumefaciens.Endogenous constitutive promoters from Chlorella spp.have also been used, for example, phytoene desaturase (PDS) from C. zofigiensis [36] and ω-3 fatty acid desaturase (FAD) promoters from C. vulgaris [69]. ) is placed upstream of an optional 5′UTR and transit peptide.This is followed by the coding sequence, which may be interrupted by an intron to enhance expression.A 2A tag can be used for multigene expression using a single promoter.The terminator containing the polyA signal is needed for mRNA transport to the ER for translation.
Inducible promoters allow for the controlled activation or repression of gene transcription in response to specific external signals or inducers.An HSP70/RbcS2 fusion promoter has been reported to contain highly light-inducible elements; however, although both promoters have been used in Chlorella spp., this effect has not been confirmed in Chlorella sp.[70].The NR promoter is inducible using nitrate [36,63], a common medium ingredient.A promoter induced by nitrate deficiency called CvNDI1 has also been recently reported [56].This might be an interesting promoter for the induction of genes associated with lipid accumulation during N starvation, but N limitation may impact protein expression if the goal is to produce recombinant proteins at high yield.Recently, a salt-inducible promoter (SIP) for C. vulgaris was developed for the expression of VHSV glycoprotein and 4.11 mg/g of wet cells was produced using this system [45,46].Lastly, an estrogen-inducible promoter system using the XVE-transcriptional activator and the CaMV35S promoter was reported by Ng et al. (2016) for the expression of lethal bacterial toxins yoeB and pezT fused to green fluorescent protein (GFP).This system proved to be effective for toxin production, since cell viability decreased to almost 0.1% within 24 h of induction for the toxins, while a minimal effect on viability was observed in the GFP-only Figure 3. Organization of genetic elements for expression in Chlorella spp.The promoter (inducible or constitutive) is placed upstream of an optional 5 ′ UTR and transit peptide.This is followed by the coding sequence, which may be interrupted by an intron to enhance expression.A 2A tag can be used for multigene expression using a single promoter.The terminator containing the polyA signal is needed for mRNA transport to the ER for translation.
Inducible promoters allow for the controlled activation or repression of gene transcription in response to specific external signals or inducers.An HSP70/RbcS2 fusion promoter has been reported to contain highly light-inducible elements; however, although both promoters have been used in Chlorella spp., this effect has not been confirmed in Chlorella sp.[70].The NR promoter is inducible using nitrate [36,63], a common medium ingredient.A promoter induced by nitrate deficiency called CvNDI1 has also been recently reported [56].This might be an interesting promoter for the induction of genes associated with lipid accumulation during N starvation, but N limitation may impact protein expression if the goal is to produce recombinant proteins at high yield.Recently, a salt-inducible promoter (SIP) for C. vulgaris was developed for the expression of VHSV glycoprotein and 4.11 mg/g of wet cells was produced using this system [45,46].Lastly, an estrogen-inducible promoter system using the XVE-transcriptional activator and the CaMV35S promoter was reported by Ng et al. (2016) for the expression of lethal bacterial toxins yoeB and pezT fused to green fluorescent protein (GFP).This system proved to be effective for toxin production, since cell viability decreased to almost 0.1% within 24 h of induction for the toxins, while a minimal effect on viability was observed in the GFP-only control [71].In the chloroplast, the endogenous rbcL and 16S promoters [43] have been effective, as well as the ribosomal RNA (rrn) promoter from C. reinhardtii [47].
Various terminators have been used in Chlorella spp.; however, the nopaline synthase (NOS) terminator has been used the most frequently, with 30 reports.Other terminators used include the 3 ′ UTRs from CaMV35S, CYC1 from S. cerevisiae, PDS, HSP70, and octopine synthase (OCS) from A. tumefaciens for nuclear transformations, while the terminator from psbA has been used in chloroplast transformants [33].Tobacco extension 3 ′ UTR (Ext3) was used as the terminator during the expression of SARS-CoV-2 receptor-binding domain (RBD) and basal fibroblast growth factor (bFGF), the bioactivity of which was confirmed by immunoassays [24].

Enhancers, Introns, and Signal Peptides
Several genetic elements have demonstrated utility in modulating or enhancing gene expression within Chlorella species (Figure 3).For instance, the incorporation of translational enhancers such as the Tobacco Mosaic Virus (TMV) Ω 5 ′ UTR has been observed to significantly increase the expression of GUS in C. ellipsoidea [68].Also, the integration of the first intron derived from the endogenous norflurazon-resistant phytoene desaturase gene has been shown to increase the expression of engineered norflurazon-resistant phytoene desaturase by 91% in C. zofingiensis [36].While the effect of intron 1 from IV2 from the potato ST-LS1 gene placed in the GUS gene was not directly studied, GUS activity was detected in this construct [48].Transit peptides for localization in the chloroplast or for secretion have been investigated.The transit peptide from RbcS was predicted by ChloroP 1.1 and fused to enhanced green fluorescent protein (EGFP) to confirm its ability to target the chloroplast in C. vulgaris [30].A synthetic extracellular transit peptide (MANKLLLLLL-LLLLPLAASG) was developed by Hawkins et al. (1999) in one of the earliest reports of Chlorella bioengineering.After 6 days, between 200 and 600 µg/L of human growth factor (hGH) was detected in the extracellular media [67].Human granulocyte colony-stimulating factor (hG-CSF) was also produced in the spent media of C. vulgaris after performing proteomic analysis of the secretome, which identified a putative cellulase and a Ras-related RABF1 protein as highly secreted proteins [56].

Model Proteins
Both basal fibroblast growth factor (bFGF) and human growth factor (hGH) have been used to study the expression of recombinant proteins in Chlorella spp.because they have readily available commercial ELISA kits that allow for their targeted quantification.SARS-CoV-2 receptor-binding domain (RBD) is also quickly becoming a model protein due to the sheer wealth of information available as a result of the coronavirus pandemic [24].The expression of SARS-CoV-2 RBD and basal fibroblast growth factor (bFGF) was confirmed by SDS-PAGE and immunoassays in C. vulgaris [24].Model proteins like EGFP and GUS are routinely used to investigate transformation efficiencies, evaluate transit peptides, or to test the efficacy of an antibiotic marker, for example (Table 3).GUS activity is absent in many organisms, making it a highly specific indicator, and GUS exhibits exceptional stability under various experimental conditions [72].The enzyme can be used with a diverse range of substrates, and the quantification of GUS activity can be achieved through fluorescent assays, spectrophotometric assays, or histochemical assays, making it a particularly useful reporter protein.
Fluorescent proteins like GFP do not need additional substrates for detection and can be monitored non-invasively through external illumination, thereby preserving host cell viability and eliminating the need for supplementary extraction steps.Additionally, GFP variants like mGFP5 can exhibit notable stability, facilitating real-time quantification, visualization, and localization of expression dynamics [73].EGFP and mCherry (a red variant) expression are also used and are clearly distinguishable from chlorophyll autofluorescence using both confocal microscopy and fluorometry [65,74], although background fluorescence in pigmented organisms is higher than traditional model systems like E. coli or yeast cells.Luciferase has also been used in C. ellipsoidea and C. vulgaris [46,75], but fluorescent proteins are generally preferred, as many of the genes and vectors used in Chlorella spp.are derived from plant studies and it is challenging to deliver the substrate luciferin to plant cells.

Recombinant Protein Production
Studies aimed at recombinant protein production in Chlorella sp.often take advantage of its edible nature (Table 4).Early on, flounder growth hormone (fGH) was expressed in C. ellipsoidea to create a feed additive for flounder fry, which increased fish growth by 25% after 30 days of feeding [32].Applications using recombinant C. vulgaris as edible vaccines have also been recently reported.C. vulgaris expressing the glycoprotein from viral hemorrhagic septicemia virus (VHSV), which infects salmonids, resulted in significantly lower mortality than in the control groups [45].In this study, the VHSV G protein comprised 4.11 mg/g wet cells, potentially the highest yield for any Chlorella system reported thus far.The same group used C. vulgaris to express VP28 from white spot syndrome virus (WSSV), which affects shrimp farms and can easily result in 100% mortality.Feeding white-legged shrimp (Litopeneus vannamei) a diet containing the VP28 expressing C. vulgaris reduced the mortality to 20% [28].Oral prophylactics are much easier to administer to large populations of farmed fish or shrimp, and if scaled, they could account for significant economic savings in these important industries.

Metabolic Engineering
While some studies have specifically aimed to produce and characterize the production of recombinant proteins, many studies have been aimed at enhancing lipid accumulation of Chlorella spp., reflecting the extensively studied use of Chlorella spp.for biodiesel production in the past (Table 6).However, protein expression plays a fundamental role in metabolic engineering.Examples of lipid engineering in Chlorella spp.include over-expression of lipid metabolism-associated proteins such as diacylglycerol acyltransferase (DGA) [35,86,87], ω-3-desaturase (FAD) [69], malic enzyme [88], and others to increase lipid accumulation in C. vulgaris, C. sorokiniana, C. protothecoides, C. minutissima, and an unidentified Chlorella sp.Other attempts to increase lipid production have looked toward increasing the phototrophic efficiency of C. vulgaris through the expression of fructose 1,6-bisphosphate adolase (FBA) in the chloroplast using a plastid signal peptide [30].The malic enzyme is a key enzyme in the regulation of lipid metabolism through its role as a producer of the reducing power needed for lipid biosynthesis.Diacylglycerol acyltransferase (DGA) P Hsp70-RbcS2 -dga-T NOS C. sorokiniana Enhance lipid production [35] Transcription factor (TF) engineering has also been used in C. vulgaris [59], C. ellipsoidea [27,93], C. sorokiniana [94], and others [92] to enhance lipid accumulation.Expression of TF GmDof4 from soybean (Glycine max) resulted in an approximately 50% increase in lipid content [27].Other TFs are known to enhance lipid accumulation in Arabidopsis thaliana, such as AtLec2, which upregulated photosynthetic proteins in C. sorokiniana, increasing the relative electron transport rate and doubling the lipid content of the cells compared to the wild type [94].Finally, expression of carbonic anhydrase from the bacterium Mesorhizobium loti resulted in higher cell densities in both C. vulgaris and C. sorokiniana when cultured with 1% CO 2 , and they also showed increased lipid content [91].Carbonic anhydrase is an important enzyme in the carbon-concentrating mechanisms used to drive carbon fixation and plays an important role in CO 2 uptake from air, where the concentration of CO 2 is rate limiting.Other efforts toward metabolic engineering in Chlorella spp.are limited, but include the knock-down of PsbO for a 10-fold increase in hydrogen production [95]; an increase in lutein production due to the expression of Vitreoscilla hemoglobin (vgb) [89]; and the production of the high-value product crocetin, a major active ingredient from saffron crocus [90].

Conclusions
Recent progress in gene expression in Chlorella spp. is establishing C. vulgaris as a promising host for the photosynthetic production of chemicals like lipids for biodiesel, hydrogen, and even some isoprenoids like lutein and crocetin.The latest demonstration of very high protein production for VHSV G for the oral vaccination of fish is an important step towards achieving more comparable yields to current model host systems.However, to advance this platform, some basic research aimed towards understanding the effectiveness of antibiotics under different growth conditions, establishing a greater number of inducible promoters, and understanding how bioprocess conditions affect protein expression is needed.One major limitation is the random nature of the current technique for nuclear transformation.However, the HR-based system for KO of NR and the recently demonstrated CRISPR-cas9 system hold promise that site-directed integration in Chlorella spp.will soon become a more established practice, fostering greater precision and reproducibility in genetic manipulation techniques.While substantial progress has been made, ongoing research holds the potential to unlock several avenues, including investigating untransformed Chlorella species, enhancing the versatility of genetic elements, developing more precise and efficient transformation methods, optimizing bioprocess conditions to enhance productivity, and exploring more biotechnological applications for Chlorella spp.

Figure 1 .
Figure 1.Cumulative tally of studies expressing recombinant proteins using Chlorella sp.

Figure 1 .
Figure 1.Cumulative tally of studies expressing recombinant proteins using Chlorella sp.

Figure 2 .
Figure 2. Comparison of transformation methods used for Chlorella spp.genome engineering.(Left) Agrobacterium-mediated transformation randomly integrates the T-DNA into the host nuclear genome.(Center) EP, PEG-mediated transformation, and MPB can be used for random integration of DNA into the nuclear genome, and in the case of MPB, into the chloroplast genome.(Right) Sitedirected methods for both nuclear and chloroplast transformation have recently been developed.Nuclear transformation into the nitrate reductase locus allows for selection of auxotrophic transformants.

Figure 2 .
Figure 2. Comparison of transformation methods used for Chlorella spp.genome engineering.(Left) Agrobacterium-mediated transformation randomly integrates the T-DNA into the host nuclear genome.(Center) EP, PEG-mediated transformation, and MPB can be used for random integration of DNA into the nuclear genome, and in the case of MPB, into the chloroplast genome.(Right) Sitedirected methods for both nuclear and chloroplast transformation have recently been developed.Nuclear transformation into the nitrate reductase locus allows for selection of auxotrophic transformants.

Figure 3 .
Figure 3. Organization of genetic elements for expression in Chlorella spp.The promoter (inducible or constitutive) is placed upstream of an optional 5′UTR and transit peptide.This is followed by the coding sequence, which may be interrupted by an intron to enhance expression.A 2A tag can be used for multigene expression using a single promoter.The terminator containing the polyA signal is needed for mRNA transport to the ER for translation.

Table 1 .
Comparison of reference genome sizes of common expression systems and well-studied microalgae from NCBI RefSeq.

Table 2 .
Antibiotics and herbicides used for selection of nuclear transformations in Chlorella spp.

Table 3 .
Studies using model proteins to investigate transformation and genetic elements.

Table 4 .
Recombinant protein expression in Chlorella spp.

Table 6 .
Studies using metabolic engineering to enhance lipid production of heterologous compounds in Chlorella spp.