Photosynth Res DOI 10.1007/s11120-014-9994-7

REVIEW

Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses Beth A. Rasala • Stephen P. Mayfield

Received: 21 January 2014 / Accepted: 3 March 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Recombinant proteins are widely used for industrial, nutritional, and medical applications. Green microalgae have attracted considerable attention recently as a biomanufacturing platform for the production of recombinant proteins for a number of reasons. These photosynthetic eukaryotic microorganisms are safe, scalable, easy to genetically modify through transformation, mutagenesis, or breeding, and inexpensive to grow. Many microalgae species are genetically transformable, but the green alga Chlamydomonas reinhardtii is the most widely used host for recombinant protein expression. An extensive suite of molecular genetic tools has been developed for C. reinhardtii over the last 25 years, including a fully sequenced genome, well-established methods for transformation, mutagenesis and breeding, and transformation vectors for high levels of recombinant protein accumulation and secretion. Here, we review recent successes in the development of C. reinhardtii as a biomanufacturing host for recombinant proteins, including antibodies and immunotoxins, hormones, industrial enzymes, an orally-active colostral protein for gastrointestinal health, and subunit vaccines. In addition, we review the biomanufacturing potential of other green algae from the genera Dunaliella and Chlorella. Keywords Biomanufacturing
Recombinant protein production
Transgenic algae
Protein therapeutics
Expression systems

B. A. Rasala
S. P. Mayfield (&) California Center for Algae Biotechnology and Division of Biological Sciences, University of California, 9500 Gilman Dr, La Jolla, San Diego, CA 92093-0368, USA e-mail: [email protected]

Introduction Biomanufacturing is the use of living organisms to convert raw materials into desirable products, and has been used for centuries, mainly for the production of foods like beer, wine, cheese, and bread. Over the last 40 years, advances in genetic engineering and biotechnology have expanded the scope of biomanufacturing, enabling the use of genetically modified organisms for production of recombinant proteins and small molecules. Nowadays, biomanufacturing impacts a broad range of industries, including healthcare, energy, agriculture, materials, and personal care. Current recombinant protein expression hosts include bacteria, yeasts, insect cell lines, plants, mammalian cell lines, and transgenic animals, and each of these production platforms has specific strengths and limitations (Demain and Vaishnav 2009; Ferrer-Miralles et al. 2009; Corchero et al. 2013). Microbial cells such as bacteria and yeasts are the preferred platform for industrial enzyme production because microbes can produce recombinant proteins at relatively low cost (Demain and Vaishnav 2009; Sanchez and Demain 2011). The bacterium Escherichia coli has been a recombinant protein production workhorse, but is best suited for producing small and structurally simple proteins. Bacteria generally do not carry out post-translational modifications such as disulfide bond formation and glycosylation and are inefficient at protein folding and secretion. Furthermore, complex proteins can often end up in insoluble inclusion bodies that necessitate expensive downstream processing (Demain and Vaishnav 2009; Ferrer-Miralles et al. 2009; Corchero et al. 2013). The yeast S. cerevisiae combines the advantages of eukaryotic expression—the ability to fold complex proteins and perform post-translational modifications—with high productivity and inexpensive media requirements associated

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with microbial systems, resulting in high yields and scalability (Celik and Calık 2012). However, S. cerevisiae tends to hyper-glycosylate recombinant proteins, which has been shown to be immunogenic to humans. Mammalian cell lines such as Chinese hamster ovary (CHO) cells are currently the preferred expression platform for large complex therapeutic proteins and those that require post-translational modifications (Zhu 2012). Production of recombinant proteins using CHO cells is a multi-billion dollar business. However, expression levels for some complex proteins can be quite low, and cell lines are often unstable (Corchero et al. 2013). Human cell lines are also now being developed to produce therapeutic proteins because murine-type glycosylation can be highly immunogenic in humans and may cause rapid clearance from circulation (Brooks 2004; Swiech et al. 2012). The growing industrial enzyme market reached $5.1 billion dollars in 2009 (Sanchez and Demain 2011), and the protein therapeutic market is valued at $99 billion (Walsh 2010). While improvements to current expression systems used for biomanufacturing recombinant proteins are ongoing, significant research is also underway to develop alternative protein production platforms that maximize the strengths of eukaryotic expression systems but minimize the aforementioned limitations. Why green algae as a biomanufacturing platform? Green algae—and green microalgae in particular—have attracted considerable attention recently as a biomanufacturing platform for the production of recombinant proteins and small molecules for a range of industries including bioenergy, biopharmaceuticals, biomaterials, nutraceuticals, agriculture and animal health, and cosmetics and personal care. Algae are photosynthetic eukaryotes that lack roots, leaves and other tissues characteristic of higher plants. Green algae include Chlorophytes and the Streptophyte algae, and contain the pigments chlorophyll a and b that give them their characteristic green hue. Green algae are a heterogeneous group estimated to consist of over 8,000 species, and can be unicellular (microalgae) or multicellular (macroalgae), flagellated or not, and live in fresh water or marine environments. There are several advantages to using green algae for biomanufacturing including low cost of production, safety, metabolic diversity, and scalability. Green algae are photosynthetic which means they convert light and CO2 into organic carbon products such as proteins, lipids and carbohydrates. Thus, whether grown in an enclosed bioreactor or outside in a 100-acre pond, the growth media requirements are little more than light, CO2, water, and simple nutrients. The advantages of photosynthetic growth are two-fold. First, media costs are reduced compared to

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conventional protein expression platforms, both in terms of economic cost and input energy costs. Second, the absence of a reduced carbon source reduces risk of contamination by bacteria or fungus, simplifying the upstream manufacturing process and allowing for agricultural-scale production. However, many green microalgae can grow heterotrophically—with a reduced carbon source in the dark—and therefore can be used in existing fermenters with FDA-approved protocols. Safety is another advantage associated with biomanufacturing using microalgae. Several species of green algae are generally recognized as safe (GRAS) for human and animal consumption. Thus, for many applications including recombinant proteins for the food, agriculture, and personal care industries, little to no bioproduct purification may be required. This simplifies downstream processing and formulation, which again results in reduced costs. Indeed, downstream processing costs can constitute up to 80 % of total manufacturing costs for conventional protein production platforms. For example, viral filters, which are used with mammalian cells, can cost $25,000 per production run, and a process-scale protein A column used for antibody purification can cost up to $1.5 million (DePalma 2009; Walsh 2010). Also, algae can be grown in full containment, so the risk of environmental contamination by a transgenic strain is low. Furthermore, because green algae are not traditional foods, the risk of contamination to the human food supply with a therapeutic protein is also low. With over 8,000 species, green algae as a group offers incredible metabolic diversity—which can be harnessed to expand bioproduct profiles and pipelines, especially for small molecules. This genetic diversity can also be exploited to optimize many other aspects of the biomanufacturing platform, including productivity, resistance to contamination, and harvesting and processing characteristics. Finally, microalgae are easily scalable, and can be expanded from a single cell to 1,000 s of liters in a matter of weeks, and to acres of open pond production within months. Thus, green microalgae combine the rapid growth and ease of cultivation inherent to many microorganisms with the photosynthetic ability of plant cells, engendering a positive economic and safety outlook. While much has been written about the potential of microalgae for the production of biofuels and small molecules, this review will focus on green microalgae for biomanufacturing of recombinant proteins. Recent studies have demonstrated the ability of green microalgae to express, fold, post-translationally modify, and secrete complex mammalian and other eukaryotic proteins (Table 1). Furthermore, promising results support the continued research and development for the production of orally available vaccines and gut-active biologics, as well as complex unique anti-cancer therapeutics.

Antibody against antrax

Immunotoxin against B-cell lymphoma

Immunotoxin against B-cell lymphoma

Anti-PA 83 anthrax IgG1

Anti-CD22-ETA sc

Anti-CD22-gelonin sc

Wound repair

Antibody mimic

Antibody mimic

Treatment for anemia

Anti-microbial

HMGB1

14FN3

SAA-10FN3

Erythropoietin

NP-1

Vaccine against white spot syndrome virus

V28

HBsAg

Vaccine against white spot syndrome virus

Surface antigen from hepatitis B virus

E2

V28

Protection against FMDV

Protection against CSFV

VP1-CTB

Subunit vaccines

MAA

Prophylaxis for enteric bacterial infections

C. reinhardtii

Stimulates vasculogenesis and angiogenesis

VEGF

Gut-active biologics

C. reinhardtii

Anti-cancer

Allophycocyanin

D. salina

C. reinhardtii

Dunaliella salina

C. reinhardtii

C. reinhardtii

C. reinhardtii

Chlorella ellipsoidea

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

Anti-radiation

Anti-cancer

TRAIL

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

Expression host

Metallothionein-2

Other protein therapeutics

Antibody against herpes simplex virus

Function

Anti-HSV glycoprotein D lsc

Antibodies and immunotoxins

Recombinant protein

Table 1 Summary of bioproducts manufactured in green algae

Nuclear

Chloroplast

Nuclear

Chloroplast

Chloroplast

Chloroplast

Nuclear

Nuclear

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Genome engineered

Eichler-Stahlberg et al. (2009)

Protein accumulated to above 5 % of total soluble protein

Orally administered VP28-transgenic algae improved survival rates in crayfish upon WSSV challenge

Protein accumulated to 3 ng/mg of protein

Protein accumulated to up to 21 % of total protein

Protein accumulated to 1.6-3.1 ng/mg of protein

Subcutaneous injections elicited an immune response in mice

Protein accumulated to 1.5-2 % of TSP

Protein accumulated to 3 % of TSP

Purified algal-MAA induced mucin secretion, demonstrating bioactivity

Feng et al. (2013)

Surzycki et al. (2009)

Geng et al. (2003)

He et al. (2007)

Sun et al. (2003)

Manuell et al. (2007a)

Chen et al. (2001)

EPO was secreted and accumulated to 100 lg/L Demonstrated anti-microbial activity against bacteria and fungus

Rasala et al. (2010)

Rasala et al. (2010)

Rasala et al. (2010)

Rasala et al. (2010)

Su et al. (2005)

Yang et al. ( 2006)

Zhang et al. (2006b)

Tran et al. (2013a)

Tran et al. (2013b)

Tran et al. (2009)

Mayfield et al. (2003)

References

Fusion of a protein that did not express alone to a wellexpressed protein enabled significant fusion protein accumulation

Protein accumulated to 3 % of TSP

Algal-HMGB1 induced chemotaxis of mouse and pig fibroblasts, demonstrating bioactivity

Protein accumulated to 2.5 % of TSP

Bioactivity demonstrated through a receptor-binding assay

Protein accumulated to 2 % of TSP

Protein accumulated to 2–3 % of TSP

Expression of two genes from a polycistronic vector

Protein accumulated to 0.43–0.67 % TSP

Increased resistance to UV-B

Bound and killed CD22-positive B-cell lymphoma cells

Soluble protein expression, 0.1–0.3 % TSP

Inhibited tumor growth in animal models

Bound and killed CD22-positive Burkitt’s lymphoma cells

Soluble protein expression

Correct assembly of light and heavy chains with disulfide bond formation

Expression of full-length IgG

Bound HSV protein

Dimerization with disulfide bond formation

Soluble protein expression

Notable results

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Malaria transmission blocking oral vaccine

Pfs25-CTB

Feed additives

Feed additives, food manufacturing, paper bleaching

Aquaculture—increased growth rates

Xylanase, a-galactosidase, phytase

b-1,4-endoxylanase

Flounder growth hormone

Sep15

Selenium supplement

Feed additives

Xylanase, a-galactosidase, phytase

Nutritional supplements

Increase phytate phosphorus utilization.

Phytase (AppA)

Industrial enzymes and enhanced animal feeds

Malaria transmission blocking vaccine

Pfs48/45

Malaria vaccine

GBSS-MSP1

Malaria transmission blocking vaccine

Malaria vaccine

GBSS-AMA1

Malaria transmission blocking vaccine

Cancer vaccine against HPV-16

E7 of HPV-16

Pfs25

Protection against S. aureus

D2-CTB

Pfs28

Treatment to prevent onset of type 1 diabetes

Function

GAD65

Recombinant protein

Table 1 continued

C. reinhardtii

C. ellipsoidea

C. reinhardtii

D. tertiolecta

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

C. reinhardtii

Expression host

Nuclear

Nuclear

Nuclear

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Chloroplast

Nuclear

Nuclear

Chloroplast

Chloroplast

Chloroplast

Genome engineered

Human selenoprotein accumulated to detectible levels

Flounder fry exposed to Chlorella-expressed fGH for 30 days exhibited a 25 % increase in both total length and width

Protein accumulated to up to 400 lg/L of algal culture

Developed an improved method for nuclear transformation that led to increased recombinant protein accumulation

Demonstrated secretion of bioactive enzyme

First report of Dunaliella chloroplast transformation

All three classes of enzymes were shown to be bioactive

All three classes of enzymes were shown to be bioactive

Oral delivery of AppA-algae to broiler chicks led to a decrease in phytate content in manure

Oral administration of mice elicited an IgA mucosal response to Pfs25 and CTB

Soluble and folded correctly

Soluble and folded correctly

Soluble and folded correctly

Immunization with transgenic starch particles led to reduced parasitemia with extended life spans upon challenge

Immunization with transgenic starch particles led to reduced parasitemia with extended life spans upon challenge

Tumor protection was demonstrated following tumor cell line challenge

Subcutaneous injections of algal lysates and purified protein elicited specific IgG responses

Protein accumulated to up to 0.12 % of TSP

Oral vaccination of mice offered protection against a lethal dose of S. aureus

Protein accumulated to up to 0.7 % of TSP

Algal-GAD65 stimulated the proliferation of spleen lymphocytes from NOD mice

Protein accumulated to 0.3 % of TSP

Notable results

Hou et al. (2013)

Kim et al. (2002)

Rasala et al. (2012)

Georgianna et al. (2013)

Georgianna et al. (2013)

Yoon et al. (2011)

Gregory et al. (2013)

Jones et al. (2013)

Gregory et al. (2012)

Gregory et al. (2012)

Dauville´e et al. (2010)

Dauville´e et al. (2010)

Demurtas et al. (2013)

Dreesen et al. (2010)

Wang et al. (2008)

References

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Photosynth Res Table 2 Comparison of nuclear and chloroplast genome engineering for recombinant protein production

Nuclear genome engineering

Chloroplast genome engineering

Electroporation, biolistic, agitation with glass beads, silicon whiskers

Biolistic

Gene Integration

Non-homologous end joining

Homologous recombination

Levels of protein accumulation Post-translational modification

Lower: as high as 0.25 % TSP reported

High: 1–21 % of total soluble protein Disulfide bond formation, phosphorylation

Protein localization

Proteins can be targeted to various locations: cytoplasm, nucleus, chloroplast, ER, mitochondria, secretion

Proteins remain in chloroplast

Gene expression machinery

Eukaryotic

Prokaryotic

Inducible gene expression

Nutrient, chemical, physiological

Light inducible

Transformation method

Disulfide bond formation, phosphorylation, glycosylation

Genetic engineering of green microalgae The green alga Chlamydomonas reinhardtii has by far the most advanced molecular genetic toolkit developed for any algae. All three genomes—nuclear, chloroplast and mitochondrial—have been sequenced and transformation protocols and vectors are available for each of these genomes (Maul 2002; Popescu and Lee 2007; Merchant et al. 2007). Recombinant proteins are typically expressed from either the nuclear or chloroplast genomes, and there are significant differences—both advantages and limitations—for proteins expressed from each genome (Table 2, Fig. 1). Nuclear genome transformation generally occurs by random insertion through non-homologous end-joining (Kindle et al. 1989; Mayfield and Kindle 1990). Nuclear genome engineering affords all the advantages associated with eukaryotic expression systems, enabling regulated (inducible) transgene expression, efficient folding of complex proteins, recombinant protein secretion, and posttranslational modifications such as disulfide bond formation and glycosylation. In addition, C. reinhardtii is a haploid organism with a well-characterized sexual life cycle (Harris et al. 2009). Thus, strain engineering and optimization through mating strategies are facile, quick, and straightforward. Limitations of nuclear genome engineering include low recombinant protein accumulation due to transgene silencing and positional effects, which are not well understood (Cerutti et al. 1997; De Wilde et al. 2000; Wu-Scharf 2000). However, recent improvements to transformation vectors have dramatically improved expression levels (Rasala et al. 2012, 2013). Furthermore, promoter engineering has led to improved expression in other systems (Alper et al. 2005; Qin et al. 2011), and may prove applicable to green algae as well. Another limitation is that targeted genome engineering via traditional

homologous recombination is very inefficient (Zorin et al. 2005), which has hindered platform optimization. Developing such technologies could allow for rapid gene knockouts and targeted transgene integration, enabling glycosylation pathway engineering and the development of protease-deficient strains, among many others. However, the generation of gene knock-downs by RNA interference, specifically artificial microRNAs (amiRNA), is possible (Schroda 2006; Zhao et al. 2007; Molna´r et al. 2007). In addition, targeted nuclear gene disruption using engineered zinc-finger nucleases has been recently reported in Chlamydomonas (Sizova et al. 2013). Furthermore, as recent advancements in targeted genome engineering using CRISPR technology (Jinek et al. 2012; Belhaj et al. 2013) or transcription activator-like effector nucleases (TALEN, Gaj et al. 2013) are transferred to green algae, greater opportunities in strain development will arise. C. reinhardtii contains a single plastid (chloroplast) that constitutes up to 70 % of the cell volume. Chloroplast genome transformation occurs through targeted integration by homologous recombination. Recombinant protein expression from the chloroplast genome is characterized by stable expression and high levels of protein accumulation, typically 1–10 % of total protein, but levels as high as 21 % have been reported (Specht et al. 2010; Gong et al. 2011). The green alga lineage arose after a primary endosymbiotic event in which a eukaryotic ancestor engulfed a photosynthetic cyanobacterium, giving rise to the plastid. Thus, the chloroplasts of green algae contain gene expression machinery that is prokaryotic in nature, including the ribosomes and translation factors (Beligni et al. 2004; Manuell et al. 2007b). However, unlike bacteria, the chloroplast contains a wide range of chaperones (Schroda 2004), protein disulfide isomerases (Danon and Mayfield 1994), and peptidylprolyl isomerases (PPIases)

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(Breiman et al. 1992) that assist with folding the complex proteins of the photosynthetic apparatus. This unique biochemical environment allows for the expression of an interesting and valuable class of therapeutic proteins— immunotoxins—that cannot be expressed easily in traditional expression systems (as discussed in more detail below). However, while recombinant proteins expressed from the chloroplast genome can form disulfide bonds, they are not glycosylated. Although C. reinhardtii is by far the most common green algae used to date for recombinant protein expression, it may not be the best strain for commercial production of recombinant proteins. Several other green algae are being considered that are more robust, more productive, or GRAS certified, including species from the genera Chlorella, Scenedesmus, and Dunaliella. Although there is not a great deal of information on recombinant protein production in these species, the information available suggests that each is capable of producing recombinant proteins at levels similar to C. reinhardtii. A direct comparison was made between C. reinhardtii and Dunaliella tertiolecta for the production of five industrial enzymes from the chloroplast genomes, and although there was some variation for each protein tested, in general bioactive recombinant proteins accumulated to similar levels in both species (Georgianna et al. 2013). Similar results were found for recombinant proteins expressed in Scenedesmus dimorphus (R. Georgiana, personal communication). Thus, other species of green algae show potential for recombinant protein production. Antibodies and immunotoxins The protein therapeutic market is valued at $99 billion and is the fastest growing segment of the $600 billion pharmaceutical industry (Walsh 2010). Monoclonal antibodies, which can be effective therapeutics against a variety of human diseases, account for one-third—or $38 billion—of the total biopharmaceutical market (Evers 2010). The first demonstration of mammalian protein production in the algal chloroplast was of a human monoclonal antibody directed against herpes simplex virus (HSV) glycoprotein D. This large single-chain (lsc) antibody, which contains the entire IgA heavy chain fused to the variable region of the light chain by a flexible linker, was expressed in the chloroplast of C. reinhardtii. HSV-lsc accumulated as a soluble protein, properly dimerized by disulfide bond formation in vivo, and bound herpes simplex virus proteins (Mayfield et al. 2003). The algal chloroplast also has the machinery to produce functional and soluble full-length human IgG1 antibodies. Genes encoding the light and heavy chain of a blocking antibody directed against protective antigen 83 (PA83) of anthrax were independently

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transformed into the chloroplast genome of C. reinhardtii. Both the heavy and light chains accumulated as soluble proteins, and importantly, two heavy chain and two light chain proteins properly assembled into a functional, disulfide-bonded full-length antibody (Tran et al. 2009). The fact that the algal chloroplast has the protein folding machinery to produce and assemble complex eukaryotic proteins, like antibodies, but possesses a translational apparatus that is prokaryotic in origin offers a unique opportunity to produce novel proteins such as immunotoxins. Immunotoxins are composed of endocytosing antibodies that are either chemically (Shen et al. 1988) or genetically (Mansfield et al. 1997) coupled to eukaryotic toxins, and can be used as anti-cancer therapies to specifically target and kill cancer cells. However, genetically linked immunotoxins cannot be produced in eukaryotic cells, such as mammalian cells or yeasts, because the toxins typically target the protein translation machinery, inhibiting host cell proliferation. On the other hand, prokaryotic protein production systems like E. coli cannot properly fold complex proteins like antibody fragments or full-length antibodies, and thus require expensive and cumbersome protein refolding and ex vivo disulfide bond formation to obtain active therapeutics. Interestingly, Tran et al. (2013b) demonstrated that the algal chloroplast can produce and assemble soluble single-gene monovalent and divalent immunotoxins, using a single-chain antibody fragment directed against CD22—a B-cell surface protein fused to the translocation and enzymatic domain of exotoxin A from Pseudomonas aeruginosa. Both monovalent and divalent versions of anti-CD22-exotoxin A immunotoxin specifically recognized and killed the CD22 positive Burkitt’s lymphoma cell line, with the divalent protein fusion being 20-fold more effective than the monovalent fusion protein. Furthermore, both immunotoxin molecules significantly inhibited tumor growth in animal models, resulting in improved mouse survival in a tumor-challenge assay (Tran et al. 2013b). Similar results were obtained when monovalent and divalent anti-CD22 single-chain antibodies were fused to a different eukaryotic toxin, gelonin, from Gelonium multiflorum (Tran et al. 2013a). Production of these fusion proteins highlights a unique application of protein production in chloroplasts, because no other protein production system appears capable of expressing these chimeric molecules (Fig. 1). Other protein therapeutics Green algae have been engineered to produce other types of protein therapeutics as well. The human metallothionein-2 gene product, which is considered to have some functional benefit after certain types of radiation exposure, was expressed in the chloroplast of C. reinhardtii and

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Fig. 1 Green microalgae as a versatile expression host for recombinant proteins. The microalgal chloroplast genome has been engineered to express a variety of therapeutic proteins and industrial enzymes. Characteristics of chloroplast engineering and recent successes are summarized (pale green boxes). Proteins that are uniquely suited for chloroplast expression—because they cannot be

easily produced in other expression systems—are highlighted. Characteristics of nuclear genome engineering and recent successes are also summarized (blue boxes). Nuclear genome engineering is best suited for proteins that require secretion, glycosylation, or protein targeting to subcellular locations other than the chloroplast

demonstrated to improve resistance to UV-B exposure compared to wild type cells (Zhang et al. 2006b). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that induces the process of apoptosis, primarily in tumor cells (Wiley et al. 1995). TRAIL was expressed in the chloroplast of C. reinhardtii and shown to accumulate to 0.43–0.67 % of total soluble protein (TSP) (Yang et al. 2006). Allophycocyanin (APC), composed of an alpha and beta subunit, is one of the photosynthetic antenna proteins in cyanobacteria and red algae, and has been reported to inhibit S-180 carcinoma in mice (Tang et al. 2004). The alpha and beta subunits of APC from the cyanobacterium Spirulina maxima were expressed in the chloroplast of C. reinhardtii using a polycistronic expression vector, and the subunits accumulated to 2–3 % of TSP (Su et al. 2005). To gain a better understanding of the versatility of the green algal chloroplast to produce human protein

therapeutics, we tested whether algal chloroplasts could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, four expressed at levels sufficient for commercial production, including two antibody mimics, vascular endothelial growth factor (VEGF), and HMGB1 (Rasala et al. 2010). Three of these expressed at 2 %-3 % of TSP, while a fourth protein accumulated to similar levels when translationally fused to a well-expressed fusion partner. Furthermore, all of the chloroplastexpressed proteins were soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivity are comparable to conventional protein expression platforms, demonstrating the utility of the C. reinhardtii chloroplast as a viable platform for human therapeutic protein production (Rasala and Mayfield 2011).

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The nuclear genomes of green algae have also been engineered for the expression of protein therapeutics. There are several advantages associated with nuclear genome engineering, including the ability to glycosylate protein therapeutics. Glycosylation of proteins occurs in the secretory pathway, as proteins travel through the endoplasmic reticulum and Golgi. Approximately, 40 % of all approved protein therapeutics are glycosylated (Walsh 2010), which is often important for protein folding, function and/or stability. For example, human erythropoietin (EPO) is a glycoprotein hormone that is used as a treatment for anemia caused by kidney failure or anticancer treatments. EPO has a retail cost of over $2 billion/kg, thus EPO may be one of the most expensive products sold today (Corchero et al. 2013). EPO is currently produced in mammalian cells, however, human EPO was expressed from the C. reinhardtii nuclear genome and successfully secreted into the culture, medium to a concentration of 100 lg/L (Eichler-Stahlberg et al. 2009). Furthermore, algal-expressed EPO displayed a molecular weight of 33 kDa, which is greater than the predicted molecular weight of the unmodified protein (19 kDa) and resembles the physiological molecular weight of human EPO, which is 34 kDa. These data suggest that algal-expressed EPO is indeed glycosylated, although the authors did not confirm this experimentally. The green microalga Chlorella ellipsoidea has also been engineered to express therapeutic proteins. A series of papers describe the nuclear transformation of C. ellipsoidea for the expression of mature rabbit neutrophil peptide 1 (NP-1) (Chen et al. 2001; Zhang et al. 2006a; Bai et al. 2013). NP-1 is a member of a class of antimicrobial peptides called alpha-defensins that are important components of the innate immune system. NP-1 has been shown to have broad-spectrum anti-microbial activity against several Gram-negative and Gram-positive bacteria, pathogenic fungi, and even certain viruses (Bai et al. 2013). Chen et al. (2001) transiently expressed NP-1 in C. ellipsoidea and demonstrated anti-microbial activity against Gram-negative E. coli, Gram-positive Bacillus subtilis, and the fungus Fusarium oxysporum. Later, the same group was able to obtain transgenic C. ellipsoidea that stably expressed NP-1 (Bai et al. 2013). NP-1 accumulated to 11.42 mg/L and the purified protein demonstrated anti-bacterial activity against E. coli at 5 mg/L. Green algae for the production and delivery of gutactive biologics For many years, green algae have been recognized as an excellent source of nutrition and have been used as both food and food ingredients in many products. Edible transgenic green algae have the potential to produce and deliver

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orally-active biologics for human and animal health and nutrition without purification. This will result in reduced costs of production, as downstream processing, like protein purification, can account for 80 % of manufacturing costs (Walsh 2010). Many proteins are orally available and bioactive in the gut including colostrum proteins. A function of colostrum—the early form of breast milk—is to orally deliver anti-microbial and protective proteins of the innate immune system to neonates while their own immune systems mature. One such colostral protein is mammary associated serum amyloid A (MAA). MAA acts as a prophylaxis to prevent pathogenic bacterial infections by stimulating the production of mucin in the small intestines, which blocks bacterial adhesion (Larson et al. 2003; Mack et al. 2003). Manuell et al. (2007a) expressed bovine MAA in the chloroplast of C. reinhardtii and demonstrated protein accumulation above 5 % of TSP (Manuell et al. 2007a). Purified algal MAA was able to stimulate the secretion of mucin3 in the human intestinal epithelial cell line HT29, demonstrating that the protein was bioactive. According to the Center for Disease Control (CDC), enteric infections caused by contaminated food and water account for an estimated 2 million deaths worldwide each year (CDC). Thus the large-scale and inexpensive production of a prophylaxis against enteric bacterial infections in an edible alga could drastically impact human health. Colostral proteins also have the potential to replace the prophylactic use of antibiotics in feed animals, which accounted for 80 % of antibiotics sold by drug makers—or nearly 30 million pounds (FDA 2009). Recently, the CDC issued a report that confirmed the link between routine use of antibiotics in livestock and growing bacterial resistance to antibiotics. Antibiotic-resistant bacteria are thought to kill almost 23,000 people a year and cause an estimated 2 million more antibiotic-resistant infections, according to the CDC. Clearly, there is a need to replace the routine use of antibiotics in feed animals, and alternatives such as orally-active biologics like algal-MAA could have a major impact if they can be successfully deployed economically. Production of recombinant subunit vaccines in green algae Transgenic algae have also gained considerable attention as a platform for the production of vaccines (Specht and Mayfield 2014). Several recombinant proteins have been produced in green algae as potential vaccine candidates against viruses, bacteria, malaria, and other communicable diseases. Recombinant protein vaccines for viral diseases have been produced in C. reinhardtii, including the VP1 protein from the foot-and-mouth disease virus, an important disease in livestock (FMDV, Sun et al. 2003). In this case, VP1 was fused to the cholera toxin B subunit (CTB),

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a potent mucosal adjuvant that can bind to intestinal epithelial surfaces. VP1-CTB was produced in the chloroplast of C. reinhardtii and accumulated to 3 % of TSP (Sun et al. 2003). Another antigenic viral protein, the E2 protein from classical swine fever virus, was produced in the Chlamydomonas chloroplast and shown to accumulate to 1.5–2 % of TSP (He et al. 2007). Subcutaneous injection of crude E2-algae extracts elicited an immune response to E2 in mice, however, oral immunization was unsuccessful. The marine green alga Dunaliella salina was transformed with a surface antigen from hepatitis B virus (HBsAg, Geng et al. 2003). Stable nuclear transformants were obtained and expression levels of HBsAg were determined to range from 1.6 to 3.1 ng/mg of protein. The White Spot Syndrome Virus (WSSV) is a pathogen that adversely impacts shrimp farms worldwide. Viral protein 28 (VP28) is the main viral envelope protein and has become a target for exploitation of WSSV subunit vaccines (Hulten et al. 2001). VP28 has been expressed from both the chloroplast of C. reinhardtii and from the nuclear genome of D. salina (Surzycki et al. 2009, Feng et al. 2013). VP28 accumulated in Chlamydomonas chloroplast to 21 % of total protein (Surzycki et al. 2009). VP28 expressed from the nuclear genome of D. salina accumulated to 3 ng/mg of total protein (Feng et al. 2013). Transgenic D. salina lysate was orally administered to crayfish, which was subsequently challenged with WSSV. Notably, Ds-VP28-vaccinated crayfish had significantly higher survival rates (59 % mortality) than the Ds-empty control group, which resulted in 100 % mortality following WSSV challenge. These data indicate that the oral administration of transgenic algae producing subunit vaccines can offer protection against viral pathogens. Subunit vaccines are also being investigated for nonviral diseases. Type 1 diabetes is an autoimmune disease that results in the destruction of insulin-producing betacells in the pancreas. Glutamic acid decarboxylase-65 (GAD65) is one of the major autoantigens for human type 1 diabetes, and studies have shown that immunizing nonobese diabetic (NOD) mice, an animal model for human type 1 diabetes, with GAD65 prevents or delays the onset of diabetes (Tisch et al. 1993). Human GAD65 was expressed in the chloroplast of C. reinhardtii to levels between 0.25 and 0.3 % of TSP (Wang et al. 2008). Algalderived hGAD65 reacted with Type 1 diabetic sera from NOD mice, and stimulated the proliferation of spleen lymphocytes from NOD mice. Staphylococcus aureus is a bacterium found in the respiratory tract and on the skin, and can cause respiratory diseases, skin infections, and food poisoning. The D2 fibronectin-binding domain of S. aureus was fused with the CTB mucosal adjuvant and expressed in the Chlamydomonas chloroplast (Dreesen et al. 2010). D2-CTB accumulated to 0.7 % of TSP, and

oral administration to mice elicited both a mucosal IgA and systemic IgG response to D2. Furthermore, oral vaccination of mice with algal-expressed D2-CTB offered protection against a lethal dose of S. aureus. This was the first demonstration of an effective algal-produced oral vaccine. Cancer vaccines are being developed for the prevention or treatment of certain types of cancers. Gardasil and Cervarix are FDA-approved cancer vaccines that protect against human papillomavirus (HPV) types 16 and 18, which are responsible for about 70 % of cervical cancer cases (NIH http://www.cancer.gov/cancertopics/factsheet/ Therapy/cancer-vaccines). Approximately half a million new cases of cervical cancer are diagnosed annually, culminating in an annual death rate approaching 300,000 (Keam and Harper 2008). The transgene encoding E7 oncoprotein of HPV-16, which is involved in malignant cell transformation, was transformed into the chloroplast of C. reinhardtii and demonstrated to reach 0.12 % of TSP (Demurtas et al. 2013). Subcutaneous injections of both transgenic algal lysate and purified E7 protein induced specific anti-E7 IgGs and E7-specific T-cell proliferation in C57BL/6 mice. Notably, high levels of tumor protection were achieved after challenge with a tumor cell line expressing the E7 protein, indicating that green microalgae are a viable platform for the production of cancer vaccines. Malaria is a mosquito-borne disease that threatens half of the world’s population. The CDC estimated that in 2010, malaria caused 219 million cases of illness and approximately 600,000 deaths (http://www.cdc.gov/malaria/). Malaria is caused by infection from parasites of the genus Plasmodium, and is transmitted by mosquitos that feed on humans. Eradication efforts have focused on both insecticides against the mosquito vector as well as vaccines against disease-causing Plasmodium (Jones and Mayfield 2013). Several recent reports have described successes in algal-produced malaria vaccines. (Dauville´e et al. 2010) fused two clinically relevant malaria antigens, AMA1 and MSP1, to a host cell starch matrix protein—granule bound starch synthase (GBSS). The nuclear genome of C. reinhardtii was transformed with the GBSS-AMA and GBSSMSP, which targeted the fusions to the starch granules in the chloroplast. Mice were immunized both orally and intraperitoneally with the transgenic starch particles and then challenged with a lethal dose of Plasmodium berghei. Both experimental strategies led to significantly reduced parasitemia and increased life span, and intraperitoneal vaccination resulted in complete protection against malaria infection (Dauville´e et al. 2010). A promising strategy for malaria eradication is to combine transmission blocking vaccines (TBVs) that prevent spread of disease with drug-based therapies to treat infected individuals. Three potential transmission blocking subunit vaccines, Pfs48/45, Pfs25, and Pfs28, were

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expressed and characterized in Chlamydomonas (Gregory et al. 2012; Jones et al. 2013). All three are naturally aglycosylated proteins with complex folding including several disulfide bonds, and have proven difficult to produce in traditional protein expression systems (Gregory et al. 2012). Importantly, algal-expressed Pfs48/45, Pfs25, and Pfs28 were shown to fold in the correct conformation required for the generation of transmission blocking antibodies (Gregory et al. 2012; Jones et al. 2013). Mice immunized with Pfs25 and Pfs28 produced antigen-specific antibodies. Furthermore, antibodies produced against algalexpressed Pfs25 completely blocked the transmission of Plasmodium oocytes (Gregory et al. 2012). To determine whether Pfs25 could be developed as an effective oral vaccine against malaria, Pfs25 was fused to the mucosal adjuvant CTB and expressed from the chloroplast genome of C. reinhardtii (Gregory et al. 2013). Mice fed freezedried algae containing Pfs25-CTB mounted a mucosal IgA response to Pfs25 and an IgA and IgG response to CTB. While mucosal IgA antibodies cannot protect against the transmission of malaria, the study does support the conclusion that the transgenic algae platform is an excellent candidate for oral vaccines against mucosal infections. Industrial enzymes and enhanced animal feeds from algae Industrial enzymes are used in many industries including food, detergents, textiles, leather, pulp and paper, and diagnostics. Recombinant DNA technology has fueled a growth in the industrial enzyme market, expanding from $1.6 billion in 1998 to $5.1 billion in 2009 (Sanchez and Demain 2011). Nowadays, 50 % of industrial enzymes are produced recombinantly, and several classes of enzymes have been successfully expressed in microalgae. Phytases are widely used as feed additives for monogastric livestock, such as poultry and swine, to increase phytate phosphorus utilization from grain. Transgenic C. reinhardtii lines were generated that produced a bioactive phytase enzyme (AppA) from E. coli (Yoon et al. 2011). Young broiler chicks fed a normal starter diet supplemented with AppAalgae had significantly lower phytate content and increased inorganic phosphate content in their manure, suggesting that algal-expressed AppA led to the breakdown of dietary phytate in the digestive tract. The study demonstrates a proof of concept of using transgenic microalgae as a direct food additive to deliver dietary enzymes without protein purification (Yoon et al. 2011). Besides phytase, other enzymes are used at industrial scale as supplements in animal feeds. Georgianna et al. (2013) transformed the chloroplast of C. reinhardtii and Dunaliella tertiolecta with 14 heterologous genes encoding important feed enzymes, including T. reesei endoxylanase,

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two b-mannosidases, four a-D-galactoside galactohydrolases (a-galactosidase), four phytases, and three cysteine proteases. Five of these enzymes were well expressed in the chloroplast of D. tertiolecta and shown to be bioactive: xylanase, two a-galactosidases, and two phytases. The chloroplast of C. reinhardtii also supported expression of the same classes of enzymes. Importantly, this report was the first demonstration of chloroplast transformation in the marine green alga D. tertiolecta (Georgianna et al. 2013). The industrial enzyme b-1,4-endoxylanase—which functions to degrade hemicellulose—is used in the manufacturing of baked goods, beverages, textiles, and pulp and paper, and could potentially be used in the future at large scale for cellulosic biofuel production (Beg et al. 2001; Polizeli et al. 2005). Many of these applications require that xylanase should be purified away from the algal biomass; therefore developing methods for industrial enzyme secretion is desirable. We engineered the nuclear genome of C. reinhardtii to express and secrete recombinant xylanase from T. reesei (Rasala et al. 2012). Xylanase was codon-optimized and fused to a Chlamydomonas secretion signal from the endogenous arlysulfatase 1 protein (SPxyn). To overcome transgene silencing that often hinders heterologous expression of genes from the nuclear genome, we transcriptionally fused SP-xyn to the selection marker, Ble. A self-processing viral peptide (2A) was inserted between Ble and SP-xyn, which led to the cleavage of SPxyn from Ble and the secretion of bioactive xylanase into the culture medium. Notably, the Ble-2A nuclear expression vector led to over 100-fold more xylanase activity than the traditional (unfused) nuclear transformation vector (Rasala et al. 2012). Another potential application for transgenic green algae is the oral delivery of growth hormones to aid in aquaculture. Oral administration of growth hormones has growth-promoting effects in fish. The flounder is an important fish for aquaculture in Asia, especially Korea, where a group engineered the expression of a flounder growth hormone (fGH) from the nuclear genome of Chlorella ellipsoidea (Kim et al. 2002). Expression levels of fGH were estimated to be over 400 lg/L of algal culture. Transgenic fGH Chlorella were fed to brine shrimp and rotifers that were then fed to flounder fry. Flounder fry exposed to Chlorella-expressed fGH for 30 days exhibited a 25 % increase in total length and width. In a similar earlier study, human growth hormone (hGH) was expressed in and secreted from Chlorella sp. (Hawkins and Nakamura 1999). hGH was reported to accumulate to 200–600 ng/ml in the culture media, although no western blots or bioactivity assays were provided. Together these studies suggest that the oral delivery of algae-expressed growth hormones for aquaculture, and potentially other applications, holds promise.

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Nutritional supplements produced in algae Selenium is required for human and animal health, and deficiencies are associated with several diseases. At least 40 lg/day of supplemented selenium is recommended for adults to maintain expression and function of endogenous selenoproteins (Rayman et al. 2008). Organic selenium has lower toxicity and higher bioavailability than inorganic selenium. Interestingly, while animals and green algae have selenoproteins, land plants and yeasts do not express these proteins. Thus, green algae could provide a vegetarian source for organic selenium supplements. Hou et al. (2013) engineered the expression of a human selenoprotein (Sep15) from the nuclear genome of C. reinhardtii to determine whether this would result in increased bioaccumulation of organic selenium in edible green algae (Hou et al. 2013). Human Sep15 accumulated in transgenic algae as determined by western blotting, although no quantitation was provided. When wild type and transgenic algae were grown in media supplemented with 5 lmol/L of inorganic selenium, wild type cells accumulated 733 lg/g of organic selenium, compared to 1,100 lg/g in the transgenic strain. Although this difference was not statistically significant, it is possible that the use of improved transformation vectors for better nuclear transgene expression may result in higher organic selenium accumulation.

Conclusions Green microalgae have been used to manufacture a variety of recombinant proteins, including antibodies, immunotoxins, growth hormones, and other biopharmaceuticals, as well as vaccines, gut-active nutraceuticals, supplements, and industrial enzymes. Many studies have demonstrated that the chloroplast supports the accumulation of complex, correctly folded and properly disulfide bonded soluble recombinant proteins that are biologically active. Furthermore, nuclear genome engineering enables protein secretion, with evidence of glycosylation of recombinant proteins, although a more in-depth analysis of green algal protein glycosylation is needed. The efficacious oral delivery of gut-active biologics, including a prophylaxis for enteric infections, oral vaccines against viral and bacterial pathogens, growth hormones for aquaculture, and enzyme supplements for enhance animal feeds—without need of protein purification—represents a significant advantage for green microalgae over traditional expression systems such as mammalian cells and E. coli. In addition to the attributes described above, the use of photosynthesis to power biomanufacturing is an intriguing idea to consider. Microalgae are extremely efficient at converting sunlight and CO2 into organic carbon, the

precursor to all recombinant proteins and metabolites. Presumably, solar-powered biomanufacturing will compare favorably to traditional protein expression platforms in terms of economics, and at large scale could be much cheaper than traditional fermentation. Capital costs for production systems could also be reduced. The absence of a reduced carbon source in the growth media should also minimize microbial contamination, thus simplifying both upstream and downstream processing. Furthermore, the ability to scale is favored for photosynthetic systems, as we have seen in modern-day agriculture. Finally, if carbon credits are ever instituted as a means of combatting climate change, then manufacturing platforms that consume CO2— like microalgae—rather than emit it like traditional fermentation will again be advantaged. Taken together, green microalgae offer many desirable traits important for a recombinant protein production platform, including low cost of production, ability to produce complex and unique proteins, scalability, and environmental and bio-safety. Recent successes in the production of many biologically active recombinant proteins in green microalgae demonstrate the potential of the system, and suggest that the time for developing commercial systems for the production of industrial bioproducts using green microalgae is now at hand. Acknowledgments This work was supported the by the Department of Energy (DE-EE0003373), the California Energy Commission (CILMSF #500-10-039) and Triton Algae Innovations. We thank Elizabeth Specht and Miller Tran for reviewing and editing the manuscript. Stephen Mayfield and Beth Rasala are founders of Triton Algae Innovations, a company that may potentially benefit from the commercialization of green algae for the production of recombinant proteins.

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