4.23 Microalgae as Bioreactors for Production of Pharmaceutical Proteins S Ma, University of Western Ontario, London, ON, Canada and Plantigen Inc., London, ON, Canada AM Jevnikar, Lawson Health Research Institute, London, ON, Canada and University of Western Ontario, London, ON, Canada NPA Hüner, University of Western Ontario, London, ON, Canada

© 2011 Elsevier B.V. All rights reserved.

4.23.1 Introduction 301 4.23.2 C. reinhardtii as Protein Expression Platforms 302 4.23.3 Pharmaceutically Relevant Proteins Produced in Transplastomic C. reinhardtii 303 4.23.4 Chlamydomonas Chloroplast Expression Systems and Strategies to Increase

Recombinant Protein Expression 304 4.23.4.1 Promoter and UTR Combinations 304 4.23.4.2 Fusion Proteins 305 4.23.4.3 Replacement of Endogenous Chloroplast Coding Regions 305 4.23.4.4 Chloroplast Codon Optimization 305 4.23.4.5 Inducible Chloroplast Gene Expression 306 4.23.5 Conclusions 306 Acknowledgment 306 References 306

Glossary bioreactors Exploitation of biological organisms for the production of foreign proteins. ferredoxin Iron–sulfur protein that accepts electrons from photosystem I. photosystem Pigment protein complexes within the thylakoid membranes of chloroplasts that transform absorbed light into electrical potential energy. Eukaryotes have two photosystems, photosystem I and

photosystem II, which mediate photosynthetic electron transport. plastid genome Chloroplasts are semi autonomous organelles with their own DNA and family of genes psbD. The chloroplast gene that encodes the D2 reaction center polypeptide of photosystem II. RuBP carboxylase–oxygenase (Rubisco) The chloroplast-localized enzyme catalyzing the initial fixation of CO2 in photosynthesis.

4.23.1 Introduction

The worldwide demand for recombinant proteins is growing faster than traditional systems can keep pace. This includes valuable pharmaceutical proteins such as monoclonal antibodies, vaccines, blood factors, hormones, growth factors, and cytokines, besides industrial enzymes and secondary metabolites [1–3]. Recombinant protein production has traditionally relied on microbial fermentation and mammalian cell culture. Although bacterial expression systems offer the potential for high-level expression of recombinant proteins, many eukaryotic proteins are not correctly folded or posttranslationally modified in Escherichia coli and, as a consequence, their biological activity and/or immunological function is greatly limited as compared to the native counterpart. On the other hand, mammalian cell expression systems enable the production of functional heterologous proteins due to their ability to perform posttranslational processing such as glycosylation, phosphorylation, protein assembly, or truncation; however, in most cases, the yield of recombinant proteins expressed in mammalian cells is low. Furthermore, conventional expression systems such as yeast are not suited for industrial-scale production of recombinant proteins at a competitive low cost due, in part, to the requirement for complicated fermentation equipment. Additionally, there are safety concerns regarding recombinant biological products derived from cell lines of human or animal origin, as cultured mammalian cells are vulnerable to potential contamination with pathogenic organisms or oncogenic DNA sequences [1–3]. The need for safe and cheap sources of large amounts of recombinant proteins has promoted the development of alternative production systems. Green microalgae are emerging as a potentially valuable new expression system for the large-scale sustainable production of recombinant proteins.

Microalgae constitute a large and diverse group of single-cell, plant-like organisms that are able to utilize energy from solar radiation and convert it into chemical energy via the process of photosynthesis. They play significant roles in ecology, accounting for about 50% of global organic carbon fixation. They are also a rich source of carbohydrates, oil, protein, enzymes and fiber, vitamins

301

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(A, C, B1, B2, and B6), and minerals (iodine, potassium, iron, magnesium, and calcium), and have provided an important food source in many countries. Additionally, they can be used as health-care products and nutritional supplements (see reviews in References 5 and 6). These properties, together with their remarkably fast growth rate and simple nutritional requirements for growth, have motivated growing interest in exploring microalgae for a source of natural products as well as for various biotechno­logical applications. One of the most promising biotechnological applications that has attracted significant attention from both academia and industry in recent years is the utilization of microalgae as a new type of bioreactor for molecular farming of high-value pharmaceutical proteins such as antibodies and vaccines [6]. Although many algal species have the potential to act as production hosts, the unicellular green alga, Chlamydomonas reinhardtii, has so far proved to be the most promising bioreactor system, as high levels of foreign proteins have been obtained via expression through its chloroplast transformation. In this article, we review the recent progress in developing C. reinhardtii as a novel production platform for recombinant pharmaceutical proteins, and discuss the technologies and strategies that could be used to increase the expression of heterologous proteins in the algal chloroplast.

4.23.2 C. reinhardtii as Protein Expression Platforms

The green alga C. reinhardtii is a well-known unicellular eukaryotic model organism that has long been used to study photosynth­esis, chloroplast biogenesis, flagella and motility, regulation of metabolism, cell–cell recognition and adhesion, and many other topics [7, 8]. Indeed, much of the information acquired of the photosynthetic apparatus of plants has been generated through studies of C. reinhardtii. C. reinhardtii has a well-studied genetics, with many known and mapped mutants available and a large supply of expression sequence data available as complementary DNA (cDNA) libraries and expressed sequence tags (ESTs). In addition, there are advanced methods and tools for genetic transformation of C. reinhardtii [9, 10]. The value of this organism has been greatly increased recently by the availability of complete sequence information of its nuclear (genomic information generated at Joint Genome Institute of the US Department of Energy accessible via the institute’s Chlamydomonas Genome Portal on http://www.jgi.doe.gov/chlamy), chloroplast (GenBank accession number BK000554), and mitochondrial (GenBank accession number NC_001638) genomes. A new value that can now be added to C. reinhardtii is its utilization as a novel expression platform for the production of human therapeutic proteins.

The use of C. reinhardtii as a heterologous protein expression system offers several advantages over the traditional bacterial fermentation or mammalian cell-culture methods. As a eukaryotic organism, C. reinhardtii offers superior protein-folding mechan­isms and posttranslational modification systems that are not present in bacteria, which is essential for the biological activity of many mammalian therapeutic proteins [11]. Furthermore, unlike most other eukaryotic algae that are refractory to genetic manipulation using recombinant DNA techniques, foreign DNA can be introduced into the genomes of the nucleus, the chloroplast, and even the mitochondria of C. reinhardtii [9, 10], and thus the organism offers both prokaryotic (chloroplast and mitochondria) and eukaryotic translation systems for a tailored expression of virtually any protein. On the other hand, unlike many other microalgae, C. reinhardtii is easy to grow. It can be cultivated photoautrophically in simple salt-based media or mixotrophically in media containing acetate as a carbon source. It was estimated that when grown in such media with a culture volume of up to 50 l, media costs for C. reinhardtii are roughly US$0.08 l–1, and that when grown at a volume greater than 100 l, these costs drop to US$0.002 l–1 [12]. Being a single-cell microorganism, C. reinhardtii has a very fast growth rate with a doubling time of 8 h under optimal growth conditions, and the length of time required between the generation of primary transformants and their scale-up to production volumes can be as short as 4–6 weeks [12]. Additionally, C. reinhardtii is rich in essential amino acids and protein, with the protein content comprising up to 25% of its dry weight [12]. These attributes, and the fact that green algae are generally regarded as safe (GRAS) by the US Food and Drug Administration (FDA) for human consumption, make C. reinhardtii particularly attractive as an expression platform, as it offers a greater promise for quickly generating large amounts of the required protein at low cost and the possibility for direct oral delivery of protein and peptide drugs. Other benefits of using C. reinhardtii as a protein production platform would include increased product safety, increased containment, and reduced cost of protein extraction and purification. Microalgae do not harbor animal pathogens, reducing the risk of accidental product contamination. As algae, in general, propagate by vegetative means, this reduces the potential for possible gene flow to related organisms and, moreover, C. reinhardtii can be grown in sealed plastic bags or sealed glass containers to ensure full containment. In case the recombinant proteins need to be purified, the use of C. reinhardtii as an expression host will greatly simplify the downstream purification processes relative to transgenic plants. The cellular population of algae is uniform in size and type, and unlike transgenic plant expression, there is no gradient of recombinant protein distribution. Furthermore, cell-wall-less strains of C. reinhardtii can be used for transformation rather than wild type. This will greatly simplify the protein purification and reduces the amount of biomass that goes toward nonproductive ends [12].

The production of foreign proteins using the chloroplast of C. reinhardtii offers substantial advantages. Compared to nuclear expression in C. reinhardtii, chloroplast-based expression results in much higher protein concentration. Furthermore, the chloroplast occupies a large proportion of the cell volume (~60%), with sufficient capacity for significant exogenous protein accumulation [12, 13]. Chloroplast transformation is based on homologous recombination, enabling site-specific integration of the foreign DNA into its genome, which ensures uniform transgene expression in transformants due to the elimination of possible position effects often observed in nuclear transgenic lines [12, 13]. Gene silencing that leads to a decrease or an elimination of transgene expression is completely absent [12, 13]. Chloroplasts also have the capacity to express multiple transgenes from a single operon (transgene stacking), and are able to perform protein modifications such as disulfide bond formation and protein folding and assembly [12, 13]. Recently, Bally et al. [14] demonstrated that plants have the ability and tendency to adapt their growth, development, and

Microalgae as Bioreactors for Production of Pharmaceutical Proteins 303

physiology to the massive foreign protein synthesis occurring in recombinant chloroplasts. There was no profound metabolic perturbation in plants when they expressed massive amounts of recombinant proteins in their chloroplasts. The recombinant proteins are synthesized at the expense of plant resident proteins. For example, RuBP carboxylase–oxygenase (Rubisco), the most abundant protein in nature, acts as a protein sink and exists naturally in quantities greater than metabolically required. Recombinant protein production replaces Rubisco as the protein sink, leading to lower Rubisco levels with rising recombinant protein accumulation, allowing for a measure of natural capacity for recombinant protein production in plants without affecting growth and development.

4.23.3 Pharmaceutically Relevant Proteins Produced in Transplastomic C. reinhardtii

There are an increasing number of therapeutic proteins that have been produced using the chloroplast of C. reinhardtii (Table 1). The expression of a large single-chain human anti herpes antibody, containing the entire immunoglobulin A (IgA) heavy chain fused to the variable region of the light chain, was the first example to test algal chloroplasts for the expression of a pharmaceutical protein [15]. The antibody was synthesized using codons optimized to reflect abundantly translated C. reinhardtii chloroplast messenger RNAs (mRNAs), and its transcription was under the control of either the rbcL or atpA promoter and 5′-untranslated region (5′-UTR) and the rbcL 3′-UTR. The completely soluble protein product accumulated to ~0.5–1% total soluble protein (TSP) in the transgenic chloroplast. Importantly, the algal chloroplast-derived single-chain antibody dimerized to form a correctly assembled, functional antibody that is capable of binding herpes simplex coat protein. These encouraging early results indicate that the algal chloroplast may be suitable for expressing a wide range of different proteins including complex molecules such as antibodies in a soluble and active form.

Since the demonstration of the algal expression of a single-chain human anti-herpes antibody [15], the chloroplast of C. reinhardtii has been increasingly investigated as a new platform for the production of therapeutic proteins. One example is the expression of foot and mouth disease virus VP1 and the B chain of cholera toxin (CTB) as a fusion protein [18]. The fusion protein was accumulated at approximately 3% TSP and was shown to retain both the binding affinity of CTB to its cell-surface receptor GM1-ganglioside and the antigenicity of VP1, supporting the possibility of using transgenic algal chloroplasts as a mucosal vaccine source. Another example is the expression of bovine albumin A3 (mammary-associated serum amyloid A (M-SAA)) in the algal chloroplast [19]. The M-SAA protein, normally found in mammalian colostrum, is a potential mucosal vaccine candidate against bacterial and viral infections in both humans and animals. M-SAA was expressed at more than 5% TSP, and in vitro cell-based assay using human gut epithelial cell line showed that the algal-derived protein was bioactive and stimulated mucin production, a protein with antibacterial and antiviral activity. Recently, Surzycki et al. [21] reported the expression of a viral protein, the white spot syndrome virus (WSSV) envelope protein VP28, in the chloroplast of C. reinhardtii at levels as high as 42% of TSP. This is the highest level of expression of the protein of interest achieved to date in algal chloroplasts. WSSV is a major shrimp pathogen, causing large economic losses, and VP28, one of the major proteins of WSSV, has been shown to be a potential WSSV vaccine candidate [22].

Human glutamic acid decarboxylase 65 (hGAD65) is a key autoantigen in type 1 diabetes, an autoimmune disease resulting from the destruction of insulin-producing β cells in the pancreas [23]. It has the potential as an important marker for the prediction and diagnosis of type 1 diabetes. Moreover, GAD65, as a diabetes-associated autoantigen, presents unique opportu­nities for the development of novel preventative therapies against the disease. However, recombinant production of hGAD65 using conventional bacterial or mammalian cell-culture-based expression systems is limited by high cost, low efficiency, and low

Table 1 Recombinant therapeutic proteins produced in chloroplasts of C. reinhardtii

Expression level Protein product Potential application (% TSP) Reference

Human IgA anti herpes antibody Therapeutic 0.5–1 [15] Single-chain scFv antibody Therapeutic NA [16] Human metallothionine-2 UV protection 0.5 [17] Cholera toxin B subunit fused to foot and mouth Animal vaccine 3 [18] disease VP1

Bovine mammary-associated serum amyloid Prophylaxis against bacterial and viral infection in 5 [19] (M-SAA) newborn mammals

Human glutamic acid decarboxylase Prevention and treatment of type 1 diabetes 0.25–0.30 [20] Infectious bursal disease virus VP2 (IBDV VP2) Vaccine 3 [21] Infectious hematopoietic necrosis virus G protein Vaccine <0.5 [21] (IHNG-G)

Infectious pancreatic necrosis virus VPS Vaccine <0.3 [21] (IPNV VP2)

Porcine circovirus type 2 (PCV2) Vaccine 0.9 [21] White spot syndrome virus VP28 Vaccine 42 [21]

304 Plant Systems

yield. The expression of hGAD65 in nuclear-transformed transgenic plants was also limited by low accumulation levels (0.04% of TSP in tobacco leaf tissues) [24]. We investigated for the first time the feasibility of using C. reinhardtii chloroplasts as an alternative expression platform for the production of hGAD65. To this end, a chloroplast transformation vector containing the full-length hGAD65 gene, under the control of the C. reinhardtii chloroplast rbcL promoter as well as rbcL 5′- and 3′-UTRs, was generated and introduced into the chloroplast genome of C. reinhardtii [20]. Transformed C. reinhardtii cells were shown to accumulate recombinant hGAD65 at levels of 0.25–0.3% algal TSP, several-fold greater compared to the levels of hGAD65 expression in transgenic plants. Immunological analysis showed that the algal-derived human protein reacted with type 1 diabetic sera from nonobese diabetic (NOD) mice serving as a model for human type 1 diabetes, and stimulated the proliferation of spleen lymphocytes from NOD mice [20]. These results demonstrate that the algal-derived GAD65 protein maintains its authentic antigenicity, indicating the potential possibility of transformed algal chloroplasts as an economical source of a recombinant auto-antigen for the treatment of type 1 diabetes.

4.23.4 Chlamydomonas Chloroplast Expression Systems and Strategies to Increase Recombinant Protein Expression

The chloroplast transformation in C. reinhardtii was established as early as in 1988 by Boynton et al. [10], which was later extended to tobacco [25]. Transformation can be achieved either by bombarding cells with DNA-coated tungsten particles [10] or by agitating cell-wall-deficient cells in the presence of glass beads and DNA [26], although the former method is more efficient and more reliable. Integration of the transforming DNA into the plastid genome occurs exclusively through site-specific homologous recombination. This is in striking contrast with the integration of transforming DNA into the nuclear genome, which occurs randomly [27]. Several approaches have been used to select cells following chloroplast transformation. Early selection methods were based on cloned chloroplast genes used to rescue photosynthetic mutants [10]. Chloroplast genes that confer resistance to antibiotics or herbicides have also been widely used [28]. However, a major breakthrough in chloroplast transformation came with the development of dominant selectable markers based on bacterial genes for antibiotic resistance, such as the aadA gene (aminoglycoside adenyl transferase) conferring resistance to spectinomycin and streptomycin [29]. As the single chloroplast of C. reinhardtii contains ~80 copies of the genome per cell, the formation of stable transformed chloroplasts requires integration of the transforming DNA into each copy of the genome. Homoplasmy can be achieved by allowing for a sufficient number of cell divisions under high selective pressure [10, 26].

There are a number of vectors available for the expression of foreign proteins in C. reinhardtii chloroplasts. In general, these chloroplast transformation vectors contain (1) the promoter and UTRs of C. reinhardtii chloroplast genes (5′-UTR and 3′-UTR) for driving the expression of the transgene; (2) a selectable marker gene (such as aadA gene conferring resistance to spectinomycin) for selection of transformants; and (3) chloroplast flanking sequences used for homologous recombination to insert the gene of interest into specific regions, such as inverted repeat regions, of the chloroplast genome.

The selective marker gene can also be placed on separate vectors or DNA molecules. Co-transformation of the chloroplast genome via particle bombardment with two plasmid constructs, one carrying the selective marker and the other carrying the transgene, is highly efficient, as up to 80% of the transformants obtained following co-transformation were found to contain the co-transformed constructs (see reviews in References 30 and 31). Promoters derived from chloroplast endogenous genes, rbcL, psbA, psbD, atpA, and 16S rRNA, are the most commonly used promoters to drive foreign gene expression, since transcripts of these genes were abundant in C. reinhardtii chloroplasts [32]. The rbcL, psbA, psbD, atpA, and 16S rRNA genes encode the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco LSU), D1 and D2 proteins of photosystem II (PS II), the α-subunit of adenosine triphosphate (ATP) synthase (ATPA), and the 16S rRNA, respectively. The 5′- and 3′-UTRs of plastid RNAs play an important role in the regulation of gene expression. While the promoter and the 5′- and 3′-UTR are essential for the regulation of gene expression, their presence in chimeric gene constructs is not always sufficient to guarantee high levels of protein accumulation in algal chloroplasts [33, 34]. Thus, reported protein expression levels from different groups remained inconsistent. We tested the expression of several other therapeutic proteins in the C. reinhardtii chloroplast in addition to GAD65 and found that some of the target proteins accumulated only to low levels (<0.25% TSP) (unpublished data). Surzycki et al. [21] examined the expression of 20 different recombinant proteins in the chloroplast of C. reinhardtii and showed variable levels of expression ranging from no expression to as high as 42% TSP. This indicates the necessity for a much greater understanding of the factors that determine the expression levels of target proteins in order to achieve economic feasibility regarding algal chloroplasts as a new, reliable, and consistent source of recombinant proteins. To this end, a number of molecular approaches have been investigated in attempts to increase the level of recombinant protein accumulation in the C. reinhardtii chloroplast. These are discussed in more detail below.

4.23.4.1 Promoter and UTR Combinations

Chloroplast protein accumulation has been shown to be impacted by transcription, RNA processing and stability, translation, and protein turnover, and the 5′-UTR of chloroplast mRNAs has key roles in each of these events [11, 35, 36]. Transcriptional efficiency was shown to be regulated by both chloroplast gene promoters as well as sequences (cis-acting RNA elements) found within the 5′-UTR [34]. Furthermore, the cis-acting RNA elements within both the 5′- and 3′-UTRs are also shown to affect transcript stability,

Microalgae as Bioreactors for Production of Pharmaceutical Proteins 305

probably through interactions with trans-acting protein factors that are encoded in the nucleus and imported into the chloroplast [37, 38]. Additionally, a number of studies have revealed that translational efficiency is a key rate-limiting step for chloroplast gene expression and that the 5′UTRs of chloroplast mRNAs have a crucial role in the regulation of chloroplast translation [11, 35, 36]. Therefore, utilization of different promoter and UTR combinations may provide an effective approach to increase the expression of recombinant proteins in algal chloroplasts. To assess the effect of various endogenous promoters and UTRs on recombinant protein expression in C. reinhardtii chloroplasts, Barnes et al. [39] created a series of chimeric genes composed of the promoter from endogenous chloroplast genes, rbcL, psbA, psbD, atpA, and 16S rRNA, 5′- and 3′-UTRs, and a green fluorescent protein (GFP) reporter gene, and transformed into the chloroplast genome. It was shown that the promoter and 5′-UTR of the atpA and psbD genes produced the highest levels of chimeric mRNA and GFP protein accumulation, while the promoter and 5′-UTR of the rbcL and psbA genes produced less mRNA and protein. No expression of the reporter protein was observed using the promoter and 5′-UTR of the 16S rRNA gene. The nature of the 3′-UTR invariably had little effect on reporter protein accumulation. Taken together, these results suggest that different chloroplast promoters and 5′-UTRs support different levels of protein synthesis.

4.23.4.2 Fusion Proteins

Genetic fusions of efficiently translated, highly abundant chloroplast proteins to an exogenous protein of interest may represent another effective strategy for high-level transgene expression in C. reinhardtii chloroplasts. Kasai et al. [40] examined the effect of coding regions of chloroplast genes on foreign gene expression using chimeric genes containing the promoter, 5′-UTR and varying lengths of protein coding regions of chloroplast genes fused with coding sequences of the bacterial β-glucuronidase (GUS) gene as a reporter gene. They demonstrated that C. reinhardtii chloroplast transformants containing either the rbcL-uidA or psbA-uidA chimeric gene, in which the uidA was fused to the coding region of the chloroplast rbcL or psbA gene, produced significantly more mRNA and protein than algal transformants containing gene constructs in which uidA was only fused to the promoter and 5′-UTR of rbcL or psbA. These results indicate that the coding region of chloroplast genes is necessary for efficient expression of foreign genes. In higher plant plastids, sequences downstream of the translation initiation codon, known as downstream box (DB), have been shown to be important determinants of translation efficiency in chloroplasts [41]. Foreign genes fused to DB regions have become an important and effective strategy for increasing recombinant protein expression in plant chloroplasts [42]. One possible disadvantage of the fusion protein approach is that recombinant proteins fused to endogenous chloroplast proteins may have reduced industrial or clinical values. Recently, Muto et al. [43] reported that the luciferase, a reporter gene, accumulated to significantly higher levels when expressed as a fusion protein with the large subunit of chloroplast Rubisco (Rubsico LSU), which is approximately 33 times better than the luciferase gene expressed alone in algal chloroplasts. To ensure that the desired recombinant protein moiety is cleavable from the fusion protein, they incorporated the 32-amino-acid N-terminal chloroplast transit peptide of the nuclear-encoded preferredoxin protein (preFd), an amino acid sequence known to be proteolytically removed from preFd upon translocation of the protein into the chloroplast. Using this strategy, they demonstrated the production of a full-length Rubisco LSU–luciferase fusion protein capable of undergoing in vivo proteolytic processing to yield a biologically active, mature reporter protein.

4.23.4.3 Replacement of Endogenous Chloroplast Coding Regions

Manuell et al. [19] demonstrated that the expression level of bovine M-SAA in the chloroplast of C. reinhardtii can be increased significantly by replacing the coding region of chloroplast psbA gene with the coding region of chloroplast codon-optimized m-saa gene, with accumulation levels beyond 5% of the TSP. By contrast, introduction of the same m-saa gene into a silent site on the chloroplast genome was shown to yield the accumulation of M-SAA to a level of only 0.25% TSP. The replacement of psbA gene rendered the M-SAA-expressing strain nonphotosynthetic; however, their photosynthetic activity can be restored by re-introducing a psbA gene into an alternative site on the chloroplast genome. There are several possibilities for this increased expression of M-SAA in the absence of the psbA gene product, D1 protein. First, a number of C. reinhardtii chloroplast proteins, including D1, have been shown to regulate translation of their own mRNAs via feedback inhibition. Elimination of the psbA gene may have abrogated this auto-inhibitory effect. Alternatively, elimination of the endogenous psbA gene and mRNA may result in increased transcription and translation of the chimeric m-saa gene and mRNA because of reduced competition with the endogenous gene for limiting transcription or translation factors [19].

4.23.4.4 Chloroplast Codon Optimization

It is well recognized that various organisms utilize certain codons in preference to others. Such preferential codon usage also occurs in chloroplasts. For example, the chloroplast of C. reinhardtii displays such codon bias, with codons containing adenine or uracil nucleotides in the third position favored over those with guanine or cytosine [12, 44]. Codon usage bias is an important factor in limiting foreign gene expression in chloroplasts [12, 44]. The adaption of foreign genes to the preferred codon usage of highly expressed chloroplast genes from Chlamydomonas may be another effective strategy for increasing recombinant protein expression in algal chloroplasts. Franklin et al. [39] demonstrated that the optimization of a GFP reporter to reflect chloroplast codon usage increased its expression at least 80-fold as compared to its nonoptimized counterpart. Similarly, Mayfield and Schultz [45] showed

306 Plant Systems

increased expression of the bacterial luciferase reporter when a chloroplast codon-optimized version of this gene was transformed into the chloroplast of C. reinhardtii. These results may indicate the necessity for codon optimization of any gene for which high levels of protein production are desired when using algal chloroplasts as an expression platform.

4.23.4.5 Inducible Chloroplast Gene Expression

Protein toxicity can also be an important factor affecting the accumulation of recombinant proteins to high levels in the algal chloroplast. Surzycki et al. [21] reported that they failed to recover a single C. reinhardtii transformant for some genes, such as DILP-2, which codes for a growth factor, due to the toxicity of the protein to host cells. Therefore, it may be highly desirable to express such proteins in a tightly controlled fashion. An inducible chloroplast gene expression system, which exploits the Nac2 chloroplast protein, has recently been developed by Surzycki et al. [46]. In C. reinhardtii, expression of the D2 component of the photosystem II (PSII), psbD, is dependent on the RNA stabilizing factor Nac2. The inducible expression system consists of the nucleus-encoded Nac2 gene fused to the copper-sensitive cytochrome c6 promoter (cyc6). The Nac2 protein is specifically required for the stable accumulation of the chloroplast psbD RNA and acts on its 5′-UTR. The cyc6 promoter is induced by copper deficiency, and is repressed in the presence of copper. The repression of psbD leads to the loss of PSII. This inducible gene expression system is applicable to any chloroplast gene by replacing its 5′-UTR with the psbD 5′-UTR in the same genetic background.

4.23.5 Conclusions

The ease and low cost to culture the algae on a very large scale, the quickness to produce chloroplast-transformed algal transfor­mants, the potential to express foreign proteins at very high levels, and the ability to assemble and fold complex mammalian proteins have made the chloroplast of C. reinhardtii an extremely attractive bioreactor for the production of high-value biopharma­ceutical proteins. An increasing number of therapeutic proteins have already been produced in the algal chloroplast, with some recombinant proteins demonstrated to accumulate to extremely high levels (42% TSP). Furthermore, in vitro experiments have shown that algal-derived recombinant proteins retain the antigenicity or biological activity of the parent protein. There is no doubt that with additional foreign proteins being produced in the chloroplast of C. reinhardtii, the value and benefits of algal chloroplasts as bioreactors will be further realized. Currently, protein expression levels reported from different laboratories remained incon­sistent, and not all recombinant proteins were found to accumulate to high levels (>1% TSP). However, this is most likely a technical issue, which could be solved by applying a combination of different approaches. Another limitation of chloroplast recombinant protein production is that like bacteria they are unable to perform glycosylation, a necessity for many pharmaceutical glycoproteins including monoclonal antibodies. It is important to note, however, that the basic building blocks, the glycans themselves, are present within the chloroplast [47]. Thus, a future goal may be to generate glycoproteins in chloroplasts with the addition of multiple steps of the glycan addition/modification pathway.

Acknowledgment

The authors gratefully acknowledge the support of the Natural Sciences and Engineering Research Council of Canada for their work on the use of C. reinhardtii chloroplasts as a novel bioreactor for the molecular farming of therapeutic proteins.

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