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High-Value Recombinant Protein Production in Microalgae

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High-Value Recombinant Protein Production in Microalgae 27 High-value Recombinant Protein Production in Microalgae Daniel J. Barrera and Stephen P. Mayfield Department of Biology, University of California–San Diego, San Diego, CA, USA Abstract Increasing interest in recombinant protein technologies for human and animal health applications has spot- lighted microalgae as a platform with the potential to meet a large impending demand. Here we describe an algae protein expression system and compare the advantages and disadvantages to other platforms currently operating on a commercial level. High-value recombinant proteins that have been produced in microalgae are presented, and strategies for developing production strains with improved commercial properties are discussed. Keywords algae; therapeutics; recombinant protein; biotechnology; genetic engineering; transformation 27.1 INTRODUCTION producing more simple proteins, such as proinsulin. Microalgae are an ideal platform for large-scale produc- Together, these two platforms represent 55% and 29% of tion of high-value products because they are fast-growing a $100 billion/year recombinant protein market (Walsh, solar-powered biofactories with minimal nutrient require- 2010). These platforms dominate the market because mam- ments. In addition, many species are generally regarded malian cells have the appropriate cellular machinery to as safe (GRAS) for human consumption, and several are properly fold, assemble, and posttranslationally modify already commercially farmed for various bioproducts rel- complex human proteins, while bacteria boast high growth evant to human or animal health (Pulz & Gross, 2004). rates, cell densities, and product yields through more inex- Recombinant proteins such as protein vaccines, therapeutic pensive cultivation techniques. Both systems are capable of antibodies, and industrial enzymes can also be produced in producing recombinant proteins on a gram-per-liter scale, microalgae where low-cost production can greatly impact but both systems lack easy cost-effective scalability, or are applicability. Furthermore, pending bioavailability, certain limited by the classes of proteins they can produce. parenterally administered therapeutic proteins could be Microalgae, on the other hand, offer scale and cost of delivered in an edible format, greatly reducing the cost production that can potentially rival that of agricultural pro- of these therapeutics. duction, once the platform is developed to produce recom- Currently, mammalian cell cultures such as Chinese ham- binant proteins at the efficiencies of these other systems. ster ovary (CHO) cells dominate commercial production for Mammalian cell culture dominates the current therapeutic complex eukaryotic therapeutic proteins (e.g., monoclonal protein market, but the end products of this system are typ- antibodies), while bacterial systems are widely used for ically only available to those patients who can afford the Handbook of Microalgal Culture: Applied Phycology and Biotechnology, Second Edition. Edited by Amos Richmond and Qiang Hu. C 2013 John Wiley & Sons, Ltd. Published 2013 by Blackwell Publishing Ltd. 532 High-value Recombinant Protein Production in Microalgae 533 extremely high price of these products. To make a compari- some microalgae (Hempel et al., 2011). Although this anti- son, costs of monoclonal antibody production are estimated body was glycosylated and thus potentially immunogenic, to be approximately $150 per gram in mammalian cells, but genetic engineering in the methylotrophic yeast Pichia only $0.05 per gram in plants (Dove, 2002). Furthermore, pastoris demonstrated that human-like glycosylation path- mammalian cell culture production facilities can cost sev- ways can be implemented in transgenic organisms (Choi eral hundred million dollars in upfront construction and et al., 2003; Hamilton et al., 2003), and presumably these equipment costs (Dove, 2002). Microalgae are a promising same genetic modifications could be made in microalgae. system due to inexpensive cultivation costs where media Also from the nucleus, transgenic Nannochloropsis oculata costs are only $0.002 per liter, and the cost of algae produc- expressing bovine lactoferricin (LFB) were able to prevent tion facilities can be a fraction of the cost of a mammalian pathogen infection of the digestive tract when fed to medaka cell culture facility. This is particularly significant for those fish (Li & Tsai, 2009). Table 27.1 lists the recombinant pro- recombinant proteins needed in massive, affordable quan- teins that have been produced from microalgae and assayed tities, such as animal feed, industrial enzymes, or vaccines for bioactivity, to date. for developing countries. Currently, the highest levels of recombinant protein Besides being highly scalable and cheap, microalgae accumulation have consistently been achieved in the have several other advantageous attributes. The nuclear, chloroplast. For example, in C. reinhardtii the mammalian- chloroplast, and mitochondrial genomes are transformable, gut mucin stimulant, mammary-associated serum amyloid and the timeline from generating initial transformants to protein (M-SAA) accumulated to 10% of TSP when grown having characterized, scaled-up production cultures is rel- heterotrophically (Manuell et al., 2007). Although there atively fast for eukaryotic cells at only a few weeks. In have been significantly more microalgae species with addition, algae cytosol and plastids both have the chap- transformed nuclear genomes, relatively few regulatory ele- erones and protein disulfide isomerases that are required ments, transformation vectors, and methods have been iden- for assembling complex therapeutic proteins derived from tified for this compartment which afford the levels of recom- higher organisms (Kim & Mayfield, 1997; Schroda, 2004). binant protein accumulation observed in chloroplasts. The chloroplast is a particularly attractive compartment for the production of certain proteins, because it is unique 27.3 GENETIC TRANSFORMATION in its ability to accumulate proteins lacking glycosylation, OF MICROALGAE which in the case of antibodies may avoid activating the Genetic transformation of photosynthetic microalgae was complement system or antibody dependent cell cytotoxic- first achieved in the chlorophytes (green algae), but has ity (Sawada-Hirai et al., 2004). since been demonstrated in rhodophytes (red algae), phaeo- phytes (brown algae), euglenoids, diatoms, and dinoflagel- 27.2 HIGH-VALUE RECOMBINANT PROTEINS lates as detailed below. Several barriers challenge exoge- PRODUCED IN MICROALGAE nous DNA before integration into an algal genome. These Several highly valuable recombinant proteins have been can include a cell wall and several additional membranes produced in microalgae from heterologous genes trans- depending on the target organelle and species being trans- formed into either the nuclear or chloroplast genomes. formed. Once integrated, the transgene has to pass the Most of these have been produced in the chloroplast of scrutiny of the host cell expression machinery as well as Chlamydomonas reinhardtii, but there have been several potential repair mechanisms and regulatory checkpoints recombinant proteins produced in other microalgae species. (Choquet et al., 1998). The following describes several The first significant therapeutic protein produced in algae methods of transgenesis along with strategies for achieving was a human single-chain antibody (Mayfield et al., 2003). and maintaining the accumulation of recombinant proteins. More recently a full-length human monoclonal antibody Many microalgae transformation techniques were first was expressed in C. reinhardtii chloroplast and was shown developed and refined in the green algae C. reinhardtii to have antigen binding activity similar to the same anti- and then applied to other algae groups. Currently, particle body expressed in the traditional CHO system (Tran et al., bombardment and electroporation are the most frequently 2009). A monoclonal antibody and its antigen were also employed methods for introducing foreign DNA. Other expressed from the nucleus of Phaeodactylum tricornu- methods include agitation in the presence of glass beads tum, and the antibody was reported to accumulate to 8% or silicon carbide whiskers, which require minimal equip- of total soluble protein (TSP) within the endoplasmic retic- ment, but generally have lower transformation rates. How- ulum, levels sufficient to reach gram-per-liter amounts in ever, a recently optimized glass bead-based technique in Table 27.1. Bioassayed microalgae-derived recombinant proteins Biotechnological Protein application Bioassay Genetic source Platform organism Compartment Reference α-HBsAg full-length IgG1 Binds hepatitis B HBsAg binding Homo sapiens Phaeodactylum Cytosol Hempel et al., mAb (CL4mAb) surface antigen ELISA tricornutum 2011 Hepatitis B virus surface Immunogen α-HBsAg binding Hepatitis B virus Phaeodactylum Cytosol Hempel et al., antigen (HBsAg) inhibition ELISA tricornutum 2011 C-terminal domain from the Immunogen Red blood cell entry Plasmodium Chlamydomonas Nuclear encoded, Dauvillee´ et al., apical major antigen inhibition assay berghei reinhardtii chloroplast 2010 AMA1 fused to a truncated and lethal dose directed granule-bound starch mouse survivability synthase (GBSS) C-terminal domain from the Immunogen Red blood cell entry Plasmodium Chlamydomonas Nuclear encoded, Dauvillee´ et al., Major Surface Protein inhibition assay falcipirum reinhardtii chloroplast 2010 (MSP1) fused to a and lethal dose
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