World J Microbiol Biotechnol DOI 10.1007/s11274-014-1714-0

REVIEW

The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins Fariba Akbari • Morteza Eskandani Ahmad Yari Khosroushahi

•

Received: 5 December 2013 / Accepted: 30 July 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of posttranslational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection

F. Akbari Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran F. Akbari  M. Eskandani Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran M. Eskandani Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran A. Y. Khosroushahi Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran A. Y. Khosroushahi (&) Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Daneshgah Street, P.O. Box 51664-14766, Tabriz, Iran e-mail: [email protected]

markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein. Keywords Codon optimization  Microalgae Dunaliella  Milking  Photobioreactor  Recombinant proteins

Introduction The noticeable merits of microalgae Dunaliella are their numerous features in producing biofuels, pigments, therapeutic and cosmetic proteins. The capability to blend the energy-capturing ability of photosynthesis with the highyield cultivation results in economical production systems (Rosenberg et al. 2008). Enormous efforts have been performed to increase the yield of Dunaliella production in agricultural scale (Courchesne et al. 2009) and several articles reviewed the production issues (Cheirsilp et al. 2011; Greenwell et al. 2010; Li et al. 2008; Williams 2007; Williams et al. 2009). However, few articles were recently published on the production of recombinant proteins in microalgae particularly Dunaliella. According to the National Center of Biotechnology Information, algae of the genus Dunaliella belongs to the class of Chlorophyceae, the order of Chlamydomonadales, and the family of Dunaliellaceae (Polle et al. 2008). Apparently, it seems that nutritional and medical application of Dunaliella produced biomolecules can be appropriate and safe for human consumptions based on the safety of these biomolecules

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(Franklin and Mayfield 2005; Gantar et al. 2008). Thus, among the microalgae, Dunaliella can consider the best and robust targets to study because of its halotolerant properties which makes it succeed in an extremely saline environment. It is also capable of biotechnological processes such as expression of foreign proteins. Dunaliella is currently utilized in b-carotene production as a suitable source of b-carotene (up to 10 % of the biomass) (BenAmotz et al. 1988, 1989; Katz et al. 1995). Dunaliella has been noted as a recombinant protein expression system in the last decade due to rapid growth and convenience cultivation as well as having the potential for post-transcriptional and post-translational modifications. Besides, Dunaliellais one of the best-studied unicellular green algae in the field of physiology, biochemistry and genetic (Oren 2005). The most important characteristics of Dunaliella in genetic and plastome engineering are listed in Table 3. The second virial coefficient, a characteristic of the interaction potential between the particles, depends only on the pair interaction between the particles. The second osmotic virial coefficients of several proteins were measured in salt solutions. The findings showed that, at low salt concentrations, protein–protein interactions can be either attractive or repulsive, possibly due to the anisotropy of the protein charge distribution. At high salt concentrations, the behavior depends on salt: In sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations so that most proteins have a second flat virial coefficient profile with increasing sodium chloride concentration. This explains the high solubility of most proteins in highly concentrated solutions of sodium chloride (Dumetz et al. 2007). On the other view, to solve the insoluble recombinant proteins produced in bacteria, the cultivation of bacteria under osmotic stress or additions of compatible solutes (for example glycine betaine) were commonly applied to protect solved proteins in peripheral space of cells. Upon Dunaliella halotolerancy, it produces high amounts of stress metabolites including glycine betaine which can facilitate protein protection in high salinity habitats (Mishra et al. 2008). Thus, the exponentially increasing demand of by-products such as recombinant proteins in recent years has made the transgenic microalgae research favorable and low cost to carry out and also a high yield system for producing bioactive compounds. Given the rapid developments in transgenic microalgae technology, the present study reviews recent progress in high-level expression of recombinant proteins, transformation methods for both nuclear and chloroplast genomes, strategies to increase recombinant protein yields, and potential research directions of interest.

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Comparison of different expression systems with Dunaliella Nowadays, several different types of expression systems exist to produce therapeutic recombinant proteins, such as bacteria, yeast, insect, mammalian cells, plant cells and micro algae. The production of recombinant proteins in bacterial and yeast-based systems can be considered as a prevalent approach because of genome’s lack of uncomplicated manipulations and their cost beneficial cultivations. However, any post-transcriptional and posttranslational modifications, including glycosylation, phosphorylation and disulfide bond formation are not performed on bacterial systems where these modifications require the correct folding of complex proteins. Besides, recombinant proteins with bacterial toxins have a considerably high contamination possibility. Although many modifications are performed in eukaryotic yeasts, the modified products are not suitable for human consumptions. The hyperglycosylation of recombinant proteins in yeast alters immunogenic epitopes of these proteins and high-mannose glycosylation also result in lower in vivo half life which decreases the therapeutic activity of these proteins (Cereghino and Cregg 2000). Although mammalian cells utilized for recombinant protein production possess various advantages, the development and maintenance of mammalian cell-based bioreactors comprise remarkable costs. Several difficulties regarding mammalian cell-based bioreactors including complex nutrient requirements, poor oxygen and nutrient distribution, waste accumulation, contamination by pathogens and high sensitivity of cells to shear stress limit using of these systems for recombinant protein production (Zhang et al. 2008, 2010). Insect cell cultures require complex nutrient media to cultivate but in comparison with mammalian, cell culture are easier and insect cells possess a high tolerance to osmolarity changes that can be considered an advantage for this system. Among the expression systems in insect cells, baculovirus system can express the high level and quality of recombinant protein but its lytic operating mechanism leads to decrease product yields due to endogenous proteases release (Ikonomou et al. 2003). Plant-based reactors considered the use of inexpensive bioreactor systems where the growth of plant cells in culture systems is less than mammalian or insect cells. Moreover, the gene flowing phenomenon in transgenic plants may be harmful for the environment (e.g., via pollen and seed dispersal). Protein glycosylation phenomenon is different between animal and plant cells; thus, allergic reactions are the major concern of plant-derived proteoglycans for human consumptions. The combination of low-cost, uncomplicated requirements, rapid growth and high potential system for post-

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transcriptional/translational processing can notably be considered as advantageous in microalgal cultivation methodology. In diverse protein production, unicellular photosynthetic green algae which are mostly classified as ‘‘generally regarded as safe’’ (GRAS), are frequently utilized because of their comfortable purification and processing of expressed proteins. Furthermore, carbon dioxide and light are indispensable elements for microalgae growth in salt-based media which are remarkably inexpensive elements. Regarding economical considerations, based on recombinant antibody production studies, functional antibody cost (per gram) is 2 $150, $0.05 and $0.002 (USD) in mammalian, plant and microalgal bioreactor systems respectively, making microalgal system very economically attractive (Mayfield et al. 2003; Mayfield and Franklin 2005). Despite the recent interest and successful transformation of microalgal species such as Chlamydomonas, Chlorella, Volvox, Haematococcus and Dunaliella genera, many obstacles still remain to overcome before microalgae can be considered in standard expression systems. Although the low expression levels of recombinant proteins in microalgal systems are primarily delayed in the usage of this system for protein production, the continuing development of genetic engineering tools for microalgae transformation has allowed the expression of fully functional antibodies (Franklin and Mayfield 2005; Jones et al. 2012; Manuell et al. 2007; Tran et al. 2009), therapeutics (Boehm 2007; Rajamani et al. 2007; Rasala et al. 2011; Rasala and Mayfield 2011; Siripornadulsil et al. 2002, 2007), and bactericides (Li and Tsai 2009) at economically viable levels.

Transformation methods for Microalgae Dunaliella Gene delivery is the first critical stage in the creation of transgenic organisms. The release of a gene to plant cells is more difficult than mammalian cells because the cell’s wall in plant cells acts as a barrier. According to Gabriel Potvin, fortunately microalgae ‘‘best of both worlds’’ green algae has contemporarily both positive properties of mammalian and plant cells (Sadka et al. 1991). To transform Dunaliella, some traditional methods are still applied in recent work. A summary of routinely used methods for transformation of Dunaliella is briefly explained in the following. (1) At biolistic particle delivery system, the method originally designed for plant transformation, target cells are bombarded with DNA-coated metallic (gold or tungsten) particles (Sanford et al. 1993; Smith et al. 1992). This was first utilized for Chlamydomonas reinhardtii transformation in 1988 (Newman et al. 1990, 1992). The biolistic

method was also successfully exploited for nuclear and chloroplast transformation of Dunaliella (Jiang et al. 2005; Tan et al. 2005). This method is not widely applied to generate a large number of nuclear transformants in microalgae due to its requirements for expensive laboratory equipments. (2) Electroporationis another method that was previously utilized for the transformation of Dunaliella by numerous researches (Geng et al. 2010, 2011; Sun et al. 2008, 2005; Wang et al. 2007). This method additionally requires specific expensive equipments. Moreover, this method of transformation efficacy is influenced by factors including field strength, pulse length, medium composition, temperature, membrane characteristics and the concentration of DNA (Brown et al. 1991; Wang et al. 2007). These variable factors result in unrepeatable results. (3) Glass beads method has been previously announced as the easiest and most effective method for Dunaliella transformation. This method is considered as an inexpensive technique in comparison with the aforementioned methods. Beside, this approach possesses some benefits like high-efficiency, simplicity, economy, controllability, and high repeatability (Feng et al. 2009), but it suffers from decrement in cell viability in transformants. To overcome the problem, the silicon carbon whisker was successfully exploited for Dunaliella transformation instead of glass beads but whisker method shows lower transformation efficiency (Potvin and Zhang 2010).

Selection markers and reporter genes Antibiotics, the main effective selectable markers, were successfully applied to select transformants, but as far as Dunaliella is resistant to most of antibiotics, fond suitable selectable markers requires extensive researches to select transformed Dunaliella in this area (Tan et al. 2005). In fact, Dunaliella does not show sensitivity to streptomycin (Str), kanamycin (Km), hygromycin (Hm) and G418 while Chloromycetin (60 lg/ml) can completely prevent the growth of Dunaliella in solid and liquid media. In addition, cat gene has been reported as a suitable selective marker for genetic engineering of Dunaliella (Wang et al. 2007). Numerous researches have previously applied some selectable genes such as the aadA gene encoding spectinomycin or streptomycin resistance, and the bar gene encoding herbicide phosphinothricin (PTT) showed resistance to select transformed cells in Dunaliella (Jiang et al. 2005). Since Dunaliella cells have been extremely sensitive to Basta, indicating the bar gene can be considered a suitable selective marker to select Dunaliella transformant cells. Dunaliella cells also show sensitivity to chloromycetin and Zeocin, but not to hygromycin where plant cells usually

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World J Microbiol Biotechnol Table 1 The sensitivity/ resistance to multiple antibiotics in Dunaliella

Selectable marker Streptomycin

Sensitivity and resistance

Concentration of antibiotic (lg/ml)

Gene

R

600

(Sun et al. 2005)

R

1,200

(Tan et al. 2005)

Kanamycin

R

600

(Sun et al. 2005)

Hygromycin

R

G418

R

1,200

(Tan et al. 2005)

600

(Sun et al. 2005;

1,200

Tan et al. 2005)

600

(Sun et al. 2005)

1,200

(Tan et al. 2005)

Chloromycetin

S

60

Chloramphenicol

S

400

Spectinomycin Spectinomycin/ Streptomycin

R S

1,200

Basta (phosphinothricin)

S

20

Zeocin

S

100

show the sensitivity for hygromycin (Sun et al. 2005; Tan et al. 2005). Moreover, several research findings indicated that selective pressure depends on cell density, and higher cell density requires an increase in selective pressure. The sensitivity and resistance to multiple antibiotics in Dunaliella are listed in Table 1. Another kind of selectable markers is based on the complementation of metabolism or photosynthetic mutants. In this method, the rescue of microalgal mutants was accomplished with wild-type gene constructs in which the cultivation conditions were considered as a transformant selection approach. This method may be particularly valuable for chloroplast transformations, where hybrid foreign DNA constructs containing wild-type genes, which their integration soccur by homologous recombination, can not only rescue microalgal mutants. It means that the gene is Knocked-out, thus allowing selection but only in targets adjacent regions for foreign DNA integration. Despite the mentioned reasons, different reporter gene expression patterns are previously reported by several researches. For example, the gus reporter gene has been successfully and transiently expressed in Dunaliella transformants but another reporter gene called lacZ, encoded for bgalactosidase, did not show successful expression in Dunaliella (Tan et al. 2005). The egfp gene, encoded for a green fluorescent protein, was then employed as a reporter gene for Dunaliella transformation, and a green fluorescence background was detected after the microscopic observation of a large amount of blank cells. Microalgal chloroplasts can be considered as an attractive platform for foreign protein expressions making marker-free systems highly desirable for the expression of recombinant therapeutic or nutritional products at high

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Reference

(Sun et al. 2005) Catgene

(Tan et al. 2005)

aadAgene

(Tan et al. 2005) (Jiang et al. 2005)

bargene

(Jiang et al. 2005; Tan et al. 2005)

Shblegene

(Tan et al. 2005)

levels. The reason is that in achieved homoplastomic transformations, 5–18 % of the total soluble protein (TSP) can consist of marker gene products, which makes the decrease on maximum yield of the target protein. Therefore, if the transformed algae are predestinated for human or animal consumptions, unnecessary DNA including genes conferring resistance to antibiotics is undesirable and removing of marker genes may be favorable to increase yield of recombinant target protein. The elimination of marker genes can be obtained by some methodologies such as homology-based excision, excision by phagesite-specific recombinases, transient cointegration of the marker gene or the cotransformation–segregation approach (Lutz et al. 2006; Lutz and Maliga 2007).

Nuclear transformation and chloroplast transformation in Dunaliella Recombinant proteins have been usually expressed in the nuclei and chloroplast genomes of algae. Each of these systems has their own special features; thus, both nuclei and chloroplast features should be considered for choosing a suitable expression system and finally it is necessary to select between these two systems to achieve the ultimate goal. Here, some properties of nuclear/chloroplast transformations in Dunaliella are described. Nuclear transformation Positional effects, RNA silencing, a prohibitively compact chromatin structure and non-conventional epigenetic

World J Microbiol Biotechnol Fig. 1 Recombinant protein production by nuclear transformation in Dunaliellasalina. a Low nuclear expression of transformed foreign proteins in Dunaliella. b The effect of a proteolytic degradation phenomenon on recombinant protein yields which caused the decrement of yield. c Co-expression of protease inhibitors with recombinant protein which caused the increase of yield. d Targeting synthesized recombinant protein in the endoplasmic reticulum or chloroplast via signal peptide

effects have been proposed as possible causes to low nuclear expression of transformed foreign proteins in Dunaliella (Fig. 1a). Low expression levels and varied wide range of protein expression were also ascribed to random integration sites of the newly introduced genes. This was also considered for influence of the chromatin structure and/or the regulatory elements on the sites of integration in the host genome (Peach and Velten 1991). To overcome these position effects, a utile approach was proposed by some researches in which the neighborly elements construct an affecting transgene expression (Kim et al. 2004; Lee et al. 2006; Van et al. 2001). In recent years, it has been clarified that a protein aceous nuclear ‘‘matrix’’ or ‘‘scaffold’’ plays a significant role in determining chromatin structure. Matrix attachment regions (MARs) are thought to be components that bind specifically to the nuclear scaffold and form the bases of loop domains. Many experiments have demonstrated that MARs not only increase the expressions of foreign genes and decrease the variation of expressions of transgenes between different transformants (Abranches et al. 2005; Allen et al. 2005; Ascenzi et al. 2003; Mankin et al. 2003; Michalowski et al. 1999) but also stabilize the transgene expressions in their progeny (Allen et al. 2005; James et al. 2002). Thus, the MARs may be considered an

effective regulatory element to improve expression levels of transgenes and stabilize the gene expression in the progeny. DMS2 and DNA fragment binds to nuclear matrices (MAR) were previously isolated from Dunaliella (Wang et al. 2005b). The effects of this fragment on the cat gene expression in stably transformed cells were successfully investigated and the results demonstrated the enhanced expression level (4.5-fold) of the cat gene in transformed Dunaliella cells (Wang et al. 2005a). A genetic screening with little influence by the position effects was designed to facilitate isolation of algal strains which efficiently express introduced recombinant protein gene. The results of this investigation showed that the accumulation levels of foreign protein in the selected strains were almost uniformly high in all transgenic clones. The selected UV-induced mutations with high express nuclear transgenes in Chlamydomonas strains showed that the increase foreign protein yields nearly 0.2 % TSP which is relatively high for nuclear expression in algae (Neupert et al. 2009, 2012). These findings may be beneficially favored to increase the recombinant protein expression in transformed Dunaliella due to the high similarity between Chlamydomonas and Dunaliella micro algae.

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The post transcription/translation processing in a nuclear transformation system can be considered as the main advantage of this system which it necessary for complex protein production despite its low yields. Sensitivity to proteases Recombinant protein yields depend on protein accumulation which is affected by the amount of protein synthesis and its degradation in transformed cells. Endogenous proteolytic enzymes essentially require correct assembling and post translational modification of proteins which may lead to degradation of recombinant proteins. These enzymes may interfere in the downstream processing of proteins and cause an inconvenience in protein purification due to total degradation or non-functional protein fragments production (Fig. 1b). Although infrequent information is available on the effect of a proteolytic degradation phenomenon on recombinant protein yields, the fundamental experiment announcing the principle factors affecting this phenomenon in microalgae has been investigated in C. reinhadtii. The conclusion of the investigation showed that protease activity is one of the main factors affecting the level of recombinant protein expression in Chlamydomonas (Surzycki et al. 2009). Some available strategies exist to minimize foreign protein degradation in plants and due to the similarities between plant and green microalgae systems, using these strategies may be beneficial to increase the recombinant protein yields (Doran et al. 2009). However, the evaluation that each factor effects on recombinant protein yield requires additional investigations (Potvin and Zhang 2010). In this section of the review, some of the most important strategies for the sensitivity of recombinant proteins were summarized to proteases. (1) Co-expression of protease inhibitors with recombinant protein has been previously applied to increase recombinant protein yields without affecting normal growth in cells (Van der Vyver et al. 2003) (Fig. 1c). (2) Since the cell endoplasmic reticulum possesses few proteases enzymes, the protein degradation can accordingly be minimized by targeting synthesized recombinant protein in the endoplasmic reticulum or chloroplast via signal peptide rather than cytosol (Conrad and Fiedler 1994, 1998; Conrad and Manteuffel 2001) (Fig. 1d). This strategy led to 104 fold increase in recombinant growth factor expression in tobacco plant cells (Wirth et al. 2004); moreover, it can be applied to recombinant special antibodies production whenever the protein secretion or its modifications in the golgi are not necessary. (3) The proteins can be expressed in the chloroplast of algae without needing post-translational modifications (Eichler-Stahlberg et al. 2009; Potvin and Zhang 2010; van Wijk 2004) while the presence of

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proteolytic pathways for protein processing in plant chloroplasts can also limit the recombinant protein accumulation resulting in the overall yield of protein in transformed cells. In addition, microalgal chloroplasts can act as an envelope in long-term storage of recombinant proteins inside the cell (Bock 2001) which may be useful to increase protein’s final yield. IV) To minimize the nuclear-expressed protein proteolytic degradations, the most efficient approach is to reserve them in chloroplast because some chloroplast proteins are encoded in the cytosol and then export to chloroplast (Faye and Daniell 2006; Jarvis 2003, 2008; Jarvis and Robinson 2004). Two different transportation mechanisms of cytosol-synthesized proteins were already described by targeting procedure. (1) The proteins are post-translationally targeted to the chloroplast through Toc and Tic complex (outer and inner apparatus respectively) of the chloroplast membranes which directly denominated targeting or the classical protein import process. (2) In indirect targeting mechanism, the protein was targeted to the endoplasmic reticulum (ER) by co-translational targeting or undergoes further process in the golgi apparatus by using the secretary pathway prior to exporting to chloroplast. Therefore, these strategies can be considered as possible approaches to increase foreign protein production yield in transformed Dunaliella due to the presence of some similarities in plant and microalgae systems. Chloroplast transformation A successful engineering in the chloroplast genome can be proposed to obtain high yields of foreign protein production in Dunaliella by considering some advantages of this expression system to express recombinant proteins which do no require post-translational modifications. Several advantages of chloroplast recombination and expression system include targeted transgene integration by homologous recombination, absence of gene silencing, polycistronic expression system, maternal inheritance of the chloroplast genome causing robust expression, envelope and protect foreign proteins from degradation (Bock 2001; Chebolu and Daniell 2007; Daniell et al. 2001; Daniell 2006; Daniell et al. 2009; Rasala et al. 2010, 2011; Rasala and Mayfield 2011). It seems this system may be an efficient method to achieve cost beneficial recombinant protein production methodology with perceiving high growth rate of this microalgae (Smith et al. 2010). The important point is to consider the most salient chloroplast genome features of the Dunaliella such as 269 kb circular plastid genome containing 102 genes with 32.1 % GC content and 65.5 % non-coding DNA regions including either intergenic or intronic DNA together with above mentioned chloroplast

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transformation advantages. In addition, the Dunaliella ptDNA sequence is already available and scientists will be able to exploit these data to develop plastid transformation vectors targeting specific regions of its ptDNA.

Table 2 GUS activity was determined according to the amount of methyl umbelliferone (MU) produced by protein extracts of transformed Dunaliella Promoter CaMV35S

Factors affecting protein expression in Dunaliella and strategies for its increase Enormous studies have recently focused on improving recombinant protein production in microalgae through studying promoter’s efficiency, the role of the untranslated region (UTR) sequences of DNA and fusion between native and recombinant peptides is to achieve a cost beneficial production mechanism and eliminate probable limitations regarding microalgae expression systems. Indeed, the regulation of recombinant protein expression system in eukaryotic cells is more complex and consist of plenty interacting elements. The extent of interaction among internal factors has not been understood completely but several strategies and mechanisms have previously been proved to increase recombinant protein yields in microalgae. Some crucial factors are reviewed following this section. Promoters Using endogenous promoters to obtain an efficient expression level of heterologous genes seems to be essential at point of molecular biology. A number of previous studies found that the endogenous promoters possess the ability to stimulate other promoters and therefore increase the expression of heterologous genes. Hence, it is highly important to select a highly active promoter to achieve a high yield recombinant protein production system (Schroda et al. 2000, 2002). A lodgment of the gene under highly active promoter can perform a crucial role in developing efficient microalgeal transformation systems particularly in Dunaliella to achieve a high yield of recombinant protein in transformed cells. Several famous promoters have been previously introduced by researchers for Dunaliella transformation which are listed in Table 2. Among different promoters, the Ubi1-X promoter is suggested as a suitable promoter to express foreign genes in Dunaliella cells (Degui Geng et al. 2003). The existence of TMVX element in the Ubi1-X promoter as a translational enhancer can increase the expression of foreign genes at in vivo/in vitro conditions. However, the expression level of the foreign genes driven by previously introduced promoters has been proved to be in a low level, usually transient expression in transformed Dunaliella cells. Besides, the RBCS2 promoter derived from Chlamydomonas reinhartii contrastingly showed higher efficiency and activity than CaMV35S promoter in

GUS activity (nmol MU mg-1 protein h-1) 4±1

Ubil

11 ± 3

Ubil- X

40 ± 3**

CaMV35S-Ubil CaMV35S-Ubil- X

2±1 10 ± 2

Asterisks denote statistically significant differences (**p B 0.01) Data show the mean ± SE

transgenic Dunaliella cells (Thanh et al. 2011; Walker et al. 2005). This, in turn, illustrates the necessity of providing some endogenous promoters to obtain high efficient expression level of foreign genes in transgenic microalgal cells. Despite very strong promoters like carbonic anhydrase 1 which have formerly been developed and exploited in transgenic microalgae, the expression of heterologous genes in transgenics represents a problematic procedure because of the potential occurrence of low activity of gene expressions. This low activity affects the growth of transformants by gene silencing and some other effects (Li et al. 2007, 2010). To overcome these obstacles caused by the permanent expression of heterologous genes, the inducible homologous promoter can be considered to create a perfect expression system in microalgae. It produces a suitable copy number of transgene to prevent the occurrence of gene silencing (Jia et al. 2012). The homologous promoter of nitrate reductase gene has previously been isolated to improve the expression of heterologous proteins in transgenic Dunaliella cells by possessing the ability to switch on/off with nitrate/ammonium respectively (Li et al. 2007). Another inducible promoter, duplicated carbonic anhydrase 1, regulates gene expression at the transcriptional level by gradient concentrations of NaCl to express heterologous genes that have previously been isolated from Dunaliella. This promoter showed a low expression level of heterologous protein but the expression was efficiently stable (Li et al. 2010). Finally, by considering abovementioned concepts, the isolation and selection of high active endogenous inducible promoter can mainly affect the recombinant protein production yield in microalgal efficient bioreactor systems such as Dunaliella. Enhancer elements Enormous investigations showed that insertion introns from native genes in heterologous sequences could improve the foreign protein yield in transformed cells

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under the control of native gene’s promoter. For instance, inserting three introns from native C. reinhardtii RBCS2 chloroplast gene into expression construct increased luciferase and erythropoietin expressions more than 400 % compared to the base level through codon optimizing manner. Although each RBCS introns individually showed a positive effect on the foreign gene expression, their integrations with physiological order and number illustrate a synergistic effect on the whole (Eichler-Stahlberg et al. 2009). Expression of recombinant genes in the nuclear transformation of C. reinhardtii also improved following the insertion of the first RBCS2 intron which has been shown to contain an enhancer element (Berthold et al. 2002). Some eukaryotic gene promoter sequences possess the consensus TATA and CAAT elements and repetitive tandem GT/AC sequences at the upstream position of homologous gene and these sequences participate in inducible regulations in Dunaliella (Lao et al. 2011; Long et al. 1989). Indeed, the existence enhancer elements in the construct can undergo the expression level of heterologous genes to transform microalgae but developing the use of these elements needs more investigations until reaching a cost beneficial and high yield production system (Table 3).

Table 3 Main precious characteristics of Dunaliella for genetic and plastome engineering Characteristics of Dunaliella

References

Sequencing of its nuclear and organelle genomes

(Smith et al. 2010)

Mutations study possibility without any requirement for further progeny analysis because of being haploid

(Primrose and Ehrlich 1981)

Appropriate selection to produce of multi-chain antibodies due to the sexual reproduction in the field culture conditions

(Mayfield et al. 2003; Mayfield and Franklin 2005)

A large scale cultivation possibility due to easy maintenance and fast growth

(Lam and Lee 2011)

Facilitated genetic manipulation particularly through nanoparticles due to lack of the rigid cell wall

(Perreault et al. 2011)

Possessing a single plastid makes it favorable selection to develop homoplasmic lines

(Wang et al. 2007)

Gene flow can’t happen in transgenic Dunaliella thus harmless to the environment

(Barzegari et al. 2010)

The ability of the recombinant proteins secretion to outside of the cell

(Kleinegris et al. 2010)

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Endogenous regulatory factors Although the expression of recombinant proteins in green algal chloroplasts can be considered a promising platform for the production of human therapeutic proteins and as far as a number of these proteins have been expressed in microalgal chloroplast, many of these proteins accumulate to significantly lower levels than endogenous chloroplast proteins do. In microalgae, some chloroplast gene products regulate the translation of their own mRNA through feedback inhibition (Coragliotti et al. 2011; Minai et al. 2006; Wostrikoff et al. 2001, 2004). This phenomenon was explained once more in heterologous genes expression when microalgae was less than expression in tobacco chloroplast due to lack of this inhibition in plants (Manuell et al. 2007). By considering the high potential of microalgal systems to produce safely and economically scaled up therapeutic proteins, the development of some methodologies to overcome this obstacle and increase the recombinant protein accumulation in chloroplast transformants seems crucially important. Among a number of approaches, the fusion exogenous protein gene with highly expressed endogenous chloroplast gene improved the accumulation of recombinant fused protein. For instance, fusing the luciferase reporter protein to the carboxy-terminal end of the large subunit of ribulose bisphosphate carboxylase showed 33-fold increase in luciferase expression compared to luciferase expressed alone. These results demonstrate the exploit of fusion proteins in algal chloroplast as a method to increase the accumulation of recombinant proteins that are difficult to express (Mayfield et al. 2003; Michelet et al. 2011; Muto et al. 2009). Codon optimization Codon optimization is a generic technique to achieve optimum expression of a foreign gene in the host’s cell system. Selection of optimum codons depends on codon usage of the host genome and the presence of several desirable and undesirable sequence motifs. The codon adaptation index (CAI) is exploited as the most important quantitative method (Xia 2007) to predict the expression level of native and heterologous genes based on organism/ organel codon usage. CAI actually measures the deviation of a given protein coding gene sequence with respect to a reference set of genes. When this parameter is considered from a reference set of highly expressed genes, the maximum expression of a heterologous gene will be assumed as dual rationale; highly expressed genes need to compete for resources (i.e. ribosomes) in fast-growing organisms and it makes sense for them to be also more accurately translated. However, a common error for optimization of heterologous gene expression in the chloroplast of microalgae, in

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particular Dunaliella, has calculated a CAI value using a codon usage table for all genes. Such optimization is incorrect because it assumes: (a) that all tRNA species are equally abundant; and (b) that translational selection does not exist in chloroplast genome where only three chloroplast genes (subunits of RNA polymerase) lack such selection (Surzycki et al. 2009). Besides, although it is popular to say that an organism has a particular codon usage, it is now known that an organism’s genes might have more than one codon usage pattern. Only a multivariate analysis method that is possible to ascertain the kinds of variation contained within the data. Thus, by considering the use of the variable codon usage even in one organism among expressed genes, it may be concluded that this variability is a procedure at the point of codon usage to regulate gene expression levels in creatures which leads some genes to be highly expressed and others to below. In summary, the codon usage optimization seems necessary to obtain optimum recombinant protein production system in microalgae in particular Dunaliella (Heitzer et al. 2007). Two main issues should be considered for this optimization; (1) the codon usage in foreign gene sequence must be optimized for common codon usage in microalgae and then (2) the optimized codons should be reoptimized again for codon usage exploited in highly expressed genes, particularly host cell (for example, Dunaliella). Transformation-associated genotypic modifications A plasmid vector for chloroplast transformation possesses two genes: the gene of interest and a selectable marker gene (an antibiotic resistance gene) that allows for selection of transformed cells (Newman et al. 1990). Some genetic elements (promoter, 50 and 30 UTRs and etc.) require homologous recombination and identification transformants to express chloroplast machinery and the correct insertion of transgene into chloroplast DNA. Thus, it has been supposed that protein levels in these strains would be accordant in each isolated transformant after transformation (Newman et al. 1990). Findings of Chlamydomonas chloroplast transformation for VP28 protein gene inserted to pBA155 and pSR229 vectors showed that the range of protein accumulation was from 20.9 to 0.88 % and 2.4 to 0.2 % TSPs, respectively (Gong et al. 2011; Jones et al. 2012; Surzycki et al. 2009). By comparing these results, it may be derived that the psbA promoter (on pBA155) is better than the atpA (on pSR229) promoter at expressing VP28 (21 vs. 2.4 %) but this conclusion depends merely on the chance of selecting a single transformed cell line for the analysis. For instance, if the transformed cell lines with 0.88 and 2.4 % were selected, the conclusion would have been reversed (Surzycki et al. 2009). By considering the high level of protein expression

levels in the Chlamydomonas chloroplast which was previously reported to be 5 % with most being \1 % (LeonBanares et al. 2004), it can be concluded that the expression of transgenic proteins may depend on a minor extent of the promoter, site of insertion or associated 30 and 50 UTRs than the nature of changes incurred during an individual transformation event. These changes could result in the formation of a transformed line having unique characteristics, or transformation-associated genotypic modification, referred to as the transformosome (Surzycki et al. 2009). Moreover, the occurrence of some other unknown genetic events in transgenic strains may possess crucial impacts on final recombinant protein yield because of recalcitrant or low yielding transformed cell lines due to specific incompatibility among genes, different level of mRNA of foreign gene in transformants, genetic elements and insertion sites. The provenance of these changes is not clearly known while the changes may result in additional insertions of the vector or DNA fragments into the nuclear genome. Moreover, inserting a transgenic gene into the chloroplast genome target site leads to a change in the function of nuclear genes involved in regulation of recombinant protein expression. If the transformosome leads to a variation of transgenic protein expression, it may affect some other properties of transformants such as growth rates or protein stability. On the other hand, protein degradation depends on an intrinsic characteristic of the protein (sensitivity to proteolytic enzymes) regardless of the nature of the promoter used or the expression level of the protein. The degradation of VP28 protein in three Chlamydomonas transgenic lines was previously proved to be different from each other which indicate that the genetic background of these strains was not the same. It presumably changed in the process of transformation which created different transformosomes (Surzycki et al. 2009). Finally, screening the transformosomes based on their potential to express and accumulate recombinant protein may provide a quick and easy way to maximize recombinant protein yield via selecting high performance transformosomes of microalgae. Milking of Dunaliella Different types of plant lipid bodies have been previously described proving that the proteins in these structures are co-induced and specifically associated with the carotenoid bodies formation. Examples include oil bodies in seeds containing primarily triacyl glycerols and special lowmolecular-weight proteins (oleosins) (Huang and Cheung 2011; Noll et al. 2000), and chromoplast lipoprotein fibrils containing carotenoids together with polar lipids and fibril in proteins (Deruere et al. 1994; Smirra et al. 1993). Likewise in mammalians, the secreted major lipid globules

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of milk containing triacyl glycerolsare covered with a membrane of polar lipids originated from the endoplasmic reticulum and monolayer of unknown hydrophilic proteins (Heid and Keenan 2005). By considering aforementioned issues, it can be concluded that globular lipid bodies have some structural similarity from plant to animal in the nature. In microalgae, the massive amount of b-carotene is arranged (Ben-Amotz et al. 1986; Ben-Amotz and Avron 1983) in minute lipid globules (100–200 nm) located in the inter thylakoid space of the chloroplast of green microalgae, which isolated globules display as a superior stability in aqueous solutions suggesting that these globules possess a stabilizing layer which prevents their aggregation and coalescence (Katz et al. 1995, 2007). Finding of some investigations showed that the isolated b-carotene globules contain exclusively b-carotene, neutral lipids, and a small amount of protein (Katz et al. 1995). The high stability of b-carotene globules from D. bardawil at in vivo/in vitro

Fig. 2 Milking of Dunaliella. There are two common secretion mechanisms in Dunaliella one via golgi apparatus and another by formed vesicles in the endoplasmic reticulum. The secretion pathway via endoplasmic reticulum seems to be suitable to increase recombinant protein yield and facilitate its harvesting and purification

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aqueous conditions may cause these structural proteins to provide a stabilizing hydrophilic layer covering the hydrophobic core in spite of their tiny dimensions and hydrophobic contents. Under induction conditions, even the metabolic pathway of b-carotene synthesis is blocked, the involved proteins in globules show over expression which suggests that these protein expression depend on induction condition and globule formation rather than directly b-carotene accumulation (Hejazi et al. 2004; Jimenez and Pick 1993). On the other hand, the involved proteins in globules did not illustrate the over expression in a related Dunaliella species that does not accumulate bcarotene. Studies on the localization of the protein at the periphery of the globules demonstrate primarily hydrophobic interactions between proteins and globules which provide the detachment of proteins from globules by a mild detergent treatment (Katz et al. 1995, 2007). Several mechanisms have been previously described for secretion

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of lipids and related lipophilic compounds towards out of the cells and their pathways. They include TAG-containing very-low-density lipid (VLDL) vesicles, TAG-containing vesicles from mammary glands, and the ATP-binding cassette (ABC) transporter-mediated export. In addition to cellular export pathways, importing fatty acids into mitochondria and peroxisomes or other organelles is considered as an intracellular transport pathway and it may be possible to utilize such pathways for the export of lipids (Radakovits et al. 2010, 2011). Even though many of the key genes that are involved in secretion phenomenon have been identified, the exact mechanisms are not generally known. The use of ABC transporters might be considered as a more straight forward approach to enabling secretion of lipids from microalgae because these transporters mediate the export of plant waxes derived from very-long-chain fatty acids, including alkanes, ketones, alcohols, aldehydes, alkyl esters, and fatty acids. One interesting characteristic of ABC transporters is their promiscuous gating properties to export from very-long-chain to medium-chain fatty acids while wax transporters have not been shown to export products that are derived from medium-chain fatty acids (Pighin et al. 2004). In summary, lipid secretion is an attractive alternative to harvesting algal biomass that could potentially lower the cost of producing microalga-derived compounds. By considering the high stability of secreted lipid globules in aqueous media and their formation locality (endoplasmic reticulum), directing the recombinant protein to endoplasmic reticulum via a signal peptide to accumulate and make vesicular and consequently secretion inside vesicles to culture media can be proposed as a suitable method to increase foreign protein production yield in under stressed culture of Dunaliella (Fig. 2). In this method, the secreted globules containing recombinant protein can be easily collected from aqueous media because of their lipid nature. Thereby, they can accumulate in surface of culture media to decrease the production process cost and provide a continuous bioreactor system by milking of Dunaliella. To achieve this goal, Dunaliella species should be selected that can produce high amount of globules under stress condition. In addition, a secretion strategy may not be the best solution when a significant number of contaminating microorganisms are present in the cultivation system because the secretion of the intermediates into the culture medium would provide a rich source of nutrient for microorganisms and thereby reducing product yield.

Conclusion Interaction of some crucial complex factors such as choice of suitable selectable markers, different transformation

methods, promoter effects, transformation associated events, sensitivity to proteases effects, protein localization efficacies and gene silencing impacts have created an inexplicit system to produce recombinant protein in Dunaliella. Thus, a comprehensive systematic study seems necessary to optimize these parameters’ effects to obtain the optimum condition for increasing recombinant protein production in microalgae. It can be concluded that all concepts must be investigated together in this review to achieve a high yield production system in microalgae, particularly in Dunaliella. Acknowledgments The financial support of the Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences is gratefully acknowledged. Conflict of interest

There is no conflict of interests to be declared.

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