plastids for the production of foreign proteins at high levels

Gene Therapy and Molecular Biology Vol 15, page 14

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Gene Ther Mol Biol Vol 15, 14-29 2013

Green factories: plastids for the production of foreign proteins at high levels Original Research Article Niaz Ahmad* and Zahid Mukhtar Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad, Pakistan

*Correspondence: Dr. Niaz Ahmad, Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad, Pakistan Postal code: 38000, Phone: +92(0)412 651 475 Ext 309, Fax: +92(0)412 651 472, Email: [email protected] Keywords: Chloroplast transformation, recombinant proteins, biopharming Abbreviations: TSP=Total soluble proteins, ptDNA= Plastid DNA, LSC=Large single copy, SSC=Small single copy, IR=Inverted repeat, UTRs =Untranslated regions

Received: 10 September 2013; Revised: 20 September 2013 Accepted: 2 October 2013; electronically published: 5 October 2013

Summary The soaring costs along with the increasing demand of recombinant proteins in different walks of life have fuelled the quest to find out alternate modes for their production, which are cheaper, safer and can deliver the modern safety standards. In this context, plants are emerging as an alternative production platform for foreign proteins. Chloroplasts – the green plastids with an in-built remarkable capacity to express foreign proteins at high levels – offer several attractive features, which make plants an exceptionally useful system for low-cost production of high-value targets at large scale. Transformation of chloroplasts, therefore, holds a great potential to meet the challenges in the areas of food, feed and medicine posed by a population on the rise. Numerous developments have been made in the field, all of which set plastids to become a centrepoint of future plant engineering efforts. The present review briefly describes ‘the state of the art’ of the technology along with its salient features whilst highlighting the latest trends in the area of chloroplast transformation.

I. Introduction The use of recombinant proteins in different spheres of life is creating a huge demand for their large-scale production. For example, the antibodies used as topical medicines are required in large doses due to their stoichiometric mechanism of action and therefore their production is also needed in huge quantities, sometimes ranging from 100-1,000 kg to cope with the demand (Twyman et al., 2012). Our current capacity to meet this challenge with the established techniques is lacking (Bosch et al., 2013). The award of US$ 100 million for research into plant-based expression systems by the US Government agency, Defense Advanced Research Projects Agency (DARPA), is the recognition of the fact that making antibodies and vaccines using plants as expression vehicles is the ‘technology of choice’ for low-cost production at a large scale (Bosch et al., 2013). Expressing proteins in plants offers a unique expression platform with low capital costs, excellent scalability, post-translational modifications and the ability to properly fold and process the recombinant proteins (see Rybicki, 2010; Lössl and Waheed, 2011 for reviews). It

has been estimated that Sanofi Pasteur, a conventional vaccine production plant in Pennsylvania (USA) would cost 150 million US$ for the production of 100 million doses of influenza vaccine in a year. Similarly Novartis, in North Carolina, USA, will cost one billion US$ to produce 300 million doses of the same vaccine in a year. On the other hand, tobacco (Nicotiana benthamiana) would produce one billion doses for 15 million US$ in one year (Penney et al., 2011). Even the costly purification requirements can be eliminated where plant tissue containing the recombinant protein/antigen is consumed directly as food material (Gunn et al., 2012). In terms of SWOT (strengths, weaknesses, opportunities and threats) the advantage of transgenic plants and their potential to mass-produce recombinant proteins becomes very distinctive over the conventional approaches (Raskin et al., 2002).

The plant-based expression system for cost-effective production of recombinant proteins is therefore more affordable as compared to conventional systems (Table 1).

Ahmad & Mukhtar: plastids for the production of foreign proteins at high levels

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Parameter Transgenic plants Bacteria Yeast Insect cells

Mammalian cells

Capital cost Low Medium Medium High Very High Operating cost Low Low Medium High Very high Speed Slow Fast Fast Medium Slow Multigene engineering

Yes Yes No No No

Glycosylation Yes but absent in chloroplasts

Absent Incorrect Yes Yes

Multimeric assembly

Yes No No No No

Protein folding

High Low Medium High High

Protein yield Low-High High Medium-high Medium Medium Scale up cost Very low High High Very high Very high Safety High Low Unknown Medium Low Storage Very cheap Costly Costly Expensive Very

expensive Distribution Easy Feasible Feasible Difficult Difficult

Table 1. Comparison of different protein expression systems

Transgenic plants are now being developed to

produce better food and nutrients. For example, golden rice – genetically engineered rice fortified with vitamin A – would help eliminate the effects of vitamin A deficiency, which results in the blindness of 124 million children around the world (Ye et al., 2000). Similarly, the development of transgenic plants with improved performance against biotic and abiotic stresses has resulted in significant increase in world food production (Herrera-Estrella et al., 2005).

Plants are unique in a sense that they possess three distinct genomes in different cellular compartments: nucleus, plastids and mitochondria; each one equipped with its own genetic system of DNA replication, repair, recombination, transcription, processing of RNA and translation (Maliga and Bock, 2011). Over the past few years, significant developments have been made to use a diverse range of plant species for the expression of recombinant proteins (Demain and Vaishnav, 2009) using different parts such as leaves, seeds, cell suspension cultures, and more recently temporary immersion bioreactors. The technical aspects of all these plant-based approaches have been discussed elsewhere (Chichester et al., 2009; Orzaez et al., 2009; Shih and Doran, 2009; Boothe et al., 2010; Komarova et al., 2010; Pogue et al., 2010; Hensel et al., 2011; Michoux et al., 2013). It is noteworthy that the majority of recombinant proteins expressed in plants have been produced via nucleus (Paul and Ma, 2011; Bosch et al., 2013; Okuzaki et al., 2013).

Expressing proteins through plant nucleus, however, has met several challenges such as poor and variable protein expression levels, unwanted DNA re-arrangements, gene silencing and the risk of out-crossing of the transgenes to the weedy relatives (Daniell, 2002). By delivering inexpensive, extraordinary expression levels, tight natural gene containment (mostly in

angiosperms) and site-specific integration of the transgenes, plastid transformation has emerged as a serious competitor of the conventional protein production platforms. This review argues that the chloroplast transformation technology has been shown as a successful alternative to conventional approaches used to engineer plants and discusses its potential for becoming a method of choice for the production of recombinant proteins at large scale.

II. Chloroplast: a bona fide organelle

The characteristic feature of plant cells is the presence of specialized organelles called plastids, which evolved from free-living prokaryotes through the process of endosymbiosis and became permanent cellular inclusions of exogenous origin (Theissen and Martin, 2006; Tveitaskog et al., 2007). Chloroplasts are observable as flat discs; usually 5-10 μm in length and 2-5 μm in width with an average surface area of 50 μm2 (Pyke, 1999). Chloroplasts are surrounded by a double-membrane envelope, which is made up of galactolipids in contrast to the rest of the cell membrane, which is phospholipid derived. Therefore, deficiency of galactolipids severely impairs chloroplast development (Jarvis et al., 2000). Chloroplasts contain a fluid-like material called stroma, which is the location of the carbon fixation reactions (Benson et al., 1950). Immersed in the stroma is an extensive network of thylakoid membranes, forming stacks known as grana (Mustardy and Garab, 2003). Thylakoids possess the necessary machinery to carry out light capture and


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energy transduction; the process of oxygenic photosynthesis in which oxygen is evolved as a by-product (Anderson and Aro, 1994). A notable feature of plastids is that they are capable of dividing within plant cells by the process of binary fission (Pyke, 1999).

Chloroplasts are green photosynthetic plastids, which are primarily responsible for the conversion of sunlight into chemical energy (Waters and Pyke, 2004). Apart from housing photosynthetic machinery, they also have got their own genome – the plastome – and the related genetic machinery, which resembles to that of the prokaryotes (Harris et al., 1994). However, the majority of the genetic material was either lost or transferred to the host-cell nucleus during the course of evolution (Martin and Herrmann, 1998; Martin et al., 1998; Bhattacharya et al., 2007; Chan et al., 2011). The plastid DNA (ptDNA) is found in the form of DNA-protein complexes called plastid nucleoids, which are attached to the inner envelope membrane of the plastids (Kuroiwa, 1991). Each nucleoid contains 10-100 copies of ptDNA (Sugiura, 1992).

It was not until early 1970s when it was demonstrated that the chloroplasts have their own genome with a size ranging from 120-160 kbp except for Epifagus virginiana (size 70 kbp) and other parasitic, non-photosynthetic plants (Bungard, 2004). The chloroplast genome exhibits a high copy number ranging from 1,000-10,000 per plant cell (Sugiura, 1992). This high copy number coupled with homologous recombination is thought to preserve the integrity of chloroplast genome by slowing down the mutation rates. The ptDNA is rich in adenine (A) and thymine (T) (~60-70% in coding region and up to 80% in non-coding regions) as compared to cytosine (C) and guanine (G). Therefore a high degree of propensity towards a codon having ‘A’ or ‘T’ at the third position exists in the plastid genetic machinery. One of the unique features of the chloroplast genome in most of the crop plants is the presence of a large inverted repeat (IR), which ranges from 6-76 kbp in length (Palmer, 1985). The size of the repeat is variable and accounts for most of the variations in the plastome (Sugiura, 1992). Any transgene inserted into IR is copied into the other by a mechanism called copy correction thereby doubling the transgene number (Khan et al., 2007).

The genes in the repeats are therefore present twice in the genome. The regions outside the repeats are called large single-copy (LSC) and small single-copy (SSC) regions. The chloroplast genome consists of 120 to 135 genes, of which 76 encode different proteins, whereas, the rest encode RNAs (Rivas et al., 2002). Genes present on the ptDNA can be divided into three groups: a) photosynthetic (~46 in number), b) involved in in-house genetic system (~21 genes plus four rRNAs) and c) involved in miscellaneous functions. The miscellaneous group represents a mixed bag including some open reading frames known as hypothetical chloroplast reading frames (ycf). Some of which are conserved across different species, while some are not. All of the proteins encoded by the ptDNA remains within the chloroplast and are not exported. Majority of

the genes are organized into operons and therefore transcribed in a polycistronic fashion (Sugiura, 1992). The genome organization of the tobacco plastome is shown in Figure 1.

Although plastids have retained many of the prokaryotic features, the gene expression mechanism in plastids is undauntedly complex. Transcription is carried out by the concerted action of two different types of RNA polymerase complexes. One of them, plastid-encoded polymerase (PEP) is encoded by four plastid genes: rpoA, rpoB, rpoC1, and rpoC2. This PEP is similar to that of E. coli polymerase and initiates transcription by recognizing -35 and -10-like conserved recognition sequences. However, it is much more specific than its bacterial counterpart. Its specificity is determined by the nuclear-encoded sigma factor.

The expression of such sigma factors is controlled by the light, and therefore PEP activity is dependent on the light-controlled sigma factors present in the nucleus. The second polymerase is a nucleus-encoded polymerase (NEP), a single component molecule that resembles to the polymerases found in bacteriophage and yeast mitochondria.

Initially, the NEP is expressed in nucleus and is imported to proplastids, where it transcribes genes essential for plastid genetic system such as components of PEP, for example. Once the PEP is assembled and other necessary components of transcription such as sigma factors and ribosomes are recruited, it starts transcribing photosynthetic genes. As soon as transcripts of PEP become available, the activity of NEP is repressed, for example, by binding of glutamyl t-RNA to NEP, so as PEP becomes the dominant RNA polymerase in mature green chloroplasts. In non-green plastids such as those present in roots where sigma factors are not available due to the light, the NEP continues to remain the dominant enzyme for transcription of photosynthetic genes.

Before the translation of messages occurs, newly synthesized transcripts undergo a series of refinement processes such as cutting of polycistrons into monocistrons, RNA editing and removal of introns to produce mature transcripts.

A plastid mRNA molecule ready for translation exhibits several features required for efficient translation such as 5' untranslated regions required for suitable context, translation initiation codon, translation termination codon, and the 3' untranslated regions needed for transcript stability. All these features, therefore, have a strong influence on the yield of final product (Hartz et al., 1991). It has been shown that protein expression levels can be significantly increased or decreased by the judicious use of such elements. For example, the levels of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) accumulation in tobacco plastids were increased by 10,000 fold by simply using a different 5' UTRs (Ye et al., 2001).

Initiation of translation in plastids begins by stepwise binding of 30S and 50S ribosomes with the help of several initiation factors imported from the nucleus into plastids at a location on the chloroplast


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genome known as ribosomal binding sites (RBS). Elongation of the polypeptide takes place with the help of t-RNA molecules, all of which are transcribed by the plastid genome. The elongation factors involved in the process are all nuclear encoded, and therefore are

imported from the cytoplasm. After reaching the termination codon, the ribosomal complex disassociates itself from the mRNA molecule and newly translated polypeptide is released.

Figure 1: Structural map of tobacco chloroplast genome. Physical map of the tobacco chloroplast genome showing the organization of different plastid genes. Genes on the inside are transcribed clockwise, whereas, genes on the outside are transcribed anticlockwise. The open reading frames of unknown functions are shown by ycf (hypothetical chloroplast reading frames) plus designation number. The map was drawn by a web-based tool, OGDRAWV1.1 (http://ogdraw.mpimp-golm.mpg.de/ssi_faqs.shtml) using the sequence information of tobacco chloroplast from GenBank under the accession number Z00044. Abbreviations: LSC = Large single-copy region, SSC= Small single-copy region, IR = Inverted repeat


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Figure 2: Schematic representation of the assembly of chloroplast transformation vectors. Chloroplast transformation vectors consist of a selection cassette and an expression cassette, both of which are placed within chloroplast sequences called flanking regions. Flanking regions are essentially plastome regions where insertion of the transgene is made. Commonly used regulatory elements for driving expression of transgene as well as selectable markers and the exploited insertion sites are shown. Abbreviations used: GOI = Gene of interest; UTR = Un-translated regions

Figure 3: Schematic representation of the events involved in chloroplast transformation by biolistic delivery system. (A)

Transformation. 4 to 6-week-old leaves are selected for transformation; DNA is coated on the surface of microparticles of gold or tungsten and then shot onto the leaf surface with a great force using a helium-driven particle delivery device (gene gun). (B) Regeneration. Leaves are then cut into small leaf discs after 48 hours of incubation in dark and put on selection media. Primary shoots generally arise within 4-6 weeks. (C) Homoplastomy. Initially few copies of plastid genome are transformed and leaf cells are called heteroplastomic. Homoplastomy, a state where all copies of chloroplast genome are transformed, is achieved by performing few rounds of regeneration under selection. The putative transgenic shoots are then further characterized for transgene integration and protein expression accordingly.


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III. Transforming chloroplasts:

the state of the art Transformation of chloroplasts consists of

series of events, which include construction of vectors tailored for high-level expression in chloroplasts (Figure 2), delivery of transgenes into chloroplast genome (Figure 3A) and then recovery of the transplastomic plants (Figure 3B, C). Since the procedural details of all these steps are beyond the scope of this article, the reader is therefore referred to the detailed protocol published on transformation of the chloroplasts by the Daniell Laboratory (Verma et al., 2008). The foremost requirement of transforming chloroplasts is the construction of expression vectors, which serves as a vehicle to integrate the transgene(s) into the plastome.

A schematic design of chloroplast transformation/expression vector is shown in Figure 2. Typically, it contains a selection cassette, an expression cassette and two flanking regions taken from the plastome where insertion of the transgenes is intended. The incorporation of ptDNA fragments in the final vector is to allow the delivery of the transgenes at the chosen site in the plastome using homologous recombination.

Once the vector has been constructed, providing all the necessary regulatory elements required for the integration of transgenes into ptDNA as well as to achieve an efficient transcription and translation, the next step is to deliver the expression cassettes to the chloroplasts. The classical approach to deliver the transgene into chloroplast is the biolistic process (Boynton et al., 1988; Svab et al., 1990) in which DNA is coated on the surface of nanoscale particles, for example, tungsten or gold and then bombarded onto the leaves with a great force (Figure 3A). Other methods include PEG-mediated transformation (Tyagi et al., 1989; Sporlein et al., 1991) and microinjection (Knoblauch et al., 1999). The PEG-mediated transformation of the chloroplasts is not very practical in terms of transformation efficiency and regeneration, which remains very poor (Koop and Schweiger, 1985). In case of microinjection, no successful regeneration has been reported yet.

After transformation of the transgenes into chloroplasts, the explant is then tissue cultured in the presence of a selection marker to recover the transplastomic plant lines. Initially, few copies of the plastome are transformed; the transformants are therefore a mixture of both transformed and wild-type ptDNA (Figure 3B), a state known as heteroplastomy. However, with repeated regeneration cycles under constant selection pressure, these non-transformed or wild-type copies are gradually bleached out, resulting in a homoplastomic (with all copies of plastome transformed) transplastomic plant line (Figure 3C)

(Maliga, 2004). The aminoglycoside antibiotics such as spectinomycin, streptomycin or kanamycin are frequently used for recovering stable homoplastomic plants. These antibiotics block protein synthesis by binding to a 30S, the smaller subunit, of a prokaryotic-type 70S ribosome or by inhibiting ribosomal translocation during translation, and therefore provide selection by preventing cell division, greening and shoot formation (Maliga, 2004). The aadA selection cassette provided in the expression vector encodes an enzyme called aminoglycoside 3''-adenylyltransferase type A. It confers resistance to spectinomycin and streptomycin by adding an ATP moiety, which results in the destruction of inhibitory activities of these antibiotics to the ribosomes.

Although organogenesis through repeated cycles of regeneration under selection pressure is the preferred strategy to recover homoplastomic lines (Ahmad et al., 2012a; Ahmad et al., 2012b; Scotti et al., 2012; Lu et al., 2013), some studies have also used somatic embryogenesis to recover homoplastomic plants (Kumar et al., 2004a; b). Use of embryogenesis offers an opportunity to extend technology to non-green plastids as well, which is a bottleneck in making technology available to other plant species.

The integration of transgenes at a pre-determined location in the plastid genome using homologous recombination is the hallmark of chloroplast transformation technology. Therefore, all types of vectors used in transformation of the chloroplasts include two fragments from the chloroplast DNA around a region, where insertion of the gene(s) is desired (Figure 4).

This homologous recombination, however, requires a prior knowledge of the sequence of the plastome region where insertion is to be made. It is indeed a barrier in the expansion of chloroplast transformation technology to those crops whose plastid genome has not been sequenced yet. Although attempts have been made to develop universal transformation vectors to maximize the potential of chloroplast transformation technology (Daniell et al., 1998; Sidorov et al., 1999; Ruf et al., 2001), the transformation efficiency, however, was compromised. For example, when flanking regions of petunia were used for chloroplast transformation in tobacco, the transformation efficiency was significantly reduced (DeGray et al., 2001).

Recently, a study reported a drastic reduction, 80% and 97% respectively, in expression levels of anthrax protective antigen (PA) and human proinsulin (Pins) fused with the cholera toxin B subunit (CTB) (CTB-Pins) in comparison to endogenous regulatory elements (Ruhlman et al., 2010).


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Figure 4: Illustration of homologous recombination in chloroplasts. Chloroplast transformation vectors are

constructed with two flanking regions of the plastome where gene(s) integration is required. DNA sequences/gene of interest and the selection marker are inserted in between these fragments. Homologous recombination between flanking fragments of vector and ptDNA then transfers transgene(s) from the vector to the plastome.

IV. Chloroplast transformation: the salient features

Chloroplast transformation offers a number of attractive advantages over conventional genetic engineering approaches such as nuclear transformation. For example, several studies have shown that chloroplasts are able to express proteins at extra ordinary levels (De Cosa et al., 2001; Oey et al., 2009a; Ruhlman et al., 2010; Michoux et al., 2011; Ahmad et al., 2012a).

This high expression level could be attributed to the high copy number, which ptDNA exhibits. For example, each chloroplast depending upon the tissue it resides in contains up to 100 ptDNA copies, giving rise to 10,000 copies per cell. Further duplication of the transgene if inserted into the inverted repeat region would double the copy number of the transgene per plant cell. This feature of chloroplast genome makes it highly beneficial for the expression of recombinant proteins in large quantities.

The other attractive feature of transforming chloroplasts is in the fact that chloroplasts are not normally transmitted through pollen and exhibit, in most of the crop plants, maternal inheritance (Bogorad, 2000). Though, very low frequency (0.01%) of leakage of plastids with pollen has been reported in tobacco plants (Svab and Maliga, 2007), this mode of inheritance provides a natural means of preventing unwanted gene flow from transgenic plants to non-transformed plants and weedy relatives.

Due to a prokaryotic origin, genes in chloroplasts are arranged in operons and therefore are transcribed in a polycistronic manner; thus providing an opportunity of engineering a whole metabolic pathway. Taking the advantage of this feature, several studies have reported successful engineering of various metabolic pathways in higher plant chloroplasts such as enhancing β-carotene and vitamin E conent (Vidi et al., 2006; Wurbs et al., 2007; Lu et al., 2013; Yabuta et al., 2013). Transformation of chloroplasts allows multi-gene genetic engineering, which means several genes can be transformed in a single transformation event. Genetic studies have shown that several traits are controlled by a number of genes. For example, nitrogen-fixation-enzyme complex from Klebsiella pneumoniae is encoded by twenty different genes (Arnold et al., 1988), whereas, polysaccharide formation gene cluster in Streptococcus is made up of sixteen genes (Cieslewicz et al., 2001). Engineering such traits via nucleus would require several transformation events and several backcrosses, which is time consuming. The situation is further aggravated by the fact that gene expression levels remain low and variable. For example, it took seven years to engineer the β-carotene biosynthetic pathway in rice to fortify vitamin A (Ye et al., 2000).

On the other hand, transformation of a complete Cry2Aa2 pathway into tobacco chloroplasts could be accomplished in a single transformation event, resulting in an expression as high as 45.1% of total soluble proteins (TSP) (De Cosa et al., 2001).


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Similarly, a recent study demonstrated a successful transformation of a 50-kbp DNA fragment into tobacco chloroplasts (Adachi et al., 2007). The introduced DNA was not only stable in chloroplasts but was successfully passed on to the next generation, demonstrating the remarkable capacity of chloroplasts to accept foreign DNA, and the extent to which metabolic pathways can be engineered in plastids.

Chloroplasts are packed into a double membrane layer, and with no protein export mechanism installed, therefore, their transformation offers a natural compartmentalization of recombinant proteins within the plant cell. This means that even toxic proteins can be expressed in chloroplasts without having lethal effects on plant phenotype. For example, plants expressing even low-levels of cholera toxin B subunit (CTB) via nucleus showed a significant growth inhibition. When the same protein was expressed in tobacco chloroplasts, the host plants did not show any symptoms of growth retardation even with 410-3,300-fold higher CTB accumulation in transplastomic plant leaves (Daniell et al., 2001).

However, the downside of this technology is that chloroplasts do not glycosylate proteins. Glycosylation – the enzymatic attachment of polysaccharide units to the proteins – is one of the common post-translational modifications, and is often considered necessary for the protein stability. However, experiments in our lab as well as others have shown it is not always a stringent requirement for proper folding of the proteins (Kim and Langridge, 2004; Huang et al., 2006; Arlen et al., 2007). For example, xylanases are single chain glycoproteins and when expressed in tobacco chloroplasts were observed to be as active as its bacterially-produced version (Leelavathi et al., 2003). In another study, Arlen et al. (2007) expressed Type I Interferon α2b (IFN-α2b), a member of human glycoprotein family (cytokines), in tobacco chloroplasts and reported that the chloroplast-made INF-α2b induced up-regulation of MHCI molecules at the level comparable to its commercially available counterpart (Arlen et al., 2007). However, more studies are needed to determine the level of effect the glycosylation may have on protein stability by taking a range of glycoproteins and then comparing them with their unglycosylated versions, for instance, by expressing them in chloroplasts.

Similarly, the stroma of chloroplasts contains many proteases (Kamiya and Shen, 2003) and some degradation of the recombinant proteins has been observed in chloroplasts (Birch-Machin et al., 2004). For example, human insulin was unstable in tobacco chloroplasts unless fused with cholera toxin B subunit (CTB) (Ruhlman et al., 2007). Similarly, VP6, the most immunogenic rotavirus subunit and a potential target for an oral subunit vaccine was observed to be rapidly lost in mature leaves and could only be stabilized after the addition of the 5′-UTRs from the gene 10 of bacteriophage T7 (T7g10) and the addition of 15 nucleotides at the 5′-end of the coding region (Inka Borchers et al., 2012). Chloroplast transformation technology is a tissue-culture-dependent technology, and

is therefore not available yet in those crops, which do not respond to current tissue culture protocols such as monocots (Cui et al., 2011), which make most of the food crops in the form of cereals (Li et al., 2010). Table 2 gives an updated list of crop plants in which successful transformation of chloroplasts has been reported.

V. Latest trends in chloroplast transformation

Since the machinery to carry out photosynthesis is present in the chloroplasts, transformation of this organelle therefore offers an invaluable tool to understand how photosynthesis works at molecular levels. Employing homologous recombination feature naturally available in plastids, an insertion or deletion mutation can be introduced to disrupt the function of an important gene and then study its effect on plant phenotype. Using this approach, the functions of a number of genes have been elucidated. However, study of essential genes for plant survival was no more successful using this approach. More recently, the development of inducible gene expression system allows ‘suspending’ the function of essential genes, which are critical to plant survival, and therefore cannot be studied by conventional approaches (Verhounig et al., 2010; Ramundo et al., 2013). Transformation of chloroplasts has become a necessary tool for plant scientists to directly test mathematical modelling for enhancing photosynthesis (Sharwood et al., 2008; Whitney et al., 2011a; Whitney et al., 2011b; Hanson et al., 2013; Parry et al., 2013; Price et al., 2013). The development of the cmtrL tobacco master line, for example, has emerged as a useful tool to rapidly analyse recombinant RuBisCOs constructed from diverse sources (Whitney et al., 2011a; Parry et al., 2013). Adaptation of high throughput cloning techniques such as Gateway® technology for the construction of expression vectors for higher plant chloroplasts would allow a speedy and ‘restriction-ligation’ independent assembly of multiple gene constructs in one simple reaction (Gottschamel et al., 2013).

Higher plant chloroplasts have been used to express difficult proteins whose expression was not otherwise possible such as cell-wall degrading enzymes (Leelavathi et al., 2003; Yu et al., 2007; Gray et al., 2009; Petersen and Bock, 2011), next-generation antibiotics (Oey et al., 2009a; b) and membrane proteins (Singh et al., 2008; Ahmad et al., 2012b). Several recent advances such as engineering of metabolic pathways in higher plant chloroplasts such as tobacco, tomato and lettuce (Vidi et al., 2006; Lu et al., 2013; Yabuta et al., 2013) and the ability to engineer the plastome of non-green plastids (Zhang et al., 2012; Lu et al., 2013) has opened the doors for engineering novel metabolic pathways in plants using plastid transformation.


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Similarly, the use of new selection markers would help to expand the technology to other crops such as cereals (Li et al., 2010). These developments set chloroplasts to become even more attractive platforms for the low cost production of high-value targets such as

therapeutics, vaccine antigens and commercial enzymes at large scale. Table 3 gives a comprehensive list of different foreign proteins expressed in chloroplasts along with their expression levels and various traits engineered using plastid transformation technology.

Crop Protein/trait Gene Reference

Alfalfa Aminoglycoside adenylyl transferase, Green fluorescent protein

aadA, gfp Wei et al. (2011)

Arabidopsis Aminoglycoside adenylyl transferase aadA Sikdar et al. (1998) Cabbage Aminoglycoside adenylyl transferase aadA, uidA Liu et al. (2007) Carrot Aminoglycoside adenylyl transferase,

Betaine aldehyde aadA, badh Kumar et al. (2004a)

Cauliflower Aminoglycoside adenylyl transferase aadA Nugent et al. (2006) Cotton Aminoglycoside transferase,

Neomycin phosphotransferase II aphA6, nptII Kumar et al. (2004b)

Eggplant Aminoglycoside adenylyl transferase aadA Singh et al. (2010a) Lettuce Aminoglycoside adenylyl transferase,

Green fluorescent protein aadA, gfp Lelivelt et al. (2005)

Oilseed rape

Aminoglycoside adenylyl transferase, Crystal protein insecticidal

aadA, cry1Aa10

Hou et al. (2003)

Petunia Aminoglycoside adenylyl transferase, β-Glucuronidase

aadA, uidA Zubko et al. (2004)

Poplar Fusion protein aadA/gfp Okumura et al. (2006) Potato Aminoglycoside adenylyl transferase,

Green fluorescent portein aadA, gfp Sidorov et al. (1999)

Rice Aminoglycoside adenylyl transferase, Green fluorescent portein

aadA, gfp Lee et al. (2006)

Soybean Aminoglycoside adenylyl transferase aadA Dufourmantel et al. (2004) Sugarbeet Aminoglycoside adenylyl transferase,

Green fluorescent portein aadA, gfp De Marchis et al. (2008)

Tobacco Aminoglycoside adenylyl transferase aadA Svab et al. (1990a) Tomato Aminoglycoside adenylyl transferase aadA Ruf et al. (2001) Wheat Neomycin phosphotransferase II nptII, gfp Cui et al. (2011)

Table 2. Crops in which plastid transformation has been achievedNote: Only first report is included in the table.


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Enzymes/protein/trait Gene Host plant Expression observed Reference

Biopharmaceuticals

Porcine post-weaning diarrhoea (PWD)

FaeG Tobacco 1% DW Kolotilin et al. (2012)

Human granulocyte colony-stimulating factor

hG-CSF Lettuce ND Sharifi Tabar et al. (2013)

Bacterial phage lytic protein

plyGBS Tobacco >70% TSP Oey et al. (2009a)

Human proinsulin CTB-Pins Tobacco Lettuce

16% TSP 2.5% TSP

Ruhlman et al. (2007)

Human serum albumin hsa Tobacco 11% TSP Fernandez-San Millan et al. (2003)

Human Somatotropin hST Tobacco 7% TSP Staub et al. (2000) IFN-γ GUS-IFN-γ Tobacco 6% TSP Leelavathi and

Reddy (2003) Insulin-like growth factor IGF-1n

IGF-1s Tobacco 32% TSP Daniell et al.

(2005) Interferon-α2b (IFN-α2b) IFN-α2b Tobacco 21% TSP Arlen et al. (2007) Monoclonal Ab Guy's 13 Tobacco ND Daniell et al.

(2005) Cardiotrophin-1 rhct1 Tobacco 5% TSP Farran et al.

(2008) α1-antitrypsin SERPINA1 Tobacco 2% TSP Nadai et al. (2009) CTB-F.IX (Coagulation factor IX)

CTB-FIX Tobacco 3.8% TSP Verma et al. (2010)

Retrocyclin-101-GFP RC101-GFP

Tobacco 38% TSP Lee et al. (2011)

Protegrin-1-GFP PG1-GFP Tobacco 26%TSP Lee et al. (2011) Endolysin Cpl-1 cpl-1 Tobacco 10%TSP Oey et al. (2009b) Endolysin Pal pal Tobacco 20% TSP Oey et al. (2009b) Thioredoxin 1 hTrx1 Lettuce 1% TSP Lim et al. (2011) Aprotinin APR Tobacco 0.5%TSP Tissot et al. (2008)

Enzymes/biomaterials

Cellulases bgl1C, cel6B, cel9A, xeg74

Tobacco 5-40% TSP Petersen and Bock (2011)

Elastin-derived polymer eg121 Tobacco ND Guda et al. (2000) Endo-1,4-Beta-glucanase celA Tobacco 10.7% TSP Gray et al. (2009) Exo-cellobiohydrolase celB Tobacco 3 % TSP Yu et al. (2007) Fibronectin extra domain A

EDA Tobacco 2 % TCP Farran et al. (2010)

Monellin monellin Tobacco 2.5% TSP Roh et al. (2006) p-Hydroxybenzoic acid ubiC Tobacco 13-18% TSP Viitanen (2004) Polyhydroxybutyrate phb operon Tobacco 18.8% DW Maclean et al.

(2007) Bohmert-Tatarev et al. ( 2011)

Trp asa2 Tobacco ND Zhang et al. (2001) Xylanase xynA Tobacco 6% TSP Leelavathi et al.

(2003) β-glucosidase Bgl1 Tobacco ND Jin et al. (2011)


24

Vaccine Antigens

Anthrax protective antigen pagA Tobacco 29% TSP Ruhlman et al. (2010)

Cholera toxin B-Proinsulin fusion protein

CTB-pins Tobacco 72% TSP Ruhlman et al. (2010)

Cholera Toxin B Subunit CTB-AMA1 CTB-MSP1

Tobacco 13% TSP 10% TSP

Davoodi-Semiromi et al. (2010)

CTB-fibronectin binding domain

CTB-D2 Chlamydomonas 23% TSP Dreesen et al. (2010)

HIV-1 Gag structural Poly-protein

Pr55gag Tobacco 8% TSP Scotti et al. (2009)

HPV Hpv16 L1 Tobacco 26%TSP Fernandez-San Millan et al. (2008)

HPV16-E7 Chlamydomonas 0.12% TSP Demurtas et al. (2013)

Human b-site APP cleaving enzyme

hBACE Tobacco 2% TSP Youm et al. (2010)

LTB fused with Hemagglutinin– Neuraminidase neutralizing Epitope

LTB-HNE Tobacco 0.5% TSP Sim et al. (2009)

Heat labile toxin B subunit fused with the heat stable toxin

LTB-ST Tobacco 2.3% TSP Rosales-Mendoza et al. (2009)

Vaccinia virus envelope A27L Tobacco 18% TSP Rigano et al. (2009)

Amoebiasis LecA Tobacco 6.3% TSP Chebolu and

Daniell (2007) Anthrax protective antigen pag Tobacco 18% TSP Koya et al. (2005) Canine parvovirus (CPV) CTB-2L21

GFP-2L21 Tobacco 22.6-31%

TSP Molina et al. (2004; 2005)

Cholera toxin CtxB Tobacco 4.1% TSP Daniell et al. (2001a)

Lyme disease OspAOspA-T

1-10% TSP Glenz (2006)

Rotavirus vp6 Tobacco 0.3-3% TSP Birch-Machin et al. (2004)

Malaria vaccine antigens CTB-AMA1 Tobacco Lettuce

13.2% TSP 7.3% TSP

Davoodi-Semiromi et al. (2010)

CTB-MSP1 Tobacco Lettuce

10.1% TSP 6.1% TSP

Davoodi-Semiromi et al. ( 2010)

Dengue virus DENV-1,2,3,4

Lettuce ND Maldaner et al. (2013)

Anti-Toxoplasma GRA4 Tobacco 6 μg/g FW Yácono et al. (2012)

Tuberculosis antigens CTB-ESAT6 CTB-Mtb72F

Tobacco 7.5% TSP 1.2% TSP

Lakshmi et al. (2013)

CTB-ESAT6 Lettuce 0.75% TSP Lakshmi et al. (2013)

Tetanus toxin TetC Tobacco 10-25% TSP Tregoning et al. (2003)

Hemorrhagic colitis EIT Tobacco 1.4% TSP Karimi et al. 2013


25

Agronomic Traits

Cytoplasmic male sterility phaA Tobacco ND Ruiz and Daniell (2005)

Disease resistance msi-99 Tobacco ND DeGray et al. (2001)

Drought tolerance tps1 (yeast) Tobacco ND Lee et al. (2003) Herbicide resistance aroA Tobacco ND Daniell et al.

(1998) Herbicide resistance bar Tobacco ND Iamtham and Day

(2000) Insect resistance cry1A(c) Tobacco 5% TSP McBride et al.

(1995) cry2Aa2 Tobacco 3% TSP Kota (1999) cry2Aa2 Operon

Tobacco 45.3% TSP De Cosa et al. (2001)

cry1Aa10 Oilseed rape ND Hou et al. (2003) cry1Ab Soyabean ND Dufourmantel et

al. (2005) cry9Aa2 Tobacco 10% TSP Chakrabarti et al.

(2006) Phytoremediation merA/merB Tobacco ND Ruiz et al. (2003)

Hussein et al. (2007)

Salt tolerance badh Carrot ND Kumar et al. (2004a)

Table 3: Heterologous expression of recombinant proteins and vaccine antigens in higher plant chloroplasts

Abbreviations: TSP: Total soluble proteins, TCP: Total cellular proteins, ND: Not determined, FW: Fresh weight,

DW: Dry weight

VI. Conclusion and future perspectives

Although a large number of proteins have been expressed in plants via chloroplast transformation technology, no chloroplast-made product has reached to the market yet. One of the major reasons for this vacuum is that the technology is heavily patented and therefore is not yet open to commercial entrepreneurs. However, the expiry of patents in the near future should result in the removal of such barriers. Other factors include time course involved in the development of transplastomic plants, costs associated with the purification of recombinant proteins, and lack of well-characterized good manufacturing practices (GMPs) and regulations for plant-made pharmaceuticals. Successful commercialization of plant-made pharmaceuticals would require the establishment of rapid and non-chromatographic techniques for the purification of recombinant proteins, facile transformation and

regeneration methodologies including new class of selection markers to be used in the recovery of transplastomic plants, and the availability of GMPs used to regulate the production of pharmaceuticals in plants. Similarly, the future attempts must focus on the transformation of non-green plastids such as roots, tubers and other storage organs, which are directly consumed up by mammals and humans.

VII. Acknowledgments The work in authors’ lab is supported by Higher Education Commission (HEC) and Punjab Agricultural Research Board (PARB). We sincerely apologise to those colleagues whose work could not be discussed here due to space limitation.

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plastids for the production of foreign proteins at high levels

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