Chloroplast Vector Systems for Biotechnology Applications

Chloroplasts are ideal hosts for expression of transgenes. Transgene integration into the chloroplast genome occurs via homologous recombination of flanking sequences used in chloroplast vectors. Identification of spacer regions to integrate transgenes and endogenous regulatory sequences that support optimal expression is the first step in construction of chloroplast vectors. Thirty-five sequenced crop chloroplast genomes provide this essential information. Various steps involved in the design and construction of chloroplast vectors, DNA delivery, and multiple rounds of selection are described. Several crop species have stably integrated transgenes conferring agronomic traits, including herbicide, insect, and disease resistance, drought and salt tolerance, and phytoremediation. Several crop chloroplast genomes have been transformed via organogenesis (cauliflower [Brassica oleracea], cabbage [Brassica capitata], lettuce [Lactuca sativa], oilseed rape [Brassica napus], petunia [Petunia hybrida], poplar [Populus spp.], potato [Solanum tuberosum], tobacco [Nicotiana tabacum], and tomato [Solanum lycopersicum]) or embryogenesis (carrot [Daucus carota], cotton [Gossypium hirsutum], rice [Oryza sativa], and soybean [Glycine max]), and maternal inheritance of transgenes has been observed. Chloroplast-derived biopharmaceutical proteins, including insulin, interferons (IFNs), and somatotropin (ST), have been evaluated by in vitro studies. Human INFα2b transplastomic plants have been evaluated in field studies. Chloroplast-derived vaccine antigens against bacterial (cholera, tetanus, anthrax, plague, and Lyme disease), viral (canine parvovirus [CPV] and rotavirus), and protozoan (amoeba) pathogens have been evaluated by immune responses, neutralizing antibodies, and pathogen or toxin challenge in animals. Chloroplasts have been used as bioreactors for production of biopolymers, amino acids, and industrial enzymes. Oral delivery of plant cells expressing proinsulin (Pins) in chloroplasts offered protection against development of insulitis in diabetic mice; such delivery eliminates expensive fermentation, purification, low temperature storage, and transportation. Chloroplast vector systems used in these biotechnology applications are described. Chloroplasts are members of a class of organelles known as plastids and are found in plant cells and eukaryotic algae. As the site of photosynthesis, chloroplasts are the primary source of the world's food productivity and they sustain life on this planet. Other important activities that occur in plastids include evolution of oxygen, sequestration of carbon, production of starch, synthesis of amino acids, fatty acids, and pigments, and key aspects of sulfur and nitrogen metabolism. Chloroplasts are generally considered as derivative of a cyanobacterial ancestor that was captured early during the evolution of a eukaryotic cell. However, the chloroplast genome is considerably reduced in size as compared to that of free-living cyanobacteria, but the genomic sequences that are still present show clear similarities (Martin et al., 2002). Land plant chloroplast genomes typically contain 110 to 120 unique genes, whereas cyanobacteria contain more than 1,500 genes. Many of the missing genes are present in the nuclear genome of the host (Martin et al., 2002). In most angiosperm plant species, plastid genes are maternally inherited (Hagemann, 2004), and therefore, transgenes in these plastids are not disseminated by pollen. This makes plastid transformation a valuable tool for the creation and cultivation of genetically modified plants that are biologically contained, thus posing lower environmental risks (Daniell, 2002, 2007). This biological containment strategy is therefore suitable for establishing the coexistence of conventional and genetically modified crops. Cytoplasmic male sterility (CMS) presents a further genetic engineering approach for transgene containment (Ruiz and Daniell, 2005). Furthermore, plant-derived therapeutic proteins are free of human pathogens and mammalian viral vectors. Therefore, plastids provide a viable alternative to conventional production systems such as microbial fermentation or mammalian cell culture. Another advantage of plastid transformation is the ability to accumulate large amounts of foreign protein (up to 46% of total leaf protein) when the transgene is stably integrated (De Cosa et al., 2001). This is due to the polyploidy of the plastid genetic system with up to 10,000 copies of the chloroplast genome in each plant cell, resulting in the ability to sustain a very high number of functional gene copies. Furthermore, site-specific integration into the chloroplast genome by homologous recombination of flanking chloroplast DNA sequences present in the chloroplast vector eliminates the concerns of position effect, frequently observed in nuclear transgenic lines (Daniell et al., 2002). Other advantages seen in chloroplast transgenic plants include the lack of transgene silencing despite the accumulation of transcripts at a level 169-fold higher than in nuclear transgenic plants (Lee et al., 2003) and accumulation of foreign proteins at levels up to 46% of total leaf protein (De Cosa et al., 2001). Chloroplast genetic engineering also offers the unique advantage of transgene stacking, i.e. simultaneous expression of multiple transgenes, creating an opportunity to produce multivalent vaccines in a single transformation step. Several heterologous operons have been expressed in transgenic chloroplasts, and polycistrons are translated without processing into monocistrons (Quesada-Vargas et al., 2005). Moreover, foreign proteins synthesized in chloroplasts are properly folded with appropriate posttranscriptional modifications, including disulfide bonds (Staub et al., 2000; Arlen et al., 2007; Ruhlman et al., 2007) and lipid modifications (Glenz et al., 2006). This article is focused on the various components of vectors used for stable protein production in transgenic chloroplasts. The chloroplast genome typically consists of basic units of double-stranded DNA of 120 to 220 kb arranged in monomeric or multimeric circles as well as in linear molecules (Palmer, 1985; Lilly et al., 2001). The chloroplast genome generally has a highly conserved organization (Raubeson and Jansen, 2005), with most land plant genomes having two identical copies of a 20- to 30-kb inverted repeat region (IRA and IRB) separating a large single copy (LSC) region and a small single copy (SSC) region. Plastid transformation is typically based on DNA delivery by the biolistic process (Daniell et al., 1990; Sanford et al., 1993) or occasionally by polyethylene glycol (PEG) treatment of protoplasts (Golds et al., 1993; O'Neill et al., 1993). This is followed by transgene integration into the chloroplast genome via homologous recombination facilitated by a RecA-type (Cerutti et al., 1992) system between the plastid-targeting sequences of the transformation vector and the targeted region of the plastid genome. Chloroplast transformation vectors are thus designed with homologous flanking sequences on either side of the transgene cassette to facilitate double recombination. Targeting sequences have no special properties other than that they are homologous to the chosen target site and are generally about 1 kb in size. Both flanking sequences are essential for homologous recombination. Transformation is accomplished by integration of the transgene into a few genome copies, followed by 25 to 30 cell divisions under selection pressure to eliminate untransformed plastids, thereby achieving a homogeneous population of plastid genomes. If the transgene is targeted into the IR region, integration in one IR is followed by the phenomenon of copy correction that duplicates the introduced transgene into the other IR as well. Transgenes have been stably integrated at several sites within the plastid genome. Transgenes were first integrated into transcriptionally silent spacer regions (Svab and Maliga, 1993). However, transcriptionally active spacer regions offer unique advantages, including insertion of transgenes without 5′ or 3′ untranslated regions (UTRs) or promoters. To the most used site of integration is the transcriptionally active region between the genes, within the in the IR regions of the chloroplast genome. The foreign gene expression levels genes integrated at this site are the (De Cosa et al., 2001). that this site is unique and highly transgene integration and Chloroplast vectors also an of that of the the thereby the copy number for homologous recombination and the of transgene is present within the flanking region and et al., 2004), and this facilitate of foreign vectors within chloroplasts (Daniell et al., the of transgene and in the first of selection et al., This is further by the first engineering by the gene at this site et al., other on engineering at other integration sites within the chloroplast genome were of transgenes between of and also processing of foreign transcripts of processing of present within flanking The of a the and genes the IR region of the tobacco chloroplast genome as flanking sequences for homologous recombination to several other plant species genome was several (Daniell et al., This was based on the high of this spacer region the higher plant chloroplast genomes. designed for transformation of the tobacco plastid genome have been used for potato and tomato plastid the homologous flanking sequences present in these vectors to the sequences of potato and tomato plastid DNA but the of transformation is lower than tobacco et al., et al., 2001). one potato and tomato chloroplast transgenic was and when compared to about tobacco chloroplast transgenic lines one et al., lower was observed when petunia flanking sequences were used to the tobacco chloroplast genome et al., that a lack of the transformation to a However, of spacer regions members of that regions are identical (Daniell et al., 2006). of spacer regions of chloroplast genomes that not a single spacer region is identical sequenced chloroplast genomes et al., 2007). Therefore, the of a vector is when a higher level of plant species but than chloroplast vectors. The for several crop chloroplast genome sequences have been at the site or for to genomic of transformation for each crop the of the selection of plastid was by in the gene et al., et al., integration and expression of the gene was first in the chloroplast genome of The gene the 3′ that and by and to chloroplast The gene was used as a in and the of transformation to more than the genes (Svab and Maliga, 1993). to the of the the was not expressed of the was of the of the gene in of transformation was first that offers selection (Svab and Maliga, 1993) by not cell and at high was observed to in other plant species Chloroplast transformation and selection for crop species Chloroplast transformation and selection for crop species The gene is alternative for plastid transformation that et al., 1993). gene with high transformation was et al., 2002). Another selection strategy a vector was used for cotton transformation for transformation was cells et al., The cotton plastid transformation vector two genes and for two enzymes. The gene was by the and gene of expression in the and in The gene was by the and of expression in the Both genes with regulatory sequences facilitated of the selection during and as well as in plastids and chloroplasts. The double transformation vector was to at more than single gene chloroplast vectors. To of genes, several have for engineering chloroplasts that are free of The gene has been as a plant-derived gene to chloroplast genomes (Daniell et al., The selection process involved of the to by the the is present in chloroplasts of a few plant species to and et al., et al., is considered as a suitable in crop The transformation of transgenic within in and selection was more than In the salt in et al., The bacterial and conferring resistance, has also been as a as an in nuclear and to the of the gene in plastid when introduced by selection for a However, the gene was not found to suitable for the selection of transplastomic when expressed at a higher level of total that selection by is by of of the gene the et al., 2001). The of to plastids was by plastid et al., In cells of tobacco leaf the of has been of the The plastid into the cell due to the plastid at several the other no on plastid Therefore, genes not used to plastid and was when genes were introduced by selection for a selection has also been for plastid transformation based on expression of the bacterial gene and Maliga, the of to is to cells that this to This selection was to on was by the site-specific et al., 2001). and have been used as plastid (Daniell and et al., 1990; et al., 1990; and Maliga, The of by et al., 1990; et al., whereas is a that of the gene in The in the of and when or and 2001). has been used to gene expression et al., and stable transformation et al., et al., et al., in chloroplasts. has also been with and used as a and and Maliga, has been used to the of delivery of foreign toxin protein with a site in between and has been used to the of and in the system et al., 2006). were with plant leaf was in the and the of the as well as various cells of and In with untransformed leaf or plant leaf no was observed. This the delivery of a foreign protein the into the Moreover, was not in in the or of with that a such as is for delivery of an of a foreign protein the into the has been used as a gene in chloroplast expression and in studies. of the plastid transformation have gene for the of transformed but such into the food a of have been to eliminate genes including via by site-specific of the and with that homologous recombination between two under et al., genes have been transplastomic tobacco via that and of of tobacco plants by et al., 2007). The vector used for plastid transformation the gene the The primary transplastomic were by were of the gene by homologous recombination within the region of the and of the of the gene to of an gene and expression of the that to and et al., 2007). of this approach facilitated of by creation of a due to the of a plastid gene et al., 2006). two and have been for plastid gene the site-specific recombination a gene by sites was expression of the protein was via the nuclear genome. The site-specific to a for the gene when with and bacterial are by of the gene was transformation of the with an gene et al., 2007). a vector used to the integration of genes et al., The approach transformation with two that target at two one a and the other a for the transplastomic that also an insertion of the The of the approach was first in et al., when the approach was in a of was tobacco has a number of chloroplasts and Maliga, of was of an gene selection et al., 2005). synthesized proteins are highly to and protection chloroplast such approach used the by the to the into The the foreign proteins thereby protein accumulation to of total protein Cosa et al., 2001). when the human was by the chloroplast 5′ and 3′ in the protein expression resulting in the of to in protein expression was seen when were in the et al., This the of regulatory sequences during and protection when is Several on transgenic chloroplasts not with transcripts were to and more than the nuclear plants when the transgene was by the 5′ or the gene the 5′ transgenic lines in the first functional in transgenic the gene transgenic lines et al., The lack of between levels and that is of than protein in transgenic chloroplasts. Several have the of 5′ However, in a few the amino sequences of the an important in synthesized proteins or and Maliga, 2001). Human was in transgenic with in expression (up to and facilitated delivery to protection against the development of insulitis in diabetic et al., 2007). is not unique to chloroplasts. in or is as a when expressed without is expression of foreign proteins have a on the plant et al., 2004), and of transplastomic plants to not In that the of is and of the expression levels or expression of foreign protein is highly the systems are well known for nuclear transgenes, most systems for plastids on nuclear transgenes, a targeted to the chloroplast expression of a transgene under the of a et al., et al., et al., 2005). expression system for plastids has been transgene in the was and 2005). is a to expression systems that not nuclear transgenes. has been the most plastid transformation system of in genetic single tobacco plant is of a and 1 of tobacco produce more than of et al., Arlen et al., 2007). offer transgene containment in to protection offered by maternal have that of transgenes in tobacco is to (Daniell, 2007; et al., 2007; and Maliga, this an ideal system for of chloroplasts as In has been via the tobacco chloroplast genome as a (Ruiz and Daniell, 2005). As a tobacco has been to more than expensive than the frequently used fermentation systems et al., tobacco eliminates of food is a and Plastid transformation in higher plants was first in tobacco and is a foreign genes have been expressed to agronomic traits, or of agronomic traits, vaccine and via the plastid genome of agronomic traits, vaccine and via the plastid genome of the plastid transformation to other species is important to this The of DNA delivery target selection and systems is for the of species in plastid transformation Plastid transformation is most by biolistic delivery of DNA into leaf but has also been via DNA by protoplasts et al., et al., 2006). In species other than petunia and oilseed leaf or plastid plants of and cotton were via embryogenesis of with the of plastid vectors. the chloroplast transformation and selection for crop have been in other plants protein production was in such as and However, the of protein was lower than the level observed in leaf chloroplasts et al., has also been in the chloroplast transformation system for tomato that plastid expression of a bacterial gene in and of the of to resulting in a of of et al., 2007). chloroplast transformation system has also been for cabbage et al., 2007). including have plastid genetic plant species not protein processing but also offer an ideal system for The of lettuce are by and the to is compared to for such as and Furthermore, lettuce is well for cultivation by systems et al., 2006). of a valuable therapeutic the in lettuce chloroplasts was et al., 2007). This is the first of expression of a therapeutic protein in an are to the of important such as and soybean in vitro embryogenesis (Daniell et al., In such transformation of the plastid genome was embryogenesis by cells or The first stable plastid transformation of cell and embryogenesis was in et al., transgenic plants were cell with the and genes. However, in the of plastid transformation the gene was and no transgenic or plants were as the selection cotton cell lines were a double vector two genes and to et al., lines were and maternal inheritance of plastid transformation was as the et al., and the gene as the transgenic soybean plants were via embryogenesis and were plastid transformation in rice was for (Lee et al., The transplastomic rice plants expressed in plastids and viable were to the transgenes to the However, transplastomic rice plants were not two of Plastid transformation of and soybean the for of the plastid genome of several that of the expression of the chloroplast expression vector the integration genes, regulatory and genes of a of regulatory and genes of used for chloroplast to of the high in the and systems between and chloroplasts, the chloroplast expression vectors are in first with plant The of the plastid transformation vector with the gene in the of expression of the with expression of the gene of of transplastomic are of transplastomic tobacco and lettuce first and and and and rounds of of transgene integration into the chloroplast genome. DNA transplastomic are by and and and Daniell, are used for of to transformed lines DNA to with in transformed The chloroplast genome is with a and transplastomic transplastomic Several transgenes have valuable agronomic traits, including and pathogen resistance, drought tolerance, salt tolerance, and chloroplast genetic engineering tobacco plants expressing an protein have against target and that against protein et al., of the in the expression levels on of total leaf protein) and in the of (De Cosa et al., 2001). In soybean plastid expressing also against et al., 2005). The an of was expressed via the chloroplast genome to high levels of expression in transgenic tobacco In with the bacterial pathogen and the pathogen in untransformed whereas transformed no et al., 2001). such as and are to plants generally of of to to drought by expressing 1 gene via nuclear transformation have of at very low levels of However, of in the chloroplasts has no the untransformed and transgenic and and in drought with to and (Lee et al., expression of in and of via plastid genetic engineering high levels of salt plants expressing in the of high of (up to the level of salt genetically modified crop plants et al., Chloroplast genetic engineering has also been used for the first to to the of plants for phytoremediation. This was accomplished by a the and genes, for and into the chloroplast genome in a single transformation integration of the into the chloroplast genome in high levels of to the when in up to (Ruiz et al., Chloroplast transgenic lines the levels in and more to than untransformed plants et al., 2007). is ideal for of is a has been for for oilseed and However, such systems are not for the of used in In various in the nuclear genome a of that are not Moreover, of sterility and the production of transgenic that transgenic to plants of the of of the with a or To of these has been via of gene for into chloroplast genome. The transgenic lines were for the male sterility (Ruiz and Daniell, 2005). of male was by of thereby the levels of fatty is for the of the Several biopharmaceutical proteins have been (Daniell, expression of a protein in chloroplasts was first for a with such as and delivery et al., Human a was expressed chloroplasts in a biologically active and (Staub et al., The key of is in the of in are treatment of and human Another important therapeutic protein that of the protein in is in to in and other at expressing have low levels of of in nuclear transgenic plants et al., 2002). the other in chloroplast transgenic expression levels of up to were observed et al., The are of the first of against viral and also by bacterial and cells in for a and The of for the treatment of is and is therefore to the of in Therefore, was expressed in tobacco chloroplasts with levels of up to of or of leaf and facilitated the first field production of a plant-derived human protein et al., 2007). in vitro biological to when for ability to cells against viral in the and to early human in Another therapeutic protein expressed in chloroplasts is human and In a the human offered protection to human against by the As to delivery and production and Daniell, 2006). vaccines expressed in plants are of a in when or these also a pathogen The ability for plant-derived vaccines to in the is a However, the vaccine in the and the in the et al., antigens against (Daniell et al., et al., et al., et al., 2005), (Daniell et al., and Daniell, and et al., have been expressed in transgenic chloroplasts the has been to an vaccine and is by The into protein and for the ability of to to the also as a and is very in several vaccine In one such of protein diabetic against development of insulitis et al., 2007). has been an of in the is not and for and by or the of is an ideal biological The human vaccine for anthrax, the of the and of the and to side with this Therefore, an expression system that provide a and vaccine is In an to produce vaccine in large and free of bacterial was expressed in transgenic tobacco chloroplasts by the gene into the chloroplast genome et al., et al., 2005). under up to of in that was in to in of with or with up to and of challenge with of the and properties of plant-derived vaccine vaccine and amino have also been expressed in chloroplasts is in small in In the gene the of to and However, in chloroplasts, is to by due to lack of integration of the gene into the tobacco chloroplast in of the and accumulation of this up to of et al., In the gene for was expressed in the chloroplasts of tobacco plants et al., in the cells to of that the was units leaf the is more than plastid transformation has been accomplished in few are that have the of chloroplast transformation to plant is the of the genome The chloroplast transformation vectors homologous flanking regions for recombination and insertion of foreign genes. Therefore, is an to chloroplast genomes to facilitate transformation of crop of the small size of the genome and of to an genome within a single is to a few crop chloroplast genomes have been sequenced and crop chloroplast genomes were In the 25 crop chloroplast genomes have been including soybean and cotton et al., et al., that spacer regions and regulatory sequences about to of the chloroplast genome and that spacer regions are not highly of chloroplast genomes that not one spacer region Therefore, chloroplast vectors for transformation of et al., 2007). Plastid transformation is a Therefore, is not to have the genome a of DNA delivery, and is essential to plastid transformation in chloroplast genome sequences of several including and have been for several of genomes has been transformed include the of expressing transgenes in plastids, in gene expression and gene systems are of chloroplasts. Moreover, is not to plants via rounds of as Furthermore, are used as the transformation target than chloroplasts that are about in size than the chloroplasts in the leaf Therefore, plastids with due to biolistic also to selection for a selection However, transformation of cotton or cells and via embryogenesis offers for and Ruhlman for with