challenges and perspectives in commercializing plastid ... challenges and perspectives in...

REVIEW

Challenges and perspectives in commercializing plastid

transformation technology

Niaz Ahmad1, *, Franck Michoux2, Andreas G. Lössl3, and Peter J. Nixon4

1 Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic

Engineering, Jhang Road, Faisalabad, Pakistan

2 Alkion Biopharma SAS, 4 rue Pierre Fontaine, 91058 Evry, France

3 Department of Applied Plant Sciences and Plant Biotechnology, University of Natural

Resources and Applied Life Sciences (BOKU), Vienna, Austria

4 Department of Life Sciences, Sir Ernst Chain Building–Wolfson Laboratories, Imperial

College, South Kensington Campus, London SW7 2AZ, UK

* Correspondence: [email protected]

Received 30 July 2016; Accepted 6 September 2016

Running head: Developments in plastid transformation technology

Editor: Christine Raines, University of Essex

Running head: Ahmad et al.

Highlight

Transformation of the plastid genome has emerged as an alternative tool to develop

transgenic plants. This review discusses recent developments and the current challenges.

Abstract

Plastid transformation has emerged as an alternative platform to generate transgenic

plants. Attractive features of this technology include specific integration of transgenes—

either individually or as operons—into the plastid genome through homologous

recombination, the potential for high-level protein expression, and transgene containment

because of the maternal inheritance of plastids. Several issues associated with nuclear

transformation such as gene silencing, variable gene expression due to the Mendelian

laws of inheritance, and epigenetic regulation have not been observed in the plastid

genome. Plastid transformation has been successfully used for the production of

therapeutics, vaccines, antigens, and commercial enzymes, and for engineering various

agronomic traits including resistance to biotic and abiotic stresses. However, these

demonstrations have usually focused on model systems such as tobacco, and the

technology per se has not yet reached the market. Technical factors limiting this

technology include the lack of efficient protocols for the transformation of cereals, poor

transgene expression in non-green plastids, a limited number of selection markers, and

the lengthy procedures required to recover fully segregated plants. This article discusses

the technology of transforming the plastid genome, the positive and negative features

compared with nuclear transformation, and the current challenges that need to be

addressed for successful commercialization.

Abbreviations: CCM, carbon concentrating mechanism; GFP, green fluorescent protein;

IR, inverted repeat; LSC, large single copy; PTM, post-translational modification; SSC,

small single copy; TIB, temporary immersion bioreactor; TSP, total soluble proteins.

Key words: Plastids, transformation of plastid genome, site-specific integration of

transgenes, gene containment, metabolic engineering, molecular farming

Introduction

Plastids are a group of organelles present in the cells of plants, algae, and some protists

that are essential for viability (Waters and Pyke, 2005). It is now widely accepted that

plastids originated around ~1.5 billion years ago when a cyanobacterial cell was engulfed

by a heterotrophic eukaryote (Bock, 2015). Over the years, the host and the incoming

bacterial cell developed a symbiotic relationship, which involved: (i) synchronization of

cell division of the cyanobacterium with that of the host cell, (ii) extensive transfer of the

incoming cell’s genetic material to the host cell nucleus and/or deletion of redundant

genetic information, (iii) evolution of mechanisms to import proteins back into the

cyanobacterium from the host cell, and (iv) the development of signalling pathways for

efficient coordination of gene expression in the plastid and in the host nucleus. The result

of this massive gene transfer to the host cell nucleus has been a drastic reduction in the

size of the plastid genome (termed plastome) of modern-day plants compared with the

genomes of free-living cyanobacteria. Analysis of the Arabidopsis chloroplast proteome

has revealed the presence of ~3000 proteins (Jarvis, 2004; Kleffmann et al., 2004), of

which only 87 are encoded on the plastome (Martin et al., 2002).

The sequence, structure, organization, and mechanisms of protein expression all

clearly indicate that the current-day plastome is a remnant of an earlier cyanobacterial-

like genome. A comparative analysis of the circular plastome of higher plants shows that

many features such as overall size, gene density, ‘AT’ content, and the presence of

inverted repeats (IR), which bifurcates the plastome into large single copy (LSC) and

small single copy (SSC) regions, are highly conserved between species (Carbonell-

Caballero et al., 2015; Wicke et al., 2011). The IR regions are mirror images of each

other, and are maintained with respect to each other by an intrinsic mechanism known as

copy correction (Khan et al., 2007).

Another interesting feature of plastids is their diversity in function. Modern-day

plants differentiate to produce more than 50 different types of cell (Pyke, 2009). The

incoming plastids have likewise been manipulated during their evolutionary journey to

carry out a range of different functions according to the type of cell/tissue in which they

reside. Therefore, in addition to photosynthesis, plastids are involved in an array of

cellular processes, including starch metabolism, sulphur metabolism, fatty acid

biosynthesis, nitrogen assimilation, and the synthesis of various amino acids (Pyke, 2009;

Van Dingenen et al., 2016; Warzecha, 2016). The study of plastid biology is therefore

essential for a holistic view of plant physiology.

The plastid genome also provides an alternative site for the stable insertion of

transgenes in plants and is also often considered an ‘environmentally friendly’ form of

plant genetic engineering, as plastid DNA (in most crops) is largely excluded from

pollen. Chloroplast transformation was first demonstrated for the unicellular green alga

Chlamydomonas reinhardtii (Boynton et al., 1988) followed by the higher plants tobacco

(Svab et al., 1990a) and tomato (Ruf et al., 2001). Since then, the chloroplast genome has

been manipulated to produce vaccine antigens, commercial enzymes, hormones,

antibodies, pharmaceuticals, and biomaterials, and engineered to provide various

agronomic traits, including resistance against various biotic (viral, fungal, and bacterial

diseases) and abiotic (e.g. salt, drought, heavy metals) factors (see Table 1). However,

the number of crop plants in which chloroplast transformation has been reported remains

disappointingly low (see Table 2) and the technology has not yet reached the field. In this

article, we will describe the process of transforming the plastid genome and its salient

advantages, followed by the challenges hampering its successful implementation to field

crops.

Transforming the plastid genome: the state of the art

One of the intriguing features of the plastome is its incredibly high copy number,

reaching levels of about 10 000 copies per mesophyll cell (Shaver et al., 2006). This high

copy number is thought to produce and maintain the photosynthetic apparatus during

plant development (Jarvis and López-Juez, 2013) and also plays an important role in

plastome stability by eliminating deleterious mutations through homologous

recombination (Garton et al., 2007; Jarvis and López-Juez, 2013).

The current procedure for experimentally transforming the plastome starts with

the construction of a transforming plasmid containing an antibiotic-resistance cassette

that functions in chloroplasts and a separate expression cassette containing the gene or

genes of interest to be expressed. Flanking these cassettes are two plastome fragments,

typically 1–2 kb in size, termed flanking regions or targeting regions (Fig. 1), which help

integrate foreign DNA at a specific location in the plastome through homologous

recombination. The plastome-transforming plasmids are usually Escherichia coli

plasmids that are unable to replicate inside the plastid. These constructs are delivered to

plastids through a gene-gun-mediated particle delivery system after coating the DNA on

to the surface of microparticles (0.4–1.0 µm) of gold or tungsten (Maliga and

Tungsuchat-Huang, 2014). Other methods such as polyethylene glycol-mediated

transformation or microinjection can also be used to deliver foreign DNA into plastids,

but the transformation and regeneration efficiency of these methods is relatively low

compared with biolistic DNA delivery (Day and Goldschmidt‐Clermont, 2011; Maliga,

2002). Subsequently, antibiotic resistance selects for transgenes that have been stably

integrated into the plastome (Maliga, 2002, 2003). The most widely used selectable

marker is the bacterial aadA gene, expressed using plastid expression regulatory

elements, which encodes the enzyme aminoglycoside 3′′-adenylyltransferase, which

detoxifies spectinomycin and streptomycin. It is a positive dominant selection marker and

has been reported to give high transformation efficiency due to selective amplification of

transformed copies of plastid DNA (Svab et al., 1990b; Svab and Maliga, 1993).

When the DNA is delivered into plastids, only a few copies of the plastome are

initially transformed, resulting in a so-called heteroplasmic state. Homoplasmy, where all

copies of the plastome contain the transgene, is achieved by subculturing the bombarded

explant in vitro under selection (Maliga and Tungsuchat-Huang, 2014). Under these

conditions, both the plastid and the plastome copy number decrease from several hundred

to much fewer. The change in overall plastid copy number is thought to allow plastids

carrying the selectable marker to divide at a much faster rate than wild-type versions

under the stringent selection conditions used (Maliga and Bock, 2011). This dilution

cycle is repeated under constant selection pressure until wild-type copies of the plastome

are lost. Once homoplasmy is achieved, the marker gene can be excised to generate

marker-free plants. Several systems are available, such as Cre/loxP, Int (integrase) phage

recombinase system, or intrinsic homologous recombination employing flanking direct

repeats (see Day and Goldschmidt‐Clermont, 2011 for a comprehensive review).

Key advantages of chloroplast transformation technology

Natural gene containment

One of the major concerns over the cultivation of transgenic plants in the field is the

outcrossing of transgenes and their dissemination into wild species. This perceived risk

stems from the observation that plants exchange their genetic material with their relatives

through pollen-mediated introgression (Stewart et al., 2003). For example, genes

encoding resistance against three herbicides—Roundup, Liberty, and Pursuit—have been

transferred from nuclear transformants of canola (Brassica napus L.) to weeds (Steward,

2000). Containment of transgenes has therefore become an important consideration when

releasing genetically modified plants into the field. In this context, transforming non-

nuclear genomes, such as those in the plastid and mitochondrion, may provide a

mechanism for natural gene containment due to their maternal mode of inheritance (Jaffe

et al., 2008). For instance, inheritance of plastids in the majority of angiosperms is

predominantly maternal (Greiner et al., 2015; Hagemann, 2004), which means that the

transgene inserted into the plastid genome of these species should in principle not be

dispersed via pollen. However, containment is not absolute, and transmission of

transgenic plastids to pollen has been measured to be in the range of 0.00024–0.008% in

tobacco (Ruf et al., 2007; Svab and Maliga, 2007) and 0.0039% in Arabidopsis thaliana

(Azhagiri and Maliga, 2007). In the case where gene containment needs to be absolute,

new methods have been developed based on in vitro propagation of transplastomic

biomass (see Transgene containment in chloroplast genome is not absolute, below, for

more details). The plastid DNA can also be transferred to the nuclear genome (Huang et

al., 2003; Sheppard et al., 2008; Wang et al., 2012), which raises the possibility that

transgenes originally inserted into the plastome could be spread like a classic nuclear

transformant (Allainguillaume et al., 2009; Gilbert, 2013).

Site-specific integration of the transgene into the plastome

As illustrated in Fig. 1, site-specific integration of a gene of interest at a predetermined

location in the plastid genome is advantageous as it avoids several issues such as gene

silencing and unwanted mutations resulting from the random integration of transgenes.

Although homologous recombination has also been reported for nuclear genomes (Vieler

et al., 2012), the standard procedure involving Agrobacterium transformation often leads

to random insertion of transgenes (Kohli et al., 2010). The foreign gene may also interact

with native nuclear genes and, as a result of this non-allelic interaction, the function of

native genes could be masked or vice versa (Scheid et al., 1991). Such potential

challenges, which are inevitable in the case of nuclear transformation (Qin et al., 2003),

have not been observed when foreign genes are introduced into the chloroplast genome

(Bock, 2015; Maliga and Bock, 2011).

Besides the insertion of foreign genes, chloroplast transformation has also been a

useful tool to probe the role of plastid-encoded proteins through the creation of knockout

and site-directed mutants (Bock, 2015).

Transformation of the chloroplast genome often yields high

expression levels

The much higher copy number displayed by the plastome favours high expression of

foreign proteins in comparison to nuclear expression. For example, expression of the β-

subunit of enterotoxigenic E. coli (LT-B) from the tobacco nuclear genome was less than

0.01% of total soluble protein (TSP) (Haq et al., 1995), whereas transformation of the

chloroplast led to a 410-fold increase in expression of the same protein (Daniell et al.,

2001a). There are now several examples of extremely high expression of foreign proteins

in tobacco chloroplasts, with levels exceeding 70% TSP (Table 1).

Expressing and confining a foreign protein to the chloroplast might also be useful

in protecting plants from the toxic effects of that protein. For example, the presence of

even small levels of cholera β-toxin in the tobacco cytosol (0.3% TSP) resulted in stunted

plant growth (Arakawa et al., 1997), whereas chloroplast transformants expressing 14-

fold higher levels were not adversely affected (Daniell et al., 2001b). Proteins that are

difficult to produce in other systems, such as antibiotics and cell-wall-degrading

enzymes, have also been successfully expressed in chloroplasts (Espinoza-Sánchez et al.,

2016; Oey et al., 2009a, 2009b; Petersen and Bock, 2011; Verma et al., 2010a) (Table 1).

However, there are examples where overexpression of foreign proteins can affect

chloroplast function and plant fitness (Scotti and Cardi, 2014). For example, expression

of tetanus toxin fragment C (TetC) at 25% TSP, but not at 10% TSP, proved detrimental

to the growth of host plants (Tregoning et al., 2003). Similarly, expression of glutathione-

S-transferase (GST) in tobacco chloroplasts at ~7% TSP induced cytoplasmic male

sterility (Ahmad et al., 2012a), whereas no such effect was observed at lower levels of

accumulation (Le Martret et al., 2011).

Gene expression in chloroplasts is highly regulated at the transcriptional,

translational, and post-translational levels (Maliga, 2003). Therefore, the accumulation of

foreign proteins can be increased or decreased several fold simply through the judicious

use of regulatory elements, such as the promoter and 5′ untranslated regions, and at the

post-translational level by manipulation of the N-terminal coding sequence of the gene of

interest (Maliga, 2003) and co-expression of a chaperone to aid protein folding (De Cosa

et al., 2001; Lin et al., 2014; Whitney et al., 2015).

Engineering complex metabolic pathways

Metabolic pathways often require the concerted action of multiple enzymes to confer a

desired trait on a plant, such as protection from biotic or abiotic stresses, improvement of

nutritional value, production of metabolites in bulk for industrial applications, or

increasing photosynthesis for improving crop yields (Bock, 2013). For example, the DNA

responsible for encoding the nitrogen-fixation enzymes in Klebsiella pneumoniae is 24

kb long and consists of 20 genes (Arnold et al., 1988), and the polysaccharide formation

gene cluster in Streptococcus is 25 kb long and composed of 16 genes (Cieslewicz et al.,

2001). Introducing these genes via nuclear transformation would require several

transformation events and several backcrosses. One of the prokaryotic characteristics of

the plastid genome is the operonal organization of genes, allowing several genes to be co-

transcribed. This feature opens up the possibility of using chloroplast transformation to

introduce natural and artificial operons into the plastome in a single transformation step.

However, post-transcriptional processes within the chloroplast, such as RNA editing,

removal of introns, processing of mRNA ends, and cleavage of larger mRNA transcripts

(reviewed by Bock, 2015). must be considered. For instance, the incorporation of small

DNA (~50 bp) elements known as intercistronic expression elements between genes

(Zhou et al., 2007) has been shown to direct the fragmentation of a larger precursor

transcript into smaller transcripts whereby translation is improved of each gene (Lu et al.,

2013; Zhou et al., 2007).

A number of novel metabolic pathways have now been successfully introduced

into plants through plastid transformation, such as the production of polyhydroxybutyrate

(Lössl et al., 2003, 2005); the enhancement of β-carotene content (Apel and Bock, 2009;

Harada et al., 2014; Wurbs et al., 2007); the introduction of the mevalonate pathway

(Kumar et al., 2012); the biosynthesis of artemisinic acid for the production of

artemisinin (Fuentes et al., 2016; Saxena et al., 2014); the increased production of

vitamin E in tobacco (Lu et al., 2013) and lettuce (Yabuta et al., 2013); the expression of

dhurrin, a cyanogenic glucoside found in Sorghum bicolor (Gnanasekaran et al., 2016);

and the production of squalene, a triterpene (Pasoreck et al., 2016) (see Table 3 for

details).

A significant amount of research is also focusing on increasing crop yields by

improving photosynthesis and reducing photorespiration (Longoni et al., 2015; Ort et al.,

2015; Sharwood et al., 2016). Ribulose-1,5-bisphosphate carboxylase/oxygenase

(Rubisco)—the enzyme that carries out CO2 fixation plus the competing oxygenase

reaction—has a relatively low turnover number for the carboxylation reaction (Carmo-

Silva et al., 2015; Occhialini et al., 2016; Whitney et al., 2015; Wilson et al., 2016).

Lower photosynthetic organisms, such as cyanobacteria, express a catalytically more

active Rubisco that is reliant on an efficient carbon concentrating mechanism (CCM) for

cell survival (Price and Howitt, 2014). Therefore, there is current interest in replacing the

higher plant Rubisco with a cyanobacterial version and its CCM (Price et al., 2013;

Raines, 2011). Recently, Lin et al. (2014) successfully swapped the tobacco plastid rbcL

gene coding for the large subunit of Rubisco with genes from the cyanobacterium

Synechococcus elongatus PCC 7942 coding for the large subunit of Rubisco. Co-

expression of the Rubisco assembly factor RbcX or the CCM-associated protein CcmM

were found to have little influence on S. elongatus PCC 7942 Rubisco assembly in leaf

chloroplasts (Occhialini et al., 2016). In the absence of a CCM, the resulting plants were

capable of growing photoautotrophically only at elevated levels of CO2 (see Carmo-Silva

et al., 2015 and Ort et al., 2015 for further studies).

Factors limiting the technology

Narrow range of transformable plant species

Routine chloroplast transformation is currently limited to dicotyledonous plants and

mainly members of the Solanaceae family (see Table 2 for details). Although

transformation of several crop plants has been successfully demonstrated, it is not yet

available for monocotyledonous plants except rice (Lee et al., 2006), where it is still

limited to ‘proof of concept’.

The major reasons for the lack of plastid transformation protocols for cereals

appear to be the recalcitrant nature of these species to existing regeneration protocols

(Ahmadabadi et al., 2007; Lee et al., 2006) and their natural resistance to the current

antibiotics used for chloroplast transformation (Fromm et al., 1987; Li et al., 2010). The

recent observation that plastids can move between cells (Bock, 2010; Stegemann and

Bock, 2009) raises the possibility of grafting donor plant tissue containing transformed

plastids on to a recipient plant tissue, thus allowing the migration of transplastomic

plastids into the untransformed stock (Stegemann et al., 2012). Using this approach,

Thyssen et al. (2012) were able to transfer plastids carrying the aadA gene and aurea

young leaf colour phenotype (barau gene) from Nicotiana sylvestris (donor) to Nicotiana

tabacum (recipient). Although incompatible nucleus–plastid combinations may result in

adverse phenotypic effects in the resulting progeny (Greiner and Bock, 2013; Pelletier

and Budar, 2007), this should not be problematic within closely related species where the

transfer of chloroplasts has been demonstrated to be functional (Bock, 2014).

Poor expression of transgenes in non-green plastids

Another major limitation is that expression of transgenes in non-green plastids is not as

efficient as in green plastids (chloroplasts). Although the plastome is relatively small, its

contribution to protein expression in photosynthetically active tissues can reach as high as

50% of the total leaf protein content. In contrast, expression levels are substantially lower

in non-green plastids (Zhang et al., 2012), which can result in poorer expression of

foreign proteins. For example, expression of the HIV antigen p24 fused with Nef (p24-

Nef) driven from the 16S rRNA promoter, Prrn, was observed to be 2.5% TSP in green

tomatoes but was not detected in ripe fruit (chromoplasts) (Zhou et al., 2008). However,

the aadA selectable marker could be expressed at ~50% the levels found in green plastids

(Ruf et al., 2001).

A genome-wide analysis of tomato fruits and potato tubers has shown that almost

all genes in non-green plastids are strongly down-regulated apart from two essential

genes, accD and clpP, involved in lipid metabolism and the plastid proteolytic

machinery, respectively (Kahlau and Bock, 2008; Valkov et al., 2009), both of which

play important functions in non-green plastids (Ohlrogge and Browse, 1995; Sakamoto,

2006). Therefore, transgene expression using the promoters and 5′-untranslated regions of

these genes may help to overcome some of the challenges related to poor gene expression

in non-green plastids.

Transgene containment in the chloroplast genome is not absolute

The attraction of using plants as ‘green factories’ lies in their scalability. Although

plastids in principle provide a degree of natural gene containment due to their maternal

mode of inheritance, this containment is not absolute, as mentioned earlier. Despite this,

it is still considered highly unlikely that transgenes from a transplastome will enter into

the germplasm of weedy relatives under field conditions (Daniell, 2007). The barriers

include sexual incompatibility between a crop and its weedy relatives, the prevalence of

selection pressure, and desynchronized flowering patterns. Even after hybridization, a

successful introgression event requires that the F1 hybrid survive to produce at least one

single backcross (BC1) hybrid. The higher the number of backcrosses, the quicker the

introgression of a gene will be. Furthermore, the incoming gene must confer a selective

advantage to the host plant; otherwise, natural rearrangements at the chromosome level

will result in its elimination from the plant genome (see Stewart et al., 2003 for a detailed

review).

In order to address potential concerns over gene containment, strategies are being

developed to deliver speed and scalability as well as absolute gene containment without

recourse to costly greenhouse facilities. One such platform is the use of temporary

immersion bioreactors (TIBs). This novel plant biomass propagation technique provides

an alternative way of obtaining rootless leafy plant biomass within a short timeframe of

just 40 days. Expression of green fluorescent protein (GFP) and TetC in leaves obtained

through TIBs was only 50% lower than that of the leaves of plants grown in pots.

However, when the accumulation of these proteins was normalized in terms of growth

area, the productivity of TIBs was around 80–100-fold higher than the plants grown in

soil (Michoux et al., 2011), suggesting that the TIB-based regeneration system could be a

potential route of obtaining high levels of recombinant proteins at low cost under

absolutely contained conditions.

Another advantage of the use of TIBs is that plant tissue is grown in the presence

of sucrose as a carbon source, which means that non-autotrophic mutants expressing high

levels of a foreign protein can still be propagated (Michoux et al., 2013). This technology

is now under further refinement and optimization to elucidate the kinetics of cell-to-

plantlet morphogenesis and accumulation of recombinant proteins.

Limited availability of inducible gene expression systems

The high level of recombinant protein expression sometimes observed with chloroplast

transformation can place a metabolic burden on the plant, leading to unintended

phenotypes such as poorer growth (Tregoning et al., 2003) and high light sensitivity

(Ahmad et al., 2012b). This makes tightly controllable expression systems highly

desirable. Although attempts have been made to repress/control the expression of plastid

genes, they have relied on the chemical induction of transgenes introduced into the

nucleus and the subsequent import of their product into plastids. Therefore, this approach

requires transformation of both cellular compartments (Buhot et al., 2006; Gottschamel et

al., 2016; Lössl et al., 2005; McBride et al., 1994; Muhlbauer and Koop, 2005). The first

‘plastid-only’ strategy for an inducible expression system used a modified form of an E.

coli theophylline-binding thiamine pyrophosphate translational riboswitch (Verhounig et

al., 2010). The synthetic riboswitch was able to deliver tight regulatory control over

transgene expression, in this case GFP, albeit with a lower than normal level of GFP

accumulation when translation was induced with theophylline (0.01─0.02% TSP; 10-fold

lower than the standard expression) (Verhounig et al., 2010).

Absence of glycosylation

Plastids are capable of carrying out many of the post-translational modifications (PTMs)

needed for the production of a physiologically active protein, such as disulphide bond

formation, lipidation, multimerization, and N-terminal methionine excision (Rigano et

al., 2012). However, chloroplasts lack the necessary machinery to carry out

glycosylation, one of the most important PTMs in eukaryotes (Paul and Ma, 2011). The

absence of glycosylation in chloroplasts may therefore represent a bottleneck for the

synthesis of those proteins that are dependent on this PTM for proper functioning.

However, experiments have shown that glycosylation is not always a stringent

requirement for protein function. For example, xylanases are single-chain glycoproteins,

ranging in size from 6–80 kDa, which are active at pH 4.5–6.5 and 40–60 °C.

Transplastomic expression of an alkali-thermostable xylanase from Bacillus subtilis

strain NG-27 in tobacco plants resulted in its accumulation to 6% TSP. The chloroplast-

expressed xylanase was shown to be as active as the native version (Leelavathi et al.,

2003). Another study expressed type I interferon α2b (IFN-α2b)—a member of the

human cytokine glycoprotein family—in tobacco chloroplasts and observed that the

chloroplast-made INF-α2b induced up-regulation of major histocompatibility complex

class I molecules and activation of natural killer cells in a similar fashion to a

commercial-grade glycosylated preparation (Arlen et al., 2007). However, more studies

are needed to determine the effect of glycosylation on protein stability by taking a range

of native glycoproteins and then comparing them with the unglycosylated versions by

expressing them in chloroplasts. Alternatively, in-vitro glycosylation can be performed

on purified chloroplast-made proteins (Strasser et al., 2014).

Degradation of foreign proteins

Plastids have evolved a complex network of biological pathways capable of maintaining

the dynamic equilibrium of the protein pool, thus allowing the cell to effectively

acclimate to environmental changes (De Marchis et al., 2012). Protein degradation plays

a crucial role in maintaining the functional integrity of the plastid proteome, for example,

through the replacement of photo-damaged photosystem II subunits such as D1 (reviewed

in Nixon et al., 2010) and the removal of unassembled subunits of photosynthetic

complexes to control protein abundance, as well as during plant senescence (Jarvis and

López-Juez, 2013).

The stroma of the chloroplast contains many proteases (Adam and Sakamoto,

2014; Sakamoto, 2006), which have the potential to degrade recombinant proteins

produced in the chloroplast (Bellucci et al., 2005; Birch-Machin et al., 2004; Elghabi et

al., 2011). Studies into the mechanism governing protein stability in plastids have

revealed that the N-terminus of a protein harbours sequences that can influence protein

stability and turnover in plastids (Apel et al., 2010). Addition of five amino acids (Met-

Ala-Ser-Ile-Ser) to the N-terminus of VP6 increased protein accumulation >16% TSP

(Borchers et al., 2012), and fusing GFP and PlyGBS residues to the N- and C- terminal

sequences of cyanovirin-N, which in vivo irreversibly inhibits fusion of HIV particles

with target cells, now permitted its accumulation (Elghabi et al., 2011). Similarly, human

insulin was rapidly lost from chloroplasts unless fused with cholera toxin β subunit

(Ruhlman et al., 2007).

Conclusion and perspectives

Despite notable achievements at the laboratory scale, chloroplast transformation

technology has not yet reached the field. Construction of chloroplast expression vectors,

their delivery into the chloroplast genome, and recovery of homoplasmic plant lines is a

relatively straightforward but lengthy process. Current attempts to streamline this process

include the adoption of high-throughput cloning methods for the construction of

chloroplast expression vectors (Gottschamel et al., 2013; Vafaee et al., 2014) and finding

new selectable markers (Bellucci et al., 2015; Li et al., 2010). Downstream technologies

are also being developed, such as protein purification from transplastomic plants (Ahmad

et al., 2012a) and growth of plant tissue in bioreactors under absolute transgene

containment (Michoux et al., 2011; 2013). The ability to transform non-green plastids

(Kumar et al., 2004a; Wurbs et al., 2007; Zhang et al., 2012) should allow this

technology to have an impact on diverse metabolic processes in plants (Warzecha, 2016).

The recent demonstrations that intact long double-stranded RNA (dsRNA) can be

produced in plastids (Jin et al., 2015; Zhang et al., 2015) has opened up new vistas to

control insect population through RNA interference (RNAi). Using viral vectors (Saxena

et al., 2016) to transiently express and target recombinant proteins to different cellular

organelles such as mitochondria, chloroplasts, and nucleus simultaneously (Majer et al.,

2015) provides another route of using plants as cellular factories.

There is increasing interest in using photoautotrophs such as cyanobacteria,

microalgae, and plants as expression platforms for synthetic biology (Scharff and Bock,

2014; Sinagawa-García et al., 2009; Verhounig et al., 2010). Chloroplast synthetic

biology is still in its infancy, but progress is being made to develop the necessary tools to

control the expression of foreign genes in the plastome. Ultimately, artificial plastomes

could be engineered to generate new types of plastid, such as a ‘nitroplast’ specialized in

nitrogen fixation. Replacing the whole genome may not be straightforward due to

homologous recombination between the resident and incoming synthetic plastid genomes,

which will result in a hybrid genome, as observed in whole-genome transplantation in

Chlamydomonas reinhardtii (O’Neill et al., 2012).

One area where chloroplast transformation holds greater immediate promise is the

production of plant-based edible vaccines (Waheed et al., 2015). Although a fairly large

number of vaccine antigens (see Lössl and Waheed, 2011; Scotti et al., 2012; Waheed et

al., 2015 for detailed reviews) have been expressed in higher plant chloroplasts (Table 1),

none of the chloroplast-made proteins has completed clinical trials. Due to the high

standards for human application, it is likely that plastid-made vaccines for veterinary

application may come to market earlier (Clarke et al., 2013). One of the possible reasons

for the delay of plastid-derived vaccines is that the technology is heavily patented. For

example, Chlorogen (St. Louis, MO, USA), now a defunct biotech start-up, acquired or

licensed a vast portfolio of intellectual property relating to chloroplast transformation.

The sale of this company’s assets may allow a new player to attempt to commercialize

this process in the future (Paul and Ma, 2011).

Acknowledgments

NA would like to thank the Higher Education Commission (Pakistan) for financial

support. FM and PN are grateful to the Biotechnology and Biological Sciences Research

Council (UK) for funding their research work. AL is grateful to the Norwegian Research

Council (GLOBVAC Program). The authors sincerely apologize to all those colleagues

whose work could not be discussed here due to space constraints.

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Table 1. Enzymes, biomaterials, vaccine antigens, and agronomic traits engineered via

transformation of the higher plant chloroplast genome

Enzymes/protein/trait Gene Host plant Expression

observed Reference

Biopharmaceuticals

α1-antitrypsin SERPINA1 Tobacco 2% TSP Nadai et al. (2009)

Aprotinin APR Tobacco 0.5%TSP Tissot et al. (2008)

Basic fibroblast growth factor

(bFGF)

bFGF Tobacco 0.1% TSP Wang et al. (2016)

Bacterial phage lytic protein plyGBS Tobacco >70% TSP Oey et al. (2009a)

Cardiotrophin-1 rhct1 Tobacco 5% TSP Farran et al. (2008)

Coagulation factor IX CTB-FIX Tobacco 3.8% TSP Verma et al.

(2010b)

Cyanovirin-N CV-N fused with

GFP or PlyGBS

Tobacco Up to 0.3% TSP Elghabi et al.

(2011)

Exendin-4 (EX4) CTB-EX4 Tobacco 14.3% TLP Kwon et al. (2013)

Endolysin Cpl-1 cpl-1 Tobacco 10%TSP Oey et al. (2009b)

Endolysin Pal pal Tobacco 20% TSP Oey et al. (2009b)

Human granulocyte colony-

stimulating factor

hG-CSF Lettuce ND Sharifi Tabar et al.

(2013)

Human proinsulin CTB-Pins Tobacco

Lettuce

16% TSP

2.5% TSP

Ruhlman et al.

(2007)

Pins-Protein A Tobacco 0.2% TSP Yarbakht et al.

(2015)

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 Ruiz (2002)

Interferon-α2b (IFN-α2b) IFN-α2b Tobacco 21% TSP Arlen et al. (2007)

Thioredoxin 1 hTrx1 Lettuce 1% TSP Lim et al. (2011)

Enzymes/biomaterials

Cellulases bgl1C, cel6B, cel9A,

xeg74

Tobacco 5–40% TSP Petersen and Bock

(2011)

CelA, CelB Tobacco 22–23 mg g-1 of

TSP, 15–21 mg

g-1 of TSP

Espinoza-Sánchez

et al. (2016)

Cel6, Cel7, EndoV,

CelKI, Cel3, TF6A,

Pga2, Vlp2

peroxidase

Tobacco ND Longoni et al.

(2015)

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)

CelB Tobacco ND Longoni et al.

(2015)

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 Bohmert-Tatarev et

al. (2011)

Trp asa2 Tobacco ND Zhang et al. (2001)

Xylanase xynA Tobacco 6% TSP Leelavathi et al.

(2003)

xyn Tobacco 35% TSP Castiglia et al.

(2016)

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

Bgl1 Tobacco 20 mg g-1 of TSP Espinoza-Sánchez

et al. (2016)

celB Tobacco 60–70% TSP Castiglia et al.

(2016)

Endo-glucanase endo Tobacco ≤2% TSP Castiglia et al.

(2016)

Pectin lyase PelA Tobacco ND Espinoza-Sánchez

et al. (2015) Manganese peroxidase MnP-2 Tobacco ND

Superoxide dismutase Cu/Zn SOD Tobacco 9% TSP Madanala et al.

(2015)

Vaccine antigens (since 2012; see Lössl and Waheed, 2011 for earlier reports)

Bacterial

Anthrax protective antigen pa Tobacco 2.5–4% TSP Gorantala et al.

(2014)

Dengue virus DENV-1,2,3,4 Lettuce ND Maldaner et al.

(2013)

EDIII Tobacco 0.8–1.6% TSP Gottschamel et al.

(2016)

Haemorrhagic colitis EIT Tobacco 1.4% TSP Karimi et al. (2013)

Lyme disease OspA:YFP Tobacco 7% TSP Michoux et al.

(2013)

Pompe disease CTB-GAA Tobacco 0.1–0.2 TLP Su et al. (2015)

Porcine post-weaning diarrhoea

(PWD)

FaeG Tobacco 1% DW Kolotilin et al.

(2012)

Toxoplasmosis GRA4 Tobacco 6 μg g–1 FW Yácono et al.

(2012)

SAG1

LiHsp83-SAG1

Tobacco

Tobacco

0.1–0.2 μg g–1

FW

50–100 μg g–1

FW

Albarracín et al.

(2015)

Albarracín et al.

(2015)

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)

Viral

Human papillomavirus GUS-E7 Tobacco 3–4% TSP Morgenfeld et al.

(2014)

HPV16 L1 Tobacco Up to 2.5% TSP Zahin et al. (2016)

Poliovirus CTB-VP1 Tobacco Native: 0.1%

TSP, Codon

optimized: 4–5%

TSP

Chan et al. (2016)

HIV/AIDS gp120, gp41 Tobacco 16 μg g–1 FW Rosales-Mendoza

et al. (2014)

HIV c4v3 Tobacco 25 μg g–1 FW Rubio-Infante et al.

(2012)

Rotavirus Vp6 Tobacco 15% TLP Inka Borchers et al.

(2012)

Agronomic Traits

Abiotic stress tolerance γ-TMT Tobacco 7.7% TLP Jin and Daniell

(2014)

Aphid/whitefly resistance Bgl-1 Tobacco ND Jin et al. (2011)

Antiviral/antibacterial/phloem-

feeding insects

pta Tobacco 9.2% TSP Jin et al. (2012)

Antiviral/antimicrobial RC101

PG1

Tobacco 32–38% TSP

17–26% TSP

Lee et al. (2011)

Cytoplasmic male sterility phaA Tobacco ND Ruiz and Daniell

(2005)

Bacterial/fungal resistance msi-99 Tobacco ND DeGray et al.

(2001)

chloroperoxidase Tobacco 10–15 μg ml–1

leaf extract

Ruhlman et al.

(2014)

msi-99 Tobacco ND Wang et al. (2015)

Drought tolerance tps1 (yeast) Tobacco ND Lee et al. (2003)

ArDH Tobacco ND Khan et al. (2015)

Herbicide resistance aroA Tobacco ND Daniell et al.

(1998)

bar Tobacco ND Iamtham and Day

(2000)

crtY Tomato/Tobacco ND Wurbs et al. (2007)

dao Tobacco ND Gisby et al. (2012)

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 Soybean ND Dufourmantel et al.

(2005)

cry9Aa2 Tobacco 10% TSP Chakrabarti et al.

(2006)

dsRNA of p450

monooxygenase, V-

ATPase and chitin

synthase-coding

genes

Tobacco (against

Helicoverpa

armigera)

ND Jin et al. (2015)

dsRNA of CPB,

ACT and SHR genes

Tobacco (against

Colorado potato

beetle

(Leptinotarsa

decemlineata)

ND Zhang et al. (2015)

cry1Ab Cabbage 4.8–11.1% TSP Liu et al. (2008)

Multiple biotic and abiotic

stresses

Simultaneous

expression of

protease inhibitors

and chitinase

Tobacco ND Chen et al. (2014)

Oxidative stress resistance Trx m Tobacco ND Rey et al. (2013)

Altered photosynthesis Cyanobacterial

Rubisco along with

assembly factors

RbcX, CcM35

replaced

endogenous

Rubisco large

subunit in tobacco

chloroplasts

Tobacco 12–18% Rubisco

of WT level

Lin et al. (2014)

Cyanobacterial

Rubisco without

RbcX or CcM35

Tobacco 10× lower

Rubisco than

WT

Occhialini et al.

(2016)

replaced tobacco

large subunit

Phytoremediation merA/merB Tobacco ND Hussein et al.

(2007); Ruiz et al.

(2003)

mt1 Tobacco ND Ruiz et al. (2011)

Salt tolerance badh Carrot ND Kumar et al.

(2004a)

ArDH Tobacco ND Khan et al. (2015)

Abbreviations: TCP, total cellular proteins; TLP, total leaf proteins; TSP, total soluble proteins; ND, not

determined; DW, dry weight; FW, fresh weight (all concentrations in w/w).

Table 2. List of plants in which chloroplast transformation has been achieved

Crop Protein/trait Gene Referencea

Alfalfa Aminoglycoside adenylyl transferase,

GFP

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. (2010)

Lettuce Aminoglycoside adenylyl transferase,

GFP

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,

GFP

aadA, gfp Sidorov et al. (1999)

Rice Aminoglycoside adenylyl transferase,

GFP

aadA, gfp Lee et al. (2006)

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

Sugar beet Aminoglycoside adenylyl transferase, aadA, gfp De Marchis et al. (2008)

GFP

Sugarcane

Tobacco Aminoglycoside adenylyl transferase aadA Svab et al. (1990a)

Tomato Aminoglycoside adenylyl transferase aadA Ruf et al. (2001)

a Only the first report is included.

Table 3. Metabolic engineering in plants using plastid transformation technology

Trait engineered Genes expressed Target site Citation

Artemisinin production Twelve genes were incorporated in tobacco

plastid genome to produce isopentenyl

pyrophosphate by an engineered mevalonate

pathway for the biosynthesis of artemisinic

acid (a precursor of artemisinin)

trnI – trnA Saxena et al. (2014)

Core enzymes involved in artemisinin

biosynthesis (FPS, ADS, CYP and CPR)

were expressed first, and the promising line

was supertransformed with genes coding for

accessory enzymes (CYB5, ADH1, ALDH1,

DBR2, DXR) to increase the expression of

artemisinic acid

trnfM – trnG Fuentes et al. (2016)

Astaxanthin Three genes, CrtW, coding for b-carotene

ketolase), CrtZ, coding for b-carotene

hydroxylase, and Idi, coding for isopentenyl

diphosphate isomerase, from marine bacteria

were expressed in lettuce chloroplasts; total

carotenoid accumulation reached 230 µg g−1

fresh weight (~ 95% carotenoids)

rbcL – accD Harada et al. (2014)

β-carotene/provitamin

A

Four different bacterial and fungal genes

were expressed in in tomato fruit; provitamin

A content increased fourfold

trnfM – trnG Wurbs et al. (2007)

Lycopene β-cyclase gene from daffodil were

expressed in tomato fruits; carotenoid

accumulation was increased 50%

trnfM – trnG Apel and Bock (2009)

Dhurrin pathway Three genes coding for two membrane-bound

cytochrome P450 enzymes (CYP79A1 and

trnfM – trnG Gnanasekaran et al.

(2016)

CYP71E1) and a soluble glucosyltransferase

(UGT85B1) were expressed in tobacco

chloroplasts to produce dhurrin, a bioactive

compound

Luciferase pathway Complete lux operon containing six genes

from Photobacterium leiognathi was

expressed in tobacco

rps12 – TrnV

trnI – trnA

Krichevsky et al.

(2010)

Mevalonate pathway Six cytosolic genes coding for the

cytoplasmic mevalonate pathway were

expressed in tobacco

trnI – trnA Kumar et al. (2012)

Engineering Rubiscoa Cyanobacterial Rubisco was assembled in

tobacco by expressing Rubisco large subunit

and small subunit coding genes along with

cofactors, transplastomic lines were much

more efficient in CO2 fixation on per unit

enzyme basis at elevated CO2

atpB – accD Occhialini et al. (2016)

Co-expression of Arabidopsis Rubisco

accumulation factor 1 (RAF1) in tobacco

improved biogenesis and accumulation of

hybrid L8AS8

t Rubisco (made of the large

subunit of Arabidopsis and the small subunit

of tobacco), as well as also led to a two-fold

increase in photosynthesis compared with the

tobacco line expressing L8AS8

t Rubisco only

atpB – accD Whitney et al. (2015)

Mutation in Methanococcoides burtonii

Rubisco (E138R, and K332E) resulted in

improved photosynthesis in tobacco

compared to the line expressing the wild-type

version of Mb Rubisco

atpB – accD Wilson et al. (2016)

Polyhydroxybutyrate Three bacterial enzymes, phbA, phbB and

phbC, were expressed in tobacco

trnN – trnR Lössl et al. (2003)

Terpene pathway Two genes coding for farnesyl diphosphate

synthase (FPS) and squalene synthase (SQS)

were expressed in tobacco chloroplast;

abnormal, massive changes in transcripts

took place

trnI – trnA Pasoreck et al. (2016)

Vitamin E Three cyanobacterial genes encoding

homogentisate phytyltransferase (HPT),

tocopherol cyclase (TCY), and γ-tocopherol

methyltransferase (TMT) were expressed in

tomato chromoplasts; tocopherol content

increased 10-fold

trnfM – trnG Lu et al. (2013)

Three Arabidopsis genes coding for

tocopherol cyclase) or γ-TMT (γ-tocopherol

methyltransferase) and TC plus TMT (TC-

TMT) as operon were expressed in lettuce

chloroplasts; tocopherol content increased

significantly

rbcL – accD Yabuta et al. (2013)

a Only recent reports are included.

Fig. 1. Schematic representation of the process of transforming the plastid genome. (A)

Basic design of a typical vector for transforming the plastid genome. Both the expression

cassette and the selection cassette are placed between the two plastid regions. These

flanking regions are taken from the wild-type plastid genome of a plant species whose

plastome is to be manipulated, to allow a crossover event take place to integrate DNA

sequences between them. Green arrows in the chloroplast expression vector represent

promoters (P) and the direction of transcription, whereas terminators (T) are indicated by

red rectangles. The untranslated regions are represented by white circles. The thin dotted

lines with arrows indicate homologous recombination. (B) Delivery of transforming

plasmids into chloroplasts in leaf cells using a particle delivery system. The plasmid

DNA is coated on the surface of the microparticles of either gold or tungsten and then

shot on to the abaxial surface of 4- to 6-week-old sterile leaves using a gene gun. The

bombarded leaves are incubated for 48 hours in the dark, cut into small discs and placed

on regeneration medium supplemented with the appropriate antibiotic and hormones.

Primary shoots generally arise within 2–3 months. (C) The process of recovering a stable

homoplasmic transplastomic plant line. Initially, only a few copies of the plastome are

transformed, and therefore the explant contains a mixture of both transformed as well as

untransformed copies, a state known as heteroplasmy. The wild-type copies (indicated by

light-coloured ovals) are sorted out gradually by repeating two or three regeneration

cycles under selection to reach homoplasmy, a state where all copies of the plastome are

transformed (indicated by dark grey ovals). (D) Summary of commonly used promoters,

terminators, untranslated regions, and plastome insertion sites used in chloroplast

transformation. GOI, gene of interest; P, promoter; T, terminator, ptDNA, plastid DNA;

UTR, untranslated regions.

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