REVI EW

The potential of transgenic green microalgae; a robust

photobioreactor to produce recombinant therapeutic proteins
Fariba Akbari

Morteza Eskandani

Ahmad Yari Khosroushahi
Received: 5 December 2013 / Accepted: 30 July 2014
Springer Science+Business Media Dordrecht 2014
Abstract Microalgae have been used in food, cosmetic,
and biofuel industries as a natural source of lipids, vita-
mins, pigments and antioxidants for a long time. Green
microalgae, as potent photobioreactors, can be considered
as an economical expression system to produce recombi-
nant therapeutical proteins at large-scale due to low cost of
production and scaling-up capitalization owning to the
inexpensive medium requirement, fast growth rate, and the
ease of manipulation. These microalgae possess all benet
eukaryotic expression systems including the ability of post-
translational modications required for proper folding and
stability of active proteins. Among the many items regar-
ded as recombinant protein production, this review com-
pares the different expression systems with green
microalgae like Dunaliella by viewing the nuclear/chlo-
roplast transformation challenges/benets, related selection
markers/reporter genes, and crucial factors/strategies
affecting the increase of foreign protein expression in
microalgae transformants. Some important factors were
discussed regarding the increase of protein yielding in
microalgae transformants including: transformation-asso-
ciated genotypic modications, endogenous regulatory
factors, promoters, codon optimization, enhancer elements,
and milking of recombinant protein.
Keywords Codon optimization Microalgae Dunaliella
Milking Photobioreactor Recombinant proteins
Introduction
The noticeable merits of microalgae Dunaliella are their
numerous features in producing biofuels, pigments, thera-
peutic and cosmetic proteins. The capability to blend the
energy-capturing ability of photosynthesis with the high-
yield cultivation results in economical production systems
(Rosenberg et al. 2008). Enormous efforts have been per-
formed to increase the yield of Dunaliella production in
agricultural scale (Courchesne et al. 2009) and several
articles reviewed the production issues (Cheirsilp et al.
2011; Greenwell et al. 2010; Li et al. 2008; Williams 2007;
Williams et al. 2009). However, few articles were recently
published on the production of recombinant proteins in
microalgae particularly Dunaliella. According to the
National Center of Biotechnology Information, algae of the
genus Dunaliella belongs to the class of Chlorophyceae,
the order of Chlamydomonadales, and the family of Du-
naliellaceae (Polle et al. 2008). Apparently, it seems that
nutritional and medical application of Dunaliella produced
biomolecules can be appropriate and safe for human con-
sumptions based on the safety of these biomolecules
F. Akbari
Drug Applied Research Center, Tabriz University of Medical
Sciences, Tabriz, Iran
F. Akbari M. Eskandani
Student Research Committee, Tabriz University of Medical
Sciences, Tabriz, Iran
M. Eskandani
Research Center for Pharmaceutical Nanotechnology, Tabriz
University of Medical Sciences, Tabriz, Iran
A. Y. Khosroushahi
Biotechnology Research Center, Tabriz University of Medical
Sciences, Tabriz, Iran
A. Y. Khosroushahi (&)
Department of Pharmacognosy, Faculty of Pharmacy, Tabriz
University of Medical Sciences, Daneshgah Street,
P.O. Box 51664-14766, Tabriz, Iran
e-mail: Yarikhosroushahia@tbzmed.ac.ir
1 3
World J Microbiol Biotechnol
DOI 10.1007/s11274-014-1714-0
(Franklin and Mayeld 2005; Gantar et al. 2008). Thus,
among the microalgae, Dunaliella can consider the best
and robust targets to study because of its halotolerant
properties which makes it succeed in an extremely saline
environment. It is also capable of biotechnological pro-
cesses such as expression of foreign proteins. Dunaliella is
currently utilized in b-carotene production as a suitable
source of b-carotene (up to 10 % of the biomass) (Ben-
Amotz et al. 1988, 1989; Katz et al. 1995).
Dunaliella has been noted as a recombinant protein
expression system in the last decade due to rapid growth
and convenience cultivation as well as having the poten-
tial for post-transcriptional and post-translational modi-
cations. Besides, Dunaliellais one of the best-studied
unicellular green algae in the eld of physiology, bio-
chemistry and genetic (Oren 2005). The most important
characteristics of Dunaliella in genetic and plastome
engineering are listed in Table 3. The second virial
coefcient, a characteristic of the interaction potential
between the particles, depends only on the pair interaction
between the particles. The second osmotic virial coef-
cients of several proteins were measured in salt solutions.
The ndings showed that, at low salt concentrations,
proteinprotein interactions can be either attractive or
repulsive, possibly due to the anisotropy of the protein
charge distribution. At high salt concentrations, the
behavior depends on salt: In sodium chloride, protein
interactions generally show little salt dependence up to
very high salt concentrations so that most proteins have a
second at virial coefcient prole with increasing sodium
chloride concentration. This explains the high solubility of
most proteins in highly concentrated solutions of sodium
chloride (Dumetz et al. 2007).
On the other view, to solve the insoluble recombinant
proteins produced in bacteria, the cultivation of bacteria
under osmotic stress or additions of compatible solutes (for
example glycine betaine) were commonly applied to pro-
tect solved proteins in peripheral space of cells. Upon
Dunaliella halotolerancy, it produces high amounts of
stress metabolites including glycine betaine which can
facilitate protein protection in high salinity habitats
(Mishra et al. 2008).
Thus, the exponentially increasing demand of by-pro-
ducts such as recombinant proteins in recent years has
made the transgenic microalgae research favorable and low
cost to carry out and also a high yield system for producing
bioactive compounds. Given the rapid developments in
transgenic microalgae technology, the present study
reviews recent progress in high-level expression of
recombinant proteins, transformation methods for both
nuclear and chloroplast genomes, strategies to increase
recombinant protein yields, and potential research direc-
tions of interest.
Comparison of different expression systems
with Dunaliella
Nowadays, several different types of expression systems
exist to produce therapeutic recombinant proteins, such as
bacteria, yeast, insect, mammalian cells, plant cells and
micro algae. The production of recombinant proteins in
bacterial and yeast-based systems can be considered as a
prevalent approach because of genomes lack of uncom-
plicated manipulations and their cost benecial cultiva-
tions. However, any post-transcriptional and post-
translational modications, including glycosylation, phos-
phorylation and disulde bond formation are not performed
on bacterial systems where these modications require the
correct folding of complex proteins. Besides, recombinant
proteins with bacterial toxins have a considerably high
contamination possibility. Although many modications
are performed in eukaryotic yeasts, the modied products
are not suitable for human consumptions. The hypergly-
cosylation of recombinant proteins in yeast alters immu-
nogenic epitopes of these proteins and high-mannose
glycosylation also result in lower in vivo half life which
decreases the therapeutic activity of these proteins (Ce-
reghino and Cregg 2000). Although mammalian cells uti-
lized for recombinant protein production possess various
advantages, the development and maintenance of mam-
malian cell-based bioreactors comprise remarkable costs.
Several difculties regarding mammalian cell-based bio-
reactors including complex nutrient requirements, poor
oxygen and nutrient distribution, waste accumulation,
contamination by pathogens and high sensitivity of cells to
shear stress limit using of these systems for recombinant
protein production (Zhang et al. 2008, 2010). Insect cell
cultures require complex nutrient media to cultivate but in
comparison with mammalian, cell culture are easier and
insect cells possess a high tolerance to osmolarity changes
that can be considered an advantage for this system.
Among the expression systems in insect cells, baculovirus
system can express the high level and quality of recombi-
nant protein but its lytic operating mechanism leads to
decrease product yields due to endogenous proteases
release (Ikonomou et al. 2003).
Plant-based reactors considered the use of inexpensive
bioreactor systems where the growth of plant cells in cul-
ture systems is less than mammalian or insect cells.
Moreover, the gene owing phenomenon in transgenic
plants may be harmful for the environment (e.g., via pollen
and seed dispersal). Protein glycosylation phenomenon is
different between animal and plant cells; thus, allergic
reactions are the major concern of plant-derived proteo-
glycans for human consumptions.
The combination of low-cost, uncomplicated require-
ments, rapid growth and high potential system for post-
World J Microbiol Biotechnol
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transcriptional/translational processing can notably be
considered as advantageous in microalgal cultivation
methodology. In diverse protein production, unicellular
photosynthetic green algae which are mostly classied as
generally regarded as safe (GRAS), are frequently uti-
lized because of their comfortable purication and pro-
cessing of expressed proteins. Furthermore, carbon dioxide
and light are indispensable elements for microalgae growth
in salt-based media which are remarkably inexpensive
elements.
Regarding economical considerations, based on recom-
binant antibody production studies, functional antibody
cost (per gram) is 2 $150, $0.05 and $0.002 (USD) in
mammalian, plant and microalgal bioreactor systems
respectively, making microalgal system very economically
attractive (Mayeld et al. 2003; Mayeld and Franklin
2005).
Despite the recent interest and successful transformation
of microalgal species such as Chlamydomonas, Chlorella,
Volvox, Haematococcus and Dunaliella genera, many
obstacles still remain to overcome before microalgae can
be considered in standard expression systems.
Although the low expression levels of recombinant
proteins in microalgal systems are primarily delayed in the
usage of this system for protein production, the continuing
development of genetic engineering tools for microalgae
transformation has allowed the expression of fully func-
tional antibodies (Franklin and Mayeld 2005; Jones et al.
2012; Manuell et al. 2007; Tran et al. 2009), therapeutics
(Boehm 2007; Rajamani et al. 2007; Rasala et al. 2011;
Rasala and Mayeld 2011; Siripornadulsil et al. 2002,
2007), and bactericides (Li and Tsai 2009) at economically
viable levels.
Transformation methods for Microalgae Dunaliella
Gene delivery is the rst critical stage in the creation of
transgenic organisms. The release of a gene to plant cells is
more difcult than mammalian cells because the cells wall
in plant cells acts as a barrier. According to Gabriel Potvin,
fortunately microalgae best of both worlds green algae
has contemporarily both positive properties of mammalian
and plant cells (Sadka et al. 1991). To transform Dunali-
ella, some traditional methods are still applied in recent
work. A summary of routinely used methods for transfor-
mation of Dunaliella is briey explained in the following.
(1) At biolistic particle delivery system, the method orig-
inally designed for plant transformation, target cells are
bombarded with DNA-coated metallic (gold or tungsten)
particles (Sanford et al. 1993; Smith et al. 1992). This was
rst utilized for Chlamydomonas reinhardtii transforma-
tion in 1988 (Newman et al. 1990, 1992). The biolistic
method was also successfully exploited for nuclear and
chloroplast transformation of Dunaliella (Jiang et al. 2005;
Tan et al. 2005). This method is not widely applied to
generate a large number of nuclear transformants in mic-
roalgae due to its requirements for expensive laboratory
equipments. (2) Electroporationis another method that was
previously utilized for the transformation of Dunaliella by
numerous researches (Geng et al. 2010, 2011; Sun et al.
2008, 2005; Wang et al. 2007). This method additionally
requires specic expensive equipments. Moreover, this
method of transformation efcacy is inuenced by factors
including eld strength, pulse length, medium composition,
temperature, membrane characteristics and the concentra-
tion of DNA (Brown et al. 1991; Wang et al. 2007). These
variable factors result in unrepeatable results. (3) Glass
beads method has been previously announced as the easiest
and most effective method for Dunaliella transformation.
This method is considered as an inexpensive technique in
comparison with the aforementioned methods. Beside, this
approach possesses some benets like high-efciency,
simplicity, economy, controllability, and high repeatability
(Feng et al. 2009), but it suffers from decrement in cell
viability in transformants. To overcome the problem, the
silicon carbon whisker was successfully exploited for Du-
naliella transformation instead of glass beads but whisker
method shows lower transformation efciency (Potvin and
Zhang 2010).
Selection markers and reporter genes
Antibiotics, the main effective selectable markers, were
successfully applied to select transformants, but as far as
Dunaliella is resistant to most of antibiotics, fond suitable
selectable markers requires extensive researches to select
transformed Dunaliella in this area (Tan et al. 2005). In
fact, Dunaliella does not show sensitivity to streptomycin
(Str), kanamycin (Km), hygromycin (Hm) and G418 while
Chloromycetin (60 lg/ml) can completely prevent the
growth of Dunaliella in solid and liquid media. In addition,
cat gene has been reported as a suitable selective marker
for genetic engineering of Dunaliella (Wang et al. 2007).
Numerous researches have previously applied some
selectable genes such as the aadA gene encoding specti-
nomycin or streptomycin resistance, and the bar gene
encoding herbicide phosphinothricin (PTT) showed resis-
tance to select transformed cells in Dunaliella (Jiang et al.
2005).
Since Dunaliella cells have been extremely sensitive to
Basta, indicating the bar gene can be considered a suitable
selective marker to select Dunaliella transformant cells.
Dunaliella cells also show sensitivity to chloromycetin and
Zeocin, but not to hygromycin where plant cells usually
World J Microbiol Biotechnol
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show the sensitivity for hygromycin (Sun et al. 2005; Tan
et al. 2005). Moreover, several research ndings indicated
that selective pressure depends on cell density, and higher
cell density requires an increase in selective pressure. The
sensitivity and resistance to multiple antibiotics in Dunal-
iella are listed in Table 1.
Another kind of selectable markers is based on the com-
plementationof metabolismor photosynthetic mutants. Inthis
method, the rescue of microalgal mutants was accomplished
with wild-type gene constructs in which the cultivation con-
ditions were considered as a transformant selection approach.
This method may be particularly valuable for chloroplast
transformations, where hybrid foreign DNA constructs con-
taining wild-type genes, which their integration soccur by
homologous recombination, can not only rescue microalgal
mutants. It means that the gene is Knocked-out, thus allowing
selection but only in targets adjacent regions for foreign DNA
integration. Despite the mentioned reasons, different reporter
gene expression patterns are previously reported by several
researches. For example, the gus reporter gene has been
successfully and transiently expressed in Dunaliella trans-
formants but another reporter gene called lacZ, encoded for b-
galactosidase, did not show successful expression in Dunali-
ella (Tan et al. 2005). The egfp gene, encoded for a green
uorescent protein, was then employed as a reporter gene for
Dunaliella transformation, and a green uorescence back-
ground was detected after the microscopic observation of a
large amount of blank cells.
Microalgal chloroplasts can be considered as an attrac-
tive platform for foreign protein expressions making mar-
ker-free systems highly desirable for the expression of
recombinant therapeutic or nutritional products at high
levels. The reason is that in achieved homoplastomic
transformations, 518 % of the total soluble protein (TSP)
can consist of marker gene products, which makes the
decrease on maximum yield of the target protein. There-
fore, if the transformed algae are predestinated for human
or animal consumptions, unnecessary DNA including
genes conferring resistance to antibiotics is undesirable and
removing of marker genes may be favorable to increase
yield of recombinant target protein. The elimination of
marker genes can be obtained by some methodologies such
as homology-based excision, excision by phagesite-specic
recombinases, transient cointegration of the marker gene or
the cotransformationsegregation approach (Lutz et al.
2006; Lutz and Maliga 2007).
Nuclear transformation and chloroplast transformation
in Dunaliella
Recombinant proteins have been usually expressed in the
nuclei and chloroplast genomes of algae. Each of these
systems has their own special features; thus, both nuclei
and chloroplast features should be considered for choosing
a suitable expression system and nally it is necessary to
select between these two systems to achieve the ultimate
goal. Here, some properties of nuclear/chloroplast trans-
formations in Dunaliella are described.
Nuclear transformation
Positional effects, RNA silencing, a prohibitively compact
chromatin structure and non-conventional epigenetic
Table 1 The sensitivity/
resistance to multiple antibiotics
in Dunaliella
Selectable marker Sensitivity and
resistance
Concentration of
antibiotic (lg/ml)
Gene Reference
Streptomycin R
R
600
1,200
(Sun et al. 2005)
(Tan et al. 2005)
Kanamycin R 600
1,200
(Sun et al. 2005)
(Tan et al. 2005)
Hygromycin R 600
1,200
(Sun et al. 2005;
Tan et al. 2005)
G418 R 600
1,200
(Sun et al. 2005)
(Tan et al. 2005)
Chloromycetin S 60 (Sun et al. 2005)
Chloramphenicol S 400 Catgene (Tan et al. 2005)
Spectinomycin R 1,200 (Tan et al. 2005)
Spectinomycin/
Streptomycin
S aadAgene (Jiang et al. 2005)
Basta
(phosphinothricin)
S 20 bargene (Jiang et al. 2005; Tan
et al. 2005)
Zeocin S 100 Shblegene (Tan et al. 2005)
World J Microbiol Biotechnol
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effects have been proposed as possible causes to low
nuclear expression of transformed foreign proteins in Du-
naliella (Fig. 1a). Low expression levels and varied wide
range of protein expression were also ascribed to random
integration sites of the newly introduced genes. This was
also considered for inuence of the chromatin structure
and/or the regulatory elements on the sites of integration in
the host genome (Peach and Velten 1991). To overcome
these position effects, a utile approach was proposed by
some researches in which the neighborly elements con-
struct an affecting transgene expression (Kim et al. 2004;
Lee et al. 2006; Van et al. 2001).
In recent years, it has been claried that a protein
aceous nuclear matrix or scaffold plays a signicant
role in determining chromatin structure. Matrix attach-
ment regions (MARs) are thought to be components that
bind specically to the nuclear scaffold and form the
bases of loop domains. Many experiments have demon-
strated that MARs not only increase the expressions of
foreign genes and decrease the variation of expressions of
transgenes between different transformants (Abranches
et al. 2005; Allen et al. 2005; Ascenzi et al. 2003; Mankin
et al. 2003; Michalowski et al. 1999) but also stabilize the
transgene expressions in their progeny (Allen et al. 2005;
James et al. 2002). Thus, the MARs may be considered an
effective regulatory element to improve expression levels
of transgenes and stabilize the gene expression in the
progeny.
DMS2 and DNA fragment binds to nuclear matrices
(MAR) were previously isolated from Dunaliella (Wang
et al. 2005b). The effects of this fragment on the cat gene
expression in stably transformed cells were successfully
investigated and the results demonstrated the enhanced
expression level (4.5-fold) of the cat gene in transformed
Dunaliella cells (Wang et al. 2005a).
A genetic screening with little inuence by the position
effects was designed to facilitate isolation of algal strains
which efciently express introduced recombinant protein
gene. The results of this investigation showed that the
accumulation levels of foreign protein in the selected
strains were almost uniformly high in all transgenic clones.
The selected UV-induced mutations with high express
nuclear transgenes in Chlamydomonas strains showed that
the increase foreign protein yields nearly 0.2 % TSP which
is relatively high for nuclear expression in algae (Neupert
et al. 2009, 2012).
These ndings may be benecially favored to increase
the recombinant protein expression in transformed Dunal-
iella due to the high similarity between Chlamydomonas
and Dunaliella micro algae.
Fig. 1 Recombinant protein
production by nuclear
transformation in
Dunaliellasalina. a Low nuclear
expression of transformed
foreign proteins in Dunaliella.
b The effect of a proteolytic
degradation phenomenon on
recombinant protein yields
which caused the decrement of
yield. c Co-expression of
protease inhibitors with
recombinant protein which
caused the increase of yield.
d Targeting synthesized
recombinant protein in the
endoplasmic reticulum or
chloroplast via signal peptide
World J Microbiol Biotechnol
1 3
The post transcription/translation processing in a nuclear
transformation system can be considered as the main
advantage of this system which it necessary for complex
protein production despite its low yields.
Sensitivity to proteases
Recombinant protein yields depend on protein accumula-
tion which is affected by the amount of protein synthesis
and its degradation in transformed cells. Endogenous pro-
teolytic enzymes essentially require correct assembling and
post translational modication of proteins which may lead
to degradation of recombinant proteins. These enzymes
may interfere in the downstream processing of proteins and
cause an inconvenience in protein purication due to total
degradation or non-functional protein fragments production
(Fig. 1b). Although infrequent information is available on
the effect of a proteolytic degradation phenomenon on
recombinant protein yields, the fundamental experiment
announcing the principle factors affecting this phenomenon
in microalgae has been investigated in C. reinhadtii. The
conclusion of the investigation showed that protease
activity is one of the main factors affecting the level of
recombinant protein expression in Chlamydomonas (Sur-
zycki et al. 2009).
Some available strategies exist to minimize foreign
protein degradation in plants and due to the similarities
between plant and green microalgae systems, using these
strategies may be benecial to increase the recombinant
protein yields (Doran et al. 2009). However, the evaluation
that each factor effects on recombinant protein yield
requires additional investigations (Potvin and Zhang 2010).
In this section of the review, some of the most important
strategies for the sensitivity of recombinant proteins were
summarized to proteases.
(1) Co-expression of protease inhibitors with recombi-
nant protein has been previously applied to increase
recombinant protein yields without affecting normal
growth in cells (Van der Vyver et al. 2003) (Fig. 1c). (2)
Since the cell endoplasmic reticulum possesses few pro-
teases enzymes, the protein degradation can accordingly be
minimized by targeting synthesized recombinant protein in
the endoplasmic reticulum or chloroplast via signal peptide
rather than cytosol (Conrad and Fiedler 1994, 1998; Con-
rad and Manteuffel 2001) (Fig. 1d). This strategy led to 10
4
fold increase in recombinant growth factor expression in
tobacco plant cells (Wirth et al. 2004); moreover, it can be
applied to recombinant special antibodies production
whenever the protein secretion or its modications in the
golgi are not necessary. (3) The proteins can be expressed
in the chloroplast of algae without needing post-transla-
tional modications (Eichler-Stahlberg et al. 2009; Potvin
and Zhang 2010; van Wijk 2004) while the presence of
proteolytic pathways for protein processing in plant chlo-
roplasts can also limit the recombinant protein accumula-
tion resulting in the overall yield of protein in transformed
cells. In addition, microalgal chloroplasts can act as an
envelope in long-term storage of recombinant proteins
inside the cell (Bock 2001) which may be useful to increase
proteins nal yield. IV) To minimize the nuclear-expres-
sed protein proteolytic degradations, the most efcient
approach is to reserve them in chloroplast because some
chloroplast proteins are encoded in the cytosol and then
export to chloroplast (Faye and Daniell 2006; Jarvis 2003,
2008; Jarvis and Robinson 2004). Two different transpor-
tation mechanisms of cytosol-synthesized proteins were
already described by targeting procedure. (1) The proteins
are post-translationally targeted to the chloroplast through
Toc and Tic complex (outer and inner apparatus respec-
tively) of the chloroplast membranes which directly
denominated targeting or the classical protein import pro-
cess. (2) In indirect targeting mechanism, the protein was
targeted to the endoplasmic reticulum (ER) by co-transla-
tional targeting or undergoes further process in the golgi
apparatus by using the secretary pathway prior to exporting
to chloroplast.
Therefore, these strategies can be considered as possible
approaches to increase foreign protein production yield in
transformed Dunaliella due to the presence of some simi-
larities in plant and microalgae systems.
Chloroplast transformation
A successful engineering in the chloroplast genome can be
proposed to obtain high yields of foreign protein produc-
tion in Dunaliella by considering some advantages of this
expression system to express recombinant proteins which
do no require post-translational modications. Several
advantages of chloroplast recombination and expression
system include targeted transgene integration by homolo-
gous recombination, absence of gene silencing, polycis-
tronic expression system, maternal inheritance of the
chloroplast genome causing robust expression, envelope
and protect foreign proteins from degradation (Bock 2001;
Chebolu and Daniell 2007; Daniell et al. 2001; Daniell
2006; Daniell et al. 2009; Rasala et al. 2010, 2011; Rasala
and Mayeld 2011).
It seems this system may be an efcient method to
achieve cost benecial recombinant protein production
methodology with perceiving high growth rate of this
microalgae (Smith et al. 2010). The important point is to
consider the most salient chloroplast genome features of
the Dunaliella such as 269 kb circular plastid genome
containing 102 genes with 32.1 % GC content and 65.5 %
non-coding DNA regions including either intergenic or
intronic DNA together with above mentioned chloroplast
World J Microbiol Biotechnol
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transformation advantages. In addition, the Dunaliella
ptDNA sequence is already available and scientists will be
able to exploit these data to develop plastid transformation
vectors targeting specic regions of its ptDNA.
Factors affecting protein expression in Dunaliella
and strategies for its increase
Enormous studies have recently focused on improving
recombinant protein production in microalgae through
studying promoters efciency, the role of the untranslated
region (UTR) sequences of DNA and fusion between native
and recombinant peptides is to achieve a cost benecial
production mechanism and eliminate probable limitations
regarding microalgae expression systems. Indeed, the regu-
lation of recombinant protein expression system in eukary-
otic cells is more complex and consist of plenty interacting
elements. The extent of interaction among internal factors
has not been understood completely but several strategies
and mechanisms have previously been proved to increase
recombinant protein yields in microalgae. Some crucial
factors are reviewed following this section.
Promoters
Using endogenous promoters to obtain an efcient
expression level of heterologous genes seems to be
essential at point of molecular biology. A number of pre-
vious studies found that the endogenous promoters possess
the ability to stimulate other promoters and therefore
increase the expression of heterologous genes. Hence, it is
highly important to select a highly active promoter to
achieve a high yield recombinant protein production sys-
tem (Schroda et al. 2000, 2002).
A lodgment of the gene under highly active promoter
can perform a crucial role in developing efcient micro-
algeal transformation systems particularly in Dunaliella to
achieve a high yield of recombinant protein in transformed
cells. Several famous promoters have been previously
introduced by researchers for Dunaliella transformation
which are listed in Table 2. Among different promoters,
the Ubi1-X promoter is suggested as a suitable promoter to
express foreign genes in Dunaliella cells (Degui Geng et al.
2003). The existence of TMVX element in the Ubi1-X
promoter as a translational enhancer can increase the
expression of foreign genes at in vivo/in vitro conditions.
However, the expression level of the foreign genes driven
by previously introduced promoters has been proved to be
in a low level, usually transient expression in transformed
Dunaliella cells. Besides, the RBCS2 promoter derived
from Chlamydomonas reinhartii contrastingly showed
higher efciency and activity than CaMV35S promoter in
transgenic Dunaliella cells (Thanh et al. 2011; Walker
et al. 2005). This, in turn, illustrates the necessity of pro-
viding some endogenous promoters to obtain high efcient
expression level of foreign genes in transgenic microalgal
cells. Despite very strong promoters like carbonic anhy-
drase 1 which have formerly been developed and exploited
in transgenic microalgae, the expression of heterologous
genes in transgenics represents a problematic procedure
because of the potential occurrence of low activity of gene
expressions. This low activity affects the growth of trans-
formants by gene silencing and some other effects (Li et al.
2007, 2010). To overcome these obstacles caused by the
permanent expression of heterologous genes, the inducible
homologous promoter can be considered to create a perfect
expression system in microalgae. It produces a suitable
copy number of transgene to prevent the occurrence of
gene silencing (Jia et al. 2012).
The homologous promoter of nitrate reductase gene has
previously been isolated to improve the expression of
heterologous proteins in transgenic Dunaliella cells by
possessing the ability to switch on/off with nitrate/ammo-
nium respectively (Li et al. 2007). Another inducible pro-
moter, duplicated carbonic anhydrase 1, regulates gene
expression at the transcriptional level by gradient concen-
trations of NaCl to express heterologous genes that have
previously been isolated from Dunaliella. This promoter
showed a low expression level of heterologous protein but
the expression was efciently stable (Li et al. 2010).
Finally, by considering abovementioned concepts, the
isolation and selection of high active endogenous inducible
promoter can mainly affect the recombinant protein pro-
duction yield in microalgal efcient bioreactor systems
such as Dunaliella.
Enhancer elements
Enormous investigations showed that insertion introns
from native genes in heterologous sequences could
improve the foreign protein yield in transformed cells
Table 2 GUS activity was determined according to the amount of
methyl umbelliferone (MU) produced by protein extracts of trans-
formed Dunaliella
Promoter GUS activity
(nmol MU mg
-1
protein h
-1
)
CaMV35S 4 1
Ubil 11 3
Ubil- X 40 3**
CaMV35S-Ubil 2 1
CaMV35S-Ubil- X 10 2
Asterisks denote statistically signicant differences (**p B 0.01)
Data show the mean SE
World J Microbiol Biotechnol
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under the control of native genes promoter. For instance,
inserting three introns from native C. reinhardtii RBCS2
chloroplast gene into expression construct increased lucif-
erase and erythropoietin expressions more than 400 %
compared to the base level through codon optimizing
manner. Although each RBCS introns individually showed
a positive effect on the foreign gene expression, their
integrations with physiological order and number illustrate
a synergistic effect on the whole (Eichler-Stahlberg et al.
2009).
Expression of recombinant genes in the nuclear trans-
formation of C. reinhardtii also improved following the
insertion of the rst RBCS2 intron which has been shown
to contain an enhancer element (Berthold et al. 2002).
Some eukaryotic gene promoter sequences possess the
consensus TATA and CAAT elements and repetitive tan-
dem GT/AC sequences at the upstream position of
homologous gene and these sequences participate in
inducible regulations in Dunaliella (Lao et al. 2011; Long
et al. 1989).
Indeed, the existence enhancer elements in the construct
can undergo the expression level of heterologous genes to
transform microalgae but developing the use of these ele-
ments needs more investigations until reaching a cost
benecial and high yield production system (Table 3).
Endogenous regulatory factors
Although the expression of recombinant proteins in green
algal chloroplasts can be considered a promising platform
for the production of human therapeutic proteins and as far
as a number of these proteins have been expressed in
microalgal chloroplast, many of these proteins accumulate
to signicantly lower levels than endogenous chloroplast
proteins do. In microalgae, some chloroplast gene products
regulate the translation of their own mRNA through feed-
back inhibition (Coragliotti et al. 2011; Minai et al. 2006;
Wostrikoff et al. 2001, 2004). This phenomenon was
explained once more in heterologous genes expression
when microalgae was less than expression in tobacco
chloroplast due to lack of this inhibition in plants (Manuell
et al. 2007). By considering the high potential of micro-
algal systems to produce safely and economically scaled up
therapeutic proteins, the development of some methodol-
ogies to overcome this obstacle and increase the recombi-
nant protein accumulation in chloroplast transformants
seems crucially important. Among a number of approa-
ches, the fusion exogenous protein gene with highly
expressed endogenous chloroplast gene improved the
accumulation of recombinant fused protein. For instance,
fusing the luciferase reporter protein to the carboxy-ter-
minal end of the large subunit of ribulose bisphosphate
carboxylase showed 33-fold increase in luciferase expres-
sion compared to luciferase expressed alone. These results
demonstrate the exploit of fusion proteins in algal chloro-
plast as a method to increase the accumulation of recom-
binant proteins that are difcult to express (Mayeld et al.
2003; Michelet et al. 2011; Muto et al. 2009).
Codon optimization
Codon optimization is a generic technique to achieve
optimum expression of a foreign gene in the hosts cell
system. Selection of optimum codons depends on codon
usage of the host genome and the presence of several
desirable and undesirable sequence motifs. The codon
adaptation index (CAI) is exploited as the most important
quantitative method (Xia 2007) to predict the expression
level of native and heterologous genes based on organism/
organel codon usage. CAI actually measures the deviation
of a given protein coding gene sequence with respect to a
reference set of genes. When this parameter is considered
from a reference set of highly expressed genes, the maxi-
mum expression of a heterologous gene will be assumed as
dual rationale; highly expressed genes need to compete for
resources (i.e. ribosomes) in fast-growing organisms and it
makes sense for them to be also more accurately translated.
However, a common error for optimization of heterologous
gene expression in the chloroplast of microalgae, in
Table 3 Main precious characteristics of Dunaliella for genetic and
plastome engineering
Characteristics of Dunaliella References
Sequencing of its nuclear and
organelle genomes
(Smith et al. 2010)
Mutations study possibility without
any requirement for further progeny
analysis because of being haploid
(Primrose and Ehrlich 1981)
Appropriate selection to produce of
multi-chain antibodies due to the
sexual reproduction in the eld
culture conditions
(Mayeld et al. 2003;
Mayeld and Franklin
2005)
A large scale cultivation possibility
due to easy maintenance and fast
growth
(Lam and Lee 2011)
Facilitated genetic manipulation
particularly through nanoparticles
due to lack of the rigid cell wall
(Perreault et al. 2011)
Possessing a single plastid makes it
favorable selection to develop
homoplasmic lines
(Wang et al. 2007)
Gene ow cant happen in transgenic
Dunaliella thus harmless to the
environment
(Barzegari et al. 2010)
The ability of the recombinant
proteins secretion to outside of the
cell
(Kleinegris et al. 2010)
World J Microbiol Biotechnol
1 3
particular Dunaliella, has calculated a CAI value using a
codon usage table for all genes. Such optimization is
incorrect because it assumes: (a) that all tRNA species are
equally abundant; and (b) that translational selection does
not exist in chloroplast genome where only three chloro-
plast genes (subunits of RNA polymerase) lack such
selection (Surzycki et al. 2009). Besides, although it is
popular to say that an organism has a particular codon
usage, it is now known that an organisms genes might
have more than one codon usage pattern. Only a multi-
variate analysis method that is possible to ascertain the
kinds of variation contained within the data. Thus, by
considering the use of the variable codon usage even in one
organism among expressed genes, it may be concluded that
this variability is a procedure at the point of codon usage to
regulate gene expression levels in creatures which leads
some genes to be highly expressed and others to below. In
summary, the codon usage optimization seems necessary to
obtain optimum recombinant protein production system in
microalgae in particular Dunaliella (Heitzer et al. 2007).
Two main issues should be considered for this optimiza-
tion; (1) the codon usage in foreign gene sequence must be
optimized for common codon usage in microalgae and then
(2) the optimized codons should be reoptimized again for
codon usage exploited in highly expressed genes, particu-
larly host cell (for example, Dunaliella).
Transformation-associated genotypic modications
A plasmid vector for chloroplast transformation possesses
two genes: the gene of interest and a selectable marker
gene (an antibiotic resistance gene) that allows for selec-
tion of transformed cells (Newman et al. 1990). Some
genetic elements (promoter, 5
0
and 3
0
UTRs and etc.)
require homologous recombination and identication
transformants to express chloroplast machinery and the
correct insertion of transgene into chloroplast DNA. Thus,
it has been supposed that protein levels in these strains
would be accordant in each isolated transformant after
transformation (Newman et al. 1990). Findings of Chla-
mydomonas chloroplast transformation for VP28 protein
gene inserted to pBA155 and pSR229 vectors showed that
the range of protein accumulation was from 20.9 to 0.88 %
and 2.4 to 0.2 % TSPs, respectively (Gong et al. 2011;
Jones et al. 2012; Surzycki et al. 2009). By comparing
these results, it may be derived that the psbA promoter (on
pBA155) is better than the atpA (on pSR229) promoter at
expressing VP28 (21 vs. 2.4 %) but this conclusion
depends merely on the chance of selecting a single trans-
formed cell line for the analysis. For instance, if the
transformed cell lines with 0.88 and 2.4 % were selected,
the conclusion would have been reversed (Surzycki et al.
2009). By considering the high level of protein expression
levels in the Chlamydomonas chloroplast which was pre-
viously reported to be 5 % with most being \1 % (Leon-
Banares et al. 2004), it can be concluded that the expres-
sion of transgenic proteins may depend on a minor extent
of the promoter, site of insertion or associated 3
0
and
5
0
UTRs than the nature of changes incurred during an
individual transformation event. These changes could
result in the formation of a transformed line having unique
characteristics, or transformation-associated genotypic
modication, referred to as the transformosome (Surzycki
et al. 2009). Moreover, the occurrence of some other
unknown genetic events in transgenic strains may possess
crucial impacts on nal recombinant protein yield because
of recalcitrant or low yielding transformed cell lines due to
specic incompatibility among genes, different level of
mRNA of foreign gene in transformants, genetic elements
and insertion sites. The provenance of these changes is not
clearly known while the changes may result in additional
insertions of the vector or DNA fragments into the nuclear
genome. Moreover, inserting a transgenic gene into the
chloroplast genome target site leads to a change in the
function of nuclear genes involved in regulation of
recombinant protein expression. If the transformosome
leads to a variation of transgenic protein expression, it may
affect some other properties of transformants such as
growth rates or protein stability. On the other hand, protein
degradation depends on an intrinsic characteristic of the
protein (sensitivity to proteolytic enzymes) regardless of
the nature of the promoter used or the expression level of
the protein. The degradation of VP28 protein in three
Chlamydomonas transgenic lines was previously proved to
be different from each other which indicate that the genetic
background of these strains was not the same. It presum-
ably changed in the process of transformation which cre-
ated different transformosomes (Surzycki et al. 2009).
Finally, screening the transformosomes based on their
potential to express and accumulate recombinant protein
may provide a quick and easy way to maximize recombi-
nant protein yield via selecting high performance trans-
formosomes of microalgae.
Milking of Dunaliella
Different types of plant lipid bodies have been previously
described proving that the proteins in these structures are
co-induced and specically associated with the carotenoid
bodies formation. Examples include oil bodies in seeds
containing primarily triacyl glycerols and special low-
molecular-weight proteins (oleosins) (Huang and Cheung
2011; Noll et al. 2000), and chromoplast lipoprotein brils
containing carotenoids together with polar lipids and bril
in proteins (Deruere et al. 1994; Smirra et al. 1993).
Likewise in mammalians, the secreted major lipid globules
World J Microbiol Biotechnol
1 3
of milk containing triacyl glycerolsare covered with a
membrane of polar lipids originated from the endoplasmic
reticulum and monolayer of unknown hydrophilic proteins
(Heid and Keenan 2005). By considering aforementioned
issues, it can be concluded that globular lipid bodies have
some structural similarity from plant to animal in the nat-
ure. In microalgae, the massive amount of b-carotene is
arranged (Ben-Amotz et al. 1986; Ben-Amotz and Avron
1983) in minute lipid globules (100200 nm) located in the
inter thylakoid space of the chloroplast of green microal-
gae, which isolated globules display as a superior stability
in aqueous solutions suggesting that these globules possess
a stabilizing layer which prevents their aggregation and
coalescence (Katz et al. 1995, 2007). Finding of some
investigations showed that the isolated b-carotene globules
contain exclusively b-carotene, neutral lipids, and a small
amount of protein (Katz et al. 1995). The high stability of
b-carotene globules from D. bardawil at in vivo/in vitro
aqueous conditions may cause these structural proteins to
provide a stabilizing hydrophilic layer covering the
hydrophobic core in spite of their tiny dimensions and
hydrophobic contents. Under induction conditions, even
the metabolic pathway of b-carotene synthesis is blocked,
the involved proteins in globules show over expression
which suggests that these protein expression depend on
induction condition and globule formation rather than
directly b-carotene accumulation (Hejazi et al. 2004;
Jimenez and Pick 1993). On the other hand, the involved
proteins in globules did not illustrate the over expression in
a related Dunaliella species that does not accumulate b-
carotene. Studies on the localization of the protein at the
periphery of the globules demonstrate primarily hydro-
phobic interactions between proteins and globules which
provide the detachment of proteins from globules by a mild
detergent treatment (Katz et al. 1995, 2007). Several
mechanisms have been previously described for secretion
Fig. 2 Milking of Dunaliella.
There are two common
secretion mechanisms in
Dunaliella one via golgi
apparatus and another by
formed vesicles in the
endoplasmic reticulum. The
secretion pathway via
endoplasmic reticulum seems to
be suitable to increase
recombinant protein yield and
facilitate its harvesting and
purication
World J Microbiol Biotechnol
1 3
of lipids and related lipophilic compounds towards out of
the cells and their pathways. They include TAG-containing
very-low-density lipid (VLDL) vesicles, TAG-containing
vesicles from mammary glands, and the ATP-binding
cassette (ABC) transporter-mediated export. In addition to
cellular export pathways, importing fatty acids into mito-
chondria and peroxisomes or other organelles is considered
as an intracellular transport pathway and it may be possible
to utilize such pathways for the export of lipids (Radako-
vits et al. 2010, 2011). Even though many of the key genes
that are involved in secretion phenomenon have been
identied, the exact mechanisms are not generally known.
The use of ABC transporters might be considered as a more
straight forward approach to enabling secretion of lipids
from microalgae because these transporters mediate the
export of plant waxes derived from very-long-chain fatty
acids, including alkanes, ketones, alcohols, aldehydes,
alkyl esters, and fatty acids. One interesting characteristic
of ABC transporters is their promiscuous gating properties
to export from very-long-chain to medium-chain fatty acids
while wax transporters have not been shown to export
products that are derived from medium-chain fatty acids
(Pighin et al. 2004). In summary, lipid secretion is an
attractive alternative to harvesting algal biomass that could
potentially lower the cost of producing microalga-derived
compounds. By considering the high stability of secreted
lipid globules in aqueous media and their formation
locality (endoplasmic reticulum), directing the recombinant
protein to endoplasmic reticulum via a signal peptide to
accumulate and make vesicular and consequently secretion
inside vesicles to culture media can be proposed as a
suitable method to increase foreign protein production
yield in under stressed culture of Dunaliella (Fig. 2). In
this method, the secreted globules containing recombinant
protein can be easily collected from aqueous media
because of their lipid nature. Thereby, they can accumulate
in surface of culture media to decrease the production
process cost and provide a continuous bioreactor system by
milking of Dunaliella. To achieve this goal, Dunaliella
species should be selected that can produce high amount of
globules under stress condition. In addition, a secretion
strategy may not be the best solution when a signicant
number of contaminating microorganisms are present in
the cultivation system because the secretion of the inter-
mediates into the culture medium would provide a rich
source of nutrient for microorganisms and thereby reducing
product yield.
Conclusion
Interaction of some crucial complex factors such as choice
of suitable selectable markers, different transformation
methods, promoter effects, transformation associated
events, sensitivity to proteases effects, protein localization
efcacies and gene silencing impacts have created an
inexplicit system to produce recombinant protein in Du-
naliella. Thus, a comprehensive systematic study seems
necessary to optimize these parameters effects to obtain
the optimum condition for increasing recombinant protein
production in microalgae. It can be concluded that all
concepts must be investigated together in this review to
achieve a high yield production system in microalgae,
particularly in Dunaliella.
Acknowledgments The nancial support of the Research Center for
Pharmaceutical Nanotechnology, Tabriz University of Medical Sci-
ences is gratefully acknowledged.
Conict of interest There is no conict of interests to be declared.
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