Appl Microbiol Biotechnol (2014) 98:4293–4300
DOI 10.1007/s00253-014-5636-4
MINI-REVIEW
Dunaliella salina as a novel host for the production
of recombinant proteins
Shuying Feng & Xuebing Li & Zhengshun Xu & Jingjiao Qi
Received: 30 October 2013 / Revised: 20 February 2014 / Accepted: 23 February 2014 / Published online: 19 March 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract Although several expression systems are currently
available for the production of recombinant proteins, they still
have some inherent disadvantages, thereby resulting in the
desire to explore a novel expression system for producing
recombinant proteins. Dunaliella salina (D. salina) has been
exploited as a novel expression system for the field of genetic
engineering because of its distinct advantages, including low
production cost, fast growth, easy culture, ease of transgenic
manipulation, and modified abilities of transcription and trans-
lation. Thus far, studies on D. salina host have made great
development and significant progress. This paper presents a
comprehensive summary of the achievements of D. salina
host from the following aspects: the advantages of D. salina
cells, transformation methods, cloning of D. salina genes, and
expression of exogenous genes into D. salina. Furthermore,
the authors identified the current main obstacles and future
application prospects for the recombinant proteins produced
by D. salina, which could be used as a basis for the future
maturation of D. salina expression system.
Keywords Dunaliella salina . Novel host . Promoter .
Enhancer . Transformation methods . Perspective
Introduction
Recombinant proteins and components are widely used in
various fields, such as antibody (Poungpair et al. 2014),
S. Feng (*) : Z. Xu : J. Qi
Medical College, Henan University of Science and Technology,
No. 6 Anhui Road, Luoyang, Henan 471003, China
e-mail: fshy001@haust.edu.cn
X. Li
College of Medical Technology and Engineering, Henan University
of Science and Technology, No. 6 Anhui Road, Luoyang,
Henan 471003, China
vaccine (Chin’ombe and Ruhanya 2013), food (Trautwein-
Schult et al. 2014), cosmetics (Miklaszewska et al. 2013),
disease diagnostics (Chidumayo et al. 2014), and pharmaceu-
tical industries (Jiang et al. 2014). Given that many valuable
proteins have yet to be produced, the field of genetic engi-
neering has attracted significant research attention. Several
expression systems, including bacteria, yeast, mammalian
cell, animal, and plant, are currently available for the produc-
tion of recombinant proteins. Each system offers advantages
and disadvantages with respect to ease of manipulation, safety
and cost of production, time required, and yields of recombi-
nant proteins (Barzegari et al. 2010).
Bacterial systems, such as Escherichia coli, are the most
commonly used expression systems for the production of
recombinant proteins. Although bacterial systems offer the
advantages of rapid cell division, low cost, and high yield,
they still have some inherent disadvantages, such as formation
of insoluble inclusion bodies, lack of effective post-
translational modifications, and possibility of bacterial toxin
contamination to protein products (Martinez-Alonso et al.
2009; Liu et al. 2013). Yeast systems have similar advantages
to bacterial systems, but they have the limitations of higher
production cost and high mannose glycosylation of protein
products (Gonçalves et al. 2013; Rabert et al. 2013). Although
mammalian expression systems possess effective capabilities
of transcription and translation modification, they have strict
cultural requirements, high cost, and presence of contamina-
tion risk to protein products (Aricescu and Owens 2013).
Transgenic animals and plants can produce proteins with very
high amounts, and the products can be easily harvested peri-
odically, but the generation of transgenic animals and plants is
a difficult process and the production period is too long
(Guan et al. 2013). Therefore, the exploration of an optimal
expression system for the production of recombinant proteins
is necessary.
With the development of microalgae, the use of microalgae
for producing valuable molecules has received a surge of
4294
interest. As a novel host, D. salina has attracted increasing
research attention and is involved in the field of genetic
engineering. In this paper, the research status of D. salina host
has been completely summarized into the following aspects:
the advantages of D. salina cells, transformation methods,
cloning of D. salina genes, and expression of exogenous
genes in D. salina. Moreover, the current problems and ob-
stacles of D. salina host were analyzed, and the future appli-
cation prospects of D. salina host were identified.
Advantages of D. salina cells
D. salina was exploited as a novel expression system because
of its many advantages (Xue et al. 2006). First, D. salina has
extremely strong adaptability to the environment and can live
in various salt concentrations from as low as 0.2 % to salt
saturation concentration of 35 %. Thus, D. salina cultures can
avoid contamination from other microorganisms, such as
some halotolerant bacteria, ciliates, and amoebae (Post et al.
1983; Butinar et al. 2005). Second, D. salina is a
photosynthetic, unicellular organism that can be easily, rapid-
ly, and inexpensively cultured, which can be achieved by
large-scale factory farming. Third, as a eukaryotic organism,
it has the modified abilities to recombinant proteins in the
levels of transcription and translation, thus facilitating the
production of crude proteins. Fourth, D. salina lacks a rigid
cell wall and is a natural protoplast that can be easily trans-
formed with the exogenous genes (Ben-Amotz and Avron
1990; Ben-Amotz 1993). Fifth, D. salina cell has a single,
large, cup-shaped chloroplast (Fig. 1, shown by arrow), which
can be used as a potentially high-efficiency expression system
for large-scale production of recombinant proteins (Wang
et al. 2009; Barzegari et al. 2010). Sixth, D. salina can
accumulate significant amounts of valuable fine components,
such as carotenoids, lipids, vitamins, and minerals, which
enable its extensive use in industrial and pharmaceutical ap-
plications (Hosseini Tafreshi and Shariati 2009). Seventh,
Fig. 1 Morphology of D. salina chloroplast (black arrow showed). The
scale bar indicates 10 μm
Appl Microbiol Biotechnol (2014) 98:4293–4300
another advantage of D. salina is the possibility of selective
milking of high-value compounds from them (Hejazi et al.
2002, 2004). Thus, recombinant proteins produced by trans-
genic D. salina can be easily extracted from the cells using
easy cell lysis procedures, and its functional effects may be
markedly enhanced than the proteins produced by other ex-
pression systems.
Cloning of D. salina genes
Promoter
Most eukaryotic algae are refractory to the expression of
transgenes after transgenic manipulation mainly because of
the unavailability of suitable regulatory elements, such as
promoters, enhancers, and terminators. Among these ele-
ments, an effective promoter has a pivotal function in the
process of gene expression. Thus, identification and cloning
of an efficient promoter are the major fundamental steps for
the utilization of D. salina host. A series of exogenous and
endogenous promoters have been developed for D. salina host
(Table 1). Among the exogenous promoters, cauliflower mo-
saic virus 35S (CaMV35S) is a typical promoter that has been
frequently used to drive the expression of foreign genes in
D. salina (Sun et al. 2005; Tan et al. 2005; Chai et al. 2012).
Other exogenous promoters, such as Ubiquitin (Ubil), Ubil-Ω,
CaMV35S-Ubil, and CaMV35S-Ubil-Ω, can also drive the
expression of foreign genes in D. salina, in which Ubil-Ω
promoter has the highest driving effect over the others (Geng
et al. 2003). However, under these promoters, the expression
of foreign genes is low or transient that needs to be addressed
in future investigations.
Endogenous promoters normally have higher driving ac-
tivity for gene expression in host cells than exogenous pro-
moters. To improve the expression levels of foreign genes,
some endogenous promoters of D. salina containing 5'
flanking region of actin gene (Jiang et al. 2005) and
glyceraldehyde-3-phosphate dehydrogenase promoter
(Jia et al. 2012) were cloned and identified, and the results
showed that the target genes, bialaphos resistance gene (bar),
and canstatin gene all have been successfully expressed in
D. salina cells. Moreover, two inducible endogenous pro-
moters have been identified, namely, the upstream region of
the nitrate reductase (NR) gene and the duplicated carbonic
anhydrase 1 (DCA1) promoter, which can provide conve-
nience for screening of transformants of D. salina. The
DCA1 promoter can drive the expression of foreign genes in
D. salina under a series of sodium chloride concentrations
(Li et al. 2010; Lv et al. 2011). The NR promoter can induce
gene expression in the presence of nitrate and inhibit gene
expression in the presence of ammonium ions in transgenic
D. salina cells (Li et al. 2007, 2008). These two inducible
Appl Microbiol Biotechnol (2014) 98:4293–4300 4295

Table 1 The used promoters for

the expression of foreign genes in Promoter Source Expression position Gene References
D. salina bioreactor
CaMV35S Cauliflower mosaic virus Nuclear expression SKTI Chai et al. 2012
Ble Sun et al. 2005
GUS Tan et al. 2005
Ubiquitin Eukaryotic cells Nuclear expression GUS Geng et al. 2003
Actin gene D. salina Nuclear expression Bar Jiang et al. 2005
DCA1 D. salina Nuclear expression Bar Li et al. 2007
GUS Li et al. 2010
NR gene Lv et al. 2011
NR D. salina Nuclear expression Bar Li et al. 2007
EGFP Li et al. 2008
GAPDH D. salina Nuclear expression Canstatin/Bar Jia et al. 2012

promoters could be powerful tools for the controllable expres-
sion of target genes in D. salina. However, the driving activ-
ities of these endogenous promoters were not compared. The
optimal promoter for D. salina remains unknown and must be
resolved before the maturation of D. salina expression system.
Enhancer
Enhancer, as a regulatory element, is an effective approach for
improving the expression levels of foreign genes because it
can overcome the “position effects” and “silence effects”
(Peach and Velten 1991). The available enhancers to
D. salina are matrix attachment regions (MARs) and 5' leader
sequence of tobacco mosaic virus RNA (Ω element). The
MAR sequence is a good candidate for increasing the expres-
sion levels of foreign genes, which has been performed in
many eukaryotes, such as tobacco (Allen et al. 1993),
Arabidopsis thaliana (Butaye et al. 2004), and Chinese ham-
ster ovary cells (Kim et al. 2004; Girod et al. 2005). The MAR
sequence has been identified from the constructed MAR li-
brary of D. salina, and the results demonstrated that MAR
sequence can improve gene expression by about 4.5-fold than
without MAR in D. salina (Wang et al. 2005). Ω element is
another effective enhancer in the expression of foreign genes in
D. salina. When Ω element is combined with promoters Ubil
and CaMV35S-Ubil, the combined promoters Ubil-Ω and
CaMV35S-Ubil-Ω have higher driving effects on foreign gene
expression than those without Ω element (Geng et al. 2003).
Other enhancers, such as the A312L 5'-UTR of Chlorella virus
PBCV-1 (Nguyen et al. 2009) and Mg-protoporphyrin IX
(Von Gromoff et al. 2006), were also deserved to be
explored which have been applied in the transformation of
Chlamydomonas reinhardtii (C. reinhardtii).
Transformation methods
Up to now, several transformation methods have been
established for the transformation of D. salina, namely, elec-
troporation (Geng et al. 2003; Sun et al. 2005), particle bom-
bardment (Tan et al. 2005), glass beads (Feng et al. 2009), and
lithium acetate/polyethylene glycol (PEG)-mediated method
(Chai et al. 2012). Each method has advantages and disad-
vantages with respect to the transformation rate, controllabil-
ity, repeatability, requirements of laboratory apparatus, and so
on (Table 2).

Table 2 The advantages and disadvantages of each transformation method for D. salina

Transformation method Electroporation Particle bombardment Glass beads LiAc/PEG

Transformation rate 2% <1 % 5.9 % 7.21 %

Specialized equipment Electroporation apparatus Particle gun, Tefzel tube Vortex mixer, glass beads LiAc, PEG
Expenses High High Low Low
Controllability Easy Difficulty Easy Easy
Physical damage to cells Severest, ~58 % survived cells Severest, ~26 % survived cells Low, ~90 % survived cells Low, almost no damage to cells
Repeatability Good Bad Good Good
Operation process Simple and easy Complex and difficulty Simple and easy Simple and easy
Specially used Nuclear Chloroplast Nuclear Nuclear
References Sun et al. 2005 Tan et al. 2005 Feng et al. 2009 Chai et al. 2012
4296
Electroporation
In electroporation, an electronic pulse is used to temporarily
disturb the bilayer lipid membrane, and DNA molecules are
allowed to enter cells. Electroporation is frequently used for
the transformation of special cells, such as protoplasts, naked
cells, cell wall-reduced mutants, and other thin-walled cells
(Amiri Yekta et al. 2013; Jiang et al. 2013). D. salina cells
have been originally successful transformed by electropora-
tion, and the results showed that the foreign genes can be
introduced into D. salina cells and retained for a considerably
long period (Geng et al. 2003). The effects of different trans-
formation parameters, including electroporation buffer, pulse
length, field strength, and DNA concentration, on the trans-
formation efficiency have been analyzed (Sun et al. 2005).
The results revealed that approximately 20 of 1×107 cells can
be transformed under the selection of 10 mg/L Zeocin, which
is significantly lower than the transformation of C. reinhardtii
by electroporation (Shimogawara et al. 1998). Thus, electro-
poration for the transformation of D. salina needs improve-
ment in many aspects.
Particle bombardment
Particle bombardment was successfully employed for the
transformation of most standard cellular expression systems
(Johnson 2013; Imamura et al. 2012). Through this method,
the foreign gene, GUS gene, was successfully transformed
and expressed in D. salina cells, which indicated that particle
bombardment is a feasible approach for the transformation of
D. salina (Tan et al. 2005). However, the transformation
efficiency of particle bombardment is the lowest (less than
1 %) than those of electroporation and glass beads methods
(Feng et al. 2009) mainly because particle bombardment
causes severe cell damage and has difficult controllability.
Particle bombardment also has some inherent disadvantages,
such as requirement of expensive laboratory apparatus, poor
repeatability, and complex operation process. So, particle
bombardment method is presently unsuitable for the transfor-
mation of D. salina to some extent. However, because of it
possessing complex integration patterns and multiple copy
insertions, particle bombardment may be a perfected method
for chloroplast transformation of D. salina in the future.
Glass beads
Glass bead method has a high transformation rate and has
been applied for the transformation of C. reinhardtii (Kindle
1990) and yeast (Costanzo and Fox 1988). To overcome the
drawbacks of particle bombardment and electroporation, glass
bead method has been successfully established for the trans-
formation of D. salina (Feng et al. 2009). Simultaneously, the
transformation parameters, including agitation periods,
Appl Microbiol Biotechnol (2014) 98:4293–4300
rotation speed, plasmid DNA amounts, and PEG concentra-
tion, have been optimized. Under the optimal transformation
conditions, the highest transformation efficiency of approxi-
mately 5.9 % was achieved by glass bead method, but this
value was somewhat lower than that reported by Kindle
(1990). Besides its high efficiency, this method also has other
advantages, such as economy, simplicity, and no requirement
for specialized equipment. Thus, glass bead method is a facile
and high-efficiency method for future genetic engineering
research of D. salina.
LiAc/PEG-mediated method
A novel transformation method known as LiAc/PEG-mediat-
ed method was recently reported by Chai et al. (2012). For
transformation efficiency, the developed LiAc/PEG-mediated
method achieved the highest transformation rate of approxi-
mately 7.21 %, which exceeded all the aforementioned
methods. The high transformation rate of LiAc-PEG-
mediated method is mainly due to its advantages, including
simple operation, less damage to host cells, and better repro-
ducibility (Table 2). Thus, this method could be used as a
powerful tool for the future application of D. salina host.
Besides the aforementioned methods, other effective transfor-
mation methods should also be exploited, such as ultrasonic
delivery (Wu et al. 2009), ultraviolet laser microbeam (Badr
et al. 2005), aerosol gene delivery (Minai-Tehrani et al. 2011),
and Agrobacterium-mediated method (Zheng et al. 2012).
Expression of exogenous genes in D. salina
Numerous research reports on the transformation of exoge-
nous genes into D. salina host exist. However, the trans-
formed exogenous genes mainly focused on the reporter genes
and selection markers, such as GUS, enhanced green fluores-
cent protein (EGFP), and Ble (Table 3). For the expression of
practical valuable proteins in D. salina host, only the hepatitis
B surface antigen (Geng et al. 2003), human canstatin
(Jia et al. 2012), soybean kunitz trypsin inhibitor (Chai et al.
2012), and white spot syndrome virus VP28 (Feng et al. 2013)
have been determined. Most of these proteins are only
exhibiting low expression in nuclear transformation of
D. salina. Thus, so far, no successful products have been
produced by D. salina host.
Chloroplast systems are also effective expression systems
in the field of algal genetic engineering because of their many
advantages, such as high expression level, feasibility of ex-
pressing multiple proteins, elimination of gene silence and
escape, and stability of transgene expression (Bock 2007;
Kittiwongwattana et al. 2007; Wang et al. 2009). Chloroplast
expression systems have been extensively used in various
species, including C. reinhardtii (Chen and Melis 2013), to-
bacco (Abdoli-Nasab et al. 2013), rice (Wu et al. 2013), cotton
Appl Microbiol Biotechnol (2014) 98:4293–4300 4297

Wang et al. 2007

Geng et al. 2003

Geng et al. 2003

Feng et al. 2013

Chai et al. 2012
(Luo et al. 2013), and wheat (Cui et al. 2011). Although

Sun et al. 2005

Jia et al. 2012

Li et al. 2008
Li et al. 2010
References D. salina has a single, large, cup-shaped chloroplast, research
on D. salina chloroplast transformation is still in its prelimi-
nary phase. The achievements have focused on the reports of
our team, which include the cloning of genes and promoter,
such as ch1L, heat shock protein 70 cDNA (hsp70), ATPase
Selective marker
Selective marker

Selective marker
alpha subunit gene (atpA), and atpA gene promoter, and the
Reporter gene
Reporter gene

Viral vaccine

Viral vaccine
Therapeutic
Therapeutic
Application

construction of expression vectors to D. salina chloroplast

transformation. The only report on chloroplast transformation
of D. salina was on foreign gene EGFP, which was success-
fully introduced into the chloroplasts of D. salina and present-
ed transient expression (Li et al. 2011). In genetic transforma-
Nuclear expression, canstatin gene fragment
Nuclear expression, no codon optimization
Nuclear expression, no codon optimization

Nuclear expression, no codon optimization

Nuclear expression, no codon optimization
Nuclear expression, no codon optimization
Nuclear expression, no codon optimization

Nuclear expression, no codon optimization

tion of microalgae, chloroplast system as the most efficient

Nuclear expression, introduced MARs

expression system and its exploitation will be an important

direction for future worthy of study.
Other expression characteristics

Conclusion and future prospects

A large number of valuable proteins are still being produced

by expression systems instead of being extracted from the
natural source. The inherent features of D. salina, such as
inexpensive culture, easy scaling-up procedure, and capability
of translational and transcription modifications, make it a
promising host for the production of foreign proteins.
40.13 nmol MU mg−1 protein h−1, transient expression

However, no successful commercial products produced by

1.6 arbitrary unit (PAT/tubulin), stable expression

0.68 % of total soluble protein, stable expression

3.04 ng/mg of soluble protein, stable expression

3.11 ng/mg of soluble protein, stable expression

D. salina exist, which indicates that this expression system

1.4 pg/107 transformed cells, stable expression

is still in its infancy. D. salina host is confronted with many

obstacles and difficulties that need to be urgently resolved and
has extensively applied prospects for the production of exog-
enous molecules.
Unknown, transient expression
Unknown, transient expression
Expression level and stability

Unknown, stable expression

Strengthening the basic research of genetic manipulation of

D. salina host The major obstacles for D. salina host are the
Table 3 The successful expression of exogenous genes in D. salina cells

unclear genome background and unavailability of effective

molecular toolkits. Thus, the basal study of molecular regula-
tory elements, which include identification and cloning of the
endogenous promoter, enhancer, and other relevant regulatory
elements, should be strengthened. The sequencing works of
nuclear genome and chloroplast genome backgrounds of
Enhanced green fluorescent protein

D. salina should be also done in the future study. A novel

Chloramphenicol acetyltransferase
Phosphinothricin acetyltransferase

Hepatitis B surface antigen gene

Soybean kunitz trypsin inhibitor

green selective marker for D. salina is highly necessary be-

cause of the potential health hazard of antibiotic selectable
Zeocin resistance protein
(under promoter DCA1)

markers. Moreover, the standard transformation procedures

essential for the high-efficiency production of foreign proteins
WSSV VP28 gene
β-Glucuronidase

in D. salina host should be established (Surzycki et al. 2009).

Description

Canstatin

Improving the expression level of recombinant proteins Another

bottleneck of D. salina host is the low or inconsistent expres-
sion of recombinant proteins in D. salina cells. Low or incon-
sistent expression may come from the competition of available
HBsAg

Can-N
EGFP

transcription and translation factors, protein localization, gene

VP28
SKTI
Gene

GUS

CAT
Bar

Ble

silencing, codon dependency, and transformation-associated

4298
events. To overcome these obstacles, some strategies could be
used in increasing the protein yields, such as codon optimiza-
tion (Rosales-Mendoza et al. 2012), transformation-associated
genotypic modifications (Surzycki et al. 2009), reduction of
sensitivity to proteases (Doran 2006), and fusion of recombi-
nant products to native proteins (Muto et al. 2009).
Exploitation of chloroplast expression system of D.
salina D. salina chloroplast system could be an effective
approach for improving the expression levels of foreign pro-
teins. Although numerous preliminary studies about chloro-
plast system of D. salina have been performed, the high-
effective production of foreign proteins by D. salina chloro-
plast system remains unfeasible. Given that many factors
affect the expression level of protein in the algal chloroplast
(Surzycki et al. 2009), the chloroplast transformation of
D. salina could be developed from the following aspects:
establishment of chloroplast transformation methods
(Ramesh et al. 2011), transgene codon optimization (Rasala
and Mayfield 2011), modification of recombinant products
(Muto et al. 2009), and strategy exertion to improve transgene
expression (Michelet et al. 2011).
Enriching the natural compounds of D. salina using gene
engineering D. salina can accumulate abundant valuable fine
components, which enable its extensively practical applica-
tions in food, cosmetics, feed, nutrition, pharmaceutical, and
biofuel fields. Through the genetic manipulation to the
D. salina, the natural compounds of D. salina cells can be
greatly enriched that can realize the using maximization
(Ramos et al. 2008; Cordero et al. 2011). Given that environ-
mental factors, such as illumination intensity, temperature, and
nutrition (Tafreshi and Shariati 2009), mainly influence the
accumulation of natural compounds, searching for the most
suitable culture conditions for transgenic D. salina cells is
worthy of future research.
Acknowledgments This work was supported by the National Natural
Science Foundation of China (No. 30900796), the Key Research Project of
Science and Technology Department of Henan Province (No.
112102110103), the Natural Science Research Project of Education De-
partment of Henan Province (No. 2010B180010), and the Young Backbone
Teacher Project of Henan Provincial Universities (No. 2012GGJS-080).
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