Akbari 2014

REVIEW

The potential of transgenic green microalgae; a robustphotobioreactor 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 benefit

eukaryotic expression systems including the ability of post-

translational modifications 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/benefits, 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 modifications, 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. EskandaniStudent Research Committee, Tabriz University of Medical


M. Eskandani

Research Center for Pharmaceutical Nanotechnology, Tabriz

University of Medical Sciences, Tabriz, Iran

A. Y. Khosroushahi

Biotechnology Research Center, Tabriz University of Medical


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: [email protected]

123

World J Microbiol Biotechnol

DOI 10.1007/s11274-014-1714-0

(Franklin and Mayfield 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 suitablesource 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 modifi-

cations. Besides, Dunaliellais one of the best-studied

unicellular green algae in the field 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

coefficient, a characteristic of the interaction potential

between the particles, depends only on the pair interaction

between the particles. The second osmotic virial coeffi-

cients of several proteins were measured in salt solutions.

The findings 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 flat virial coefficient profile 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 beneficial cultiva-

tions. However, any post-transcriptional and post-

translational modifications, including glycosylation, phos-

phorylation and disulfide bond formation are not performed

on bacterial systems where these modifications require the

correct folding of complex proteins. Besides, recombinant

proteins with bacterial toxins have a considerably high

contamination possibility. Although many modifications

are performed in eukaryotic yeasts, the modified 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 difficulties 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 flowing 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-


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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 classified as

generally regarded as safe (GRAS), are frequently uti-

lized because of their comfortable purification 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 (Mayfield et al. 2003; Mayfield 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 Mayfield 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 Mayfield 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 first critical stage in the creation of

transgenic organisms. The release of a gene to plant cells is

more difficult 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 briefly 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

first 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 specific expensive equipments. Moreover, this

method of transformation efficacy is influenced by factors

including field 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 benefits like high-efficiency,

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 efficiency (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 thegrowth 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


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show the sensitivity for hygromycin (Sun et al. 2005; Tan

et al. 2005). Moreover, several research findings 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-

plementation of metabolism or photosynthetic mutants. In this

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

fluorescent protein, was then employed as a reporter gene for

Dunaliella transformation, and a green fluorescence 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-specific

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 finally 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)


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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 influence 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 clarified that a protein

aceous nuclear matrix or scaffold plays a significant

role in determining chromatin structure. Matrix attach-

ment regions (MARs) are thought to be components that

bind specifically 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 influence by the position

effects was designed to facilitate isolation of algal strains

which efficiently 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 findings may be beneficially 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 proteinproduction 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


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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 modification 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 purification 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 beneficial 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 104

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 modifications in the

golgi are not necessary. (3) The proteins can be expressed

in the chloroplast of algae without needing post-transla-

tional modifications (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 final yield. IV) To minimize the nuclear-expres-

sed protein proteolytic degradations, the most efficient

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 modifications. 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 Mayfield 2011).

It seems this system may be an efficient method to

achieve cost beneficial 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


123

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 specific 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 efficiency, the role of the untranslated

region (UTR) sequences of DNA and fusion between native

and recombinant peptides is to achieve a cost beneficial

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 efficient

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 efficient 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 toexpress foreign genes in Dunaliella cells (Degui Geng et al.

2003). The existence of TMVX element in the Ubi1-Xpromoter 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 efficiency 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 efficient

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 efficiently 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 efficient 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 ofmethyl 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 significant differences (**p B 0.01)

Data show the mean SE


123

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 first 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

beneficial 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 significantly 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 difficult to express (Mayfield 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 andplastome 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 field

culture conditions

(Mayfield et al. 2003;

Mayfield 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 flow 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)


123

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 modifications

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, 50 and 30UTRs and etc.)require homologous recombination and identification

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 30 and50UTRs than the nature of changes incurred during anindividual transformation event. These changes could

result in the formation of a transformed line having unique

characteristics, or transformation-associated genotypic

modification, 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 final recombinant protein yield because

of recalcitrant or low yielding transformed cell lines due to

specific 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 specifically 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 fibrils

containing carotenoids together with polar lipids and fibril

in proteins (Deruere et al. 1994; Smirra et al. 1993).

Likewise in mammalians, the secreted major lipid globules


123

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 isarranged (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 globulescontain exclusively b-carotene, neutral lipids, and a smallamount 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

purification


123

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

identified, 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 significant

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

efficacies 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 financial support of the Research Center forPharmaceutical Nanotechnology, Tabriz University of Medical Sci-

ences is gratefully acknowledged.

Conflict of interest There is no conflict of interests to be declared.

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The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteinsAbstractIntroductionComparison of different expression systems with DunaliellaTransformation methods for Microalgae DunaliellaSelection markers and reporter genesNuclear transformation and chloroplast transformation in DunaliellaNuclear transformationSensitivity to proteases

Chloroplast transformation

Factors affecting protein expression in Dunaliella and strategies for its increasePromotersEnhancer elementsEndogenous regulatory factorsCodon optimizationTransformation-associated genotypic modificationsMilking of Dunaliella

ConclusionAcknowledgmentsReferences

Akbari 2014

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