Advances in genetic engineering of marine algae.
ABSTRACT Algae are a component of bait sources for animal aquaculture, and they produce abundant valuable compounds for the chemical industry and human health. With today's fast growing demand for algae biofuels and the profitable market for cosmetics and pharmaceuticals made from algal natural products, the genetic engineering of marine algae has been attracting increasing attention as a crucial systemic technology to address the challenge of the biomass feedstock supply for sustainable industrial applications and to modify the metabolic pathway for the more efficient production of high-value products. Nevertheless, to date, only a few marine algae species can be genetically manipulated. In this article, an updated account of the research progress in marine algal genomics is presented along with methods for transformation. In addition, vector construction and gene selection strategies are reviewed. Meanwhile, a review on the progress of bioreactor technologies for marine algae culture is also revisited.
- SourceAvailable from: Boris Zorin[Show abstract] [Hide abstract]
ABSTRACT: Microalgae are considered a promising source for various high value products, such as carotenoids, ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The unicellular green alga Lobosphaera (Parietochloris) incisa is an outstanding candidate for the efficient phototrophic production of arachidonic acid (AA), an essential ω-6 PUFA for infant brain development and a widely used ingredient in the baby formula industry. Although phototrophic production of such algal products has not yet been established, estimated costs are considered to be 2-5 times higher than competing heterotrophic production costs. This alga accumulates unprecedented amounts of AA within triacylglycerols and the molecular pathway of AA biosynthesis in L. incisa has been previously elucidated. Thus, progress in transformation and metabolic engineering of this high value alga could be exploited for increasing the efficient production of AA at competitive prices. We describe here the first successful transformation of L. incisa using the ble gene as a selection marker, under the control of the endogenous RBCS promoter. Furthermore, we have succeeded in the functional complementation of the L. incisa mutant strain P127, containing a mutated, inactive version of the delta-5 (Δ5) fatty acid desaturase gene. A copy of the functional Δ5 desaturase gene, linked to the ble selection marker, was transformed into the P127 mutant. The resulting transformants selected for zeocine resistant, had AA biosynthesis partially restored, indicating the functional complementation of the mutant strain with the wild-type gene. The results of this study present a platform for the successful genetic engineering of L. incisa and its long-chain PUFA metabolism.PLoS ONE 01/2014; 9(8):e105223. · 3.53 Impact Factor
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ABSTRACT: Genetic engineering in microalgae is gaining attraction but nuclear transformation methods available so far are either inefficient or require special equipment. In this study, we employ positively charged nanoparticles, 3-aminopropyl-functionalized magnesium phyllosilicate (aminoclay, approximate unit cell composition of [H2N(CH2)3]8Si8Mg6O12(OH)4), for nuclear transformation into eukaryotic microalgae. TEM and EDX analysis of the process of transformation reveals that aminoclay coats negatively-charged DNA biomolecules and forms a self-assembled hybrid nanostructure. Subsequently, when this nanostructure is mixed with microalgal cells and plated onto selective agar plates with high friction force, cell wall is disrupted facilitating delivery of plasmid DNA into the cell and ultimately to the nucleus. This method is not only simple, inexpensive, and non-toxic to cells but also provides efficient transformation (5.03×102 transformants/µg DNA), second only to electroporation which needs advanced instrumentation. We present optimized parameters for efficient transformation including pre-treatment, friction force, concentration of foreign DNA/aminoclay, and plasticity of agar plates. It is also confirmed the successful integration and stable expression of foreign gene in Chlamydomonas reinhardtii through molecular methods.PLoS ONE 01/2014; 9(7):e101018. · 3.53 Impact Factor
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ABSTRACT: Single-celled microorganisms such as diatoms and coccolithophores produce inorganic microparticles with genetically controlled hierarchical nanopatterns. Besides serving as paradigms to inspire new routes for materials synthesis, biominerals themselves, particularly diatom biosilica, are increasingly utilized as templates for the synthesis of novel functional materials. Over the past decade, a large variety of methods have been established that allow not only for the attachment or coating of desired materials onto diatom biosilica but also for complete chemical conversion without altering the characteristic micro- and nanoscale morphology. Examples include the synthesis of materials for photonics (surface-enhanced Raman spectroscopy,SERS, extraordinary optical transmission, EOT), ultraresponsive and sensitive gas sensors, gas storage materials, and highly active catalysts. More recently, emerging insight into the cellular mechanisms of biosilica formation has enabled the in vivo functionalization of diatom biosilica through advanced cultivation techniques and genetic engineering. As a naturally renewable material, biominerals hold the promise of serving as an inexpensive and easily available resource for a future nanotechnology-based industry.For further resources related to this article, please visit the WIREs website.Conflict of interest: The authors have declared no conflicts of interest for this article.Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology 08/2014; · 5.68 Impact Factor
Research review paper
Advances in genetic engineering of marine algae
Song Qina,⁎,1, Hanzhi Linb,c,1, Peng Jiangb
aYantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, Shandong, China
bKey Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong, China
cGraduate University of Chinese Academy of Sciences, Beijing 100049, Beijing, China
a b s t r a c t a r t i c l ei n f o
Available online 24 May 2012
Algae are a component of bait sources for animal aquaculture, and they produce abundant valuable compounds
for the chemical industry and human health. With today's fast growing demand for algae biofuels and the prof-
itable market for cosmetics and pharmaceuticals made from algal natural products, the genetic engineering of
marine algae has been attracting increasing attention as a crucial systemic technology to address the challenge
of the biomass feedstock supply for sustainable industrial applications and to modify the metabolic pathway
for the more efficient production of high-value products. Nevertheless, to date, only a few marine algae species
can begeneticallymanipulated. In thisarticle, anupdatedaccountofthe research progress inmarine algal geno-
mics is presented along with methods for transformation. In addition, vector construction and gene selection
strategies are reviewed.Meanwhile,a review on the progress ofbioreactor technologies for marinealgae culture
is also revisited.
© 2012 Elsevier Inc. All rights reserved.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Marine algal genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Cyanobacterial genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.Eukaryotic algal genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods for transformation in marine algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Trans-conjugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.Natural transformation and induced transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.Biolistic transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.Glass beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6. Silicon carbon whiskers method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7.Microinjection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.8.Artificial transposon method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.9. Recombinant eukaryotic algal viruses as transformation vectors
Agrobacterium tumefaciens-mediated genetic transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vector construction and gene selection strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Vector construction: promoter selection and codon usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.Reporter and marker genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.Gene copy number and homology-dependent gene silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. RNA interference technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Current progress of photobioreactor technologies for marine algae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Marine algal photobioreactor: co-development of the ‘open’ and the ‘closed’
5.2.Photoioreactor for marine microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .
Biotechnology Advances 30 (2012) 1602–1613
⁎ Corresponding author at: No. 17 Chunhui Road, Laishan District, Yantai 264003, Shandong Province, China. Tel.: +86 535 2109005.
E-mail addresses: email@example.com, firstname.lastname@example.org (S. Qin).
1These authors contributed equally to this work.
0734-9750/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/biotechadv
Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Key elements for evaluating marine algal expression systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.Crop protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.Biosafety assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Photobioreactor for marine macroalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1609
Marine algae, including marine cyanobacteria, marine eukaryotic
dating back three billion years and distributing from the polar region to
tropical areas and from nutrient-rich coastal seas to oligotrophic open
oceans. They are photoautotrophs unified primarily by their lack of
roots, leaves, and other organs that characterize higher plants and
scopic individualcellsofmicroalgaetohugeplantsthat aregreaterthan
30 m long and are called macroalgae Macrocystis. Marine algae are
responsible for approximately 40%–50% of the photosynthesis that
or cyanophyta) and eukaryotic taxa. Marine algae are a component of
the bait sources for animal aquaculture and produce abundant valuable
compounds for the chemical industry and human health, including
oils (e.g., triglyceride), polysaccharides (e.g., algin, agar), pigments
(e.g., phycobiliproteins, carotenoids), and also potential new pharma-
ceuticals (Apt and Behrens, 1999; Chisti, 2007, Lin et al., 2011,
Witvrouw and DeClercq, 1997). Recently, a microbial platform that
can simultaneously degrade, uptake, and metabolize alginate was
established based on new discovered enzymes for alginate transport
and metabolis, which enables bioethanol production directly from
brown macroalgae via a consolidated process (Wargacki et al., 2012).
The ancient cyanobateria are the last common ancestor of all oxygenic
photosynthetic lineages, which have the closet evolutionary relation-
ship with heliobacteria and other anaerobic photoautotrophs (Xiong
et al., 2000), while photosynthetic eukaryotes acquired their photosyn-
thetic properties from endosymbiosis with cyanobacteria (Gray, 1992;
Reyes-Prieto and Bhattacharya, 2007). The green algae are primitive
members of the kingdom Plantae from which land plants evolved ap-
proximately 500 million years ago (Parker et al., 2008, Wise, 2006).
Due to the algal complex and the unique genetic and evolutionary
scheme, the genetic engineering of algae must be considered to apply
both of the methodologies from prokaryotic microorganisms and
plants. Since the end of the 1970s, marine algal genetic engineering
began being implemented in the model system cyanobacterial strain
Synechococcus 7002, which can be transformed by exogenously adding
homologous DNA carrying a selectable marker (Buzby et al., 1985;
Matsunaga and Takeyama, 1995; Stevens and Porter, 1980). In the late
1980s, several eukaryotic marine microalgae and seaweeds were suc-
cessfully transformed by different transformation methods, e.g., micro-
injection in the marine macro-green alga Acetabalaria sp. (Neuhaus
et al., 1986), plasmid vectors in the marine diatom Cyclotella cryptica
(Dunahay et al., 1995), and gene gun or the biolistic method in the
macro-red alga Eucheuma sp. (Kurtzman and Cheney, 1991) and
brown alga Laminaria japonica (Qin et al., 1998). Entering the new
century, the trend of the genetic engineering of marine algae has been
to apply the transgenic marine algae as cell factories and marine biore-
actors (León-Bañares et al., 2004; Qin et al., 2005; Zaslavskaia et al.,
2001). To date, the most successful algal genetic transformation system
is still the model system of the eukaryotic freshwater green alga
Chlamydomonas reinhardtii (Grossman, 2000) whose nucleus and chlo-
roplast transformations have reached promising commercial relevance
(Franklin and Mayfield, 2004, 2005). Although C. reinhardtii may not be
have the potential for applications in other algal species (Hannon et al.,
With today's rapidly growing demand and development for bio-
energy from algae and the profitable market for cosmetics and phar-
maceuticals from algal natural products, the genetic engineering of
marine algae has been attracting an increasing amount of attention as
a crucial systemic technology to overcome the biomass problem in
industrial applications (John et al., 2011), to modify the metabolic
pathwayforhigh-valueproducts(Schmidtet al.,2010),and toengineer
the bio-bricks and design the artificial photoautotroph in the rising and
promising field of synthetic biology (Heidorn et al., 2011, Muers, 2012).
Nevertheless, to date, only a few marine algae species have been genet-
research progress in marine algal genomics is presented as well as
methods for transformation. In addition, vector construction and gene
selection strategies are reviewed. Meanwhile, a review of the progress
of bioreactor technologies for marine algae culture is also revisited.
2. Marine algal genomics
Genomes are fundamental for genetic manipulation and further ge-
netic engineering, which not only provide the location and the distribu-
of elements that can improve genetic engineering, including cis-acting
elements, trans-acting factors, and other regulatory elements.
2.1. Cyanobacterial genomics
In evolutionary terms, chloroplasts are cyanobacteria (Allen et al.,
2011). The emerging field of marine algal genomics first began with
publications of the three genomes of the smallest known oxygen-
evolving autotroph Prochlorococcus (Dufresne et al., 2003, Rocap et al.,
2003). To date, over 20 cyanobacterial genomes have been released.
Marine cyanobacteria possess several traits in their genome that are
different from other algae. Examples of these differences include their
out-membrane light harvesting antenna, a two-component signal
transduction system, and their autotrophic metabolism. Many cyano-
bacteria havewater-soluble, light-harvestingprotein-pigmentcomplex
phycobilisomes, which can reach a width of 40 nm (Yi et al., 2005) and
ments (chromophores) are phycobilins as opposed to chlorophylls and
covalently bind to their apoproteins. In marine Synechococcus spp, the
genes for the metabolism of phycobiliproteins are concentratedly
distributed in several operons or gene clusters. The cyanobacteria can
intimately attune to ambient light conditions with shifts in the levels
of their phycobilisome composition, i.e., chromatic acclimation (Kehoe
and Gutu, 2006), which can be achieved by the regulations of a two-
component signal system (Gutu and Kehoe, 2012). However, in
Prochlorococcus, most of the genes for phycobiliproteins disappear,
and only a small set of genes for phycoerythrin Type III and their reduc-
tases is conserved in the genome, which suggests that the genes for
phycobilierythrin are being lost through selection in the evolutionary
process (Ting et al., 2001). Compared to the complicated environments
S. Qin et al. / Biotechnology Advances 30 (2012) 1602–1613
marine cyanobacteria may not require an entire, large set of the two-
component (hik & rer genes) signal system. Most genomes of the
marine cyanobacteria that are currently available harbor a very limited
repertoire of hik and rer genes, including only five to six potential
hik genes and seven to 11 potential Rer genes, as compared to the 13
to95 potential hiksand23to94potentialRersoffreshwaterandterres-
trial strains (Ashby and Houmard, 2006; Mary and Vaulot, 2003). In
the N metabolism of the assimilation of ammonium into organic N
compounds, as in other cyanobacteria, marine Synechococcus and
Prochlorococcus strains use the glutamine synthetase glutamate
ever, several Prochlorococcus genomes do not contain genes for the
transport systems of nitrate, nitrite, cyanate, and urea, which are pre-
sent in freshwater cyanobacteria. They also do not contain the coding
for a nitrate/nitrite permease that was recently discovered in a marine
Synechococcus (Dufresne et al., 2003; El Alaoui et al., 2001), which
may be the most surprising discovery regarding substrate utilization
(Scanlan et al., 2009).
2.2. Eukaryotic algal genomics
The emerging field of marine eukaryotic algal genomics first began
with the discovery of the complete sequences for all three remnant
nucleomorph chromosomes of the cryptomonad Guillardia theta
(Douglas et al., 2001). Since then, numerous genomes have been se-
quenced or are in progress for many marine algae. The draft genome
and transcriptome data from red alga Cyanophora paradoxa which
provides evidence for a single origin of the primary plastid in the
Plantae were sequenced and analyzed showing that this basally diverg-
ing algal genome retains ancestral features of starch biosynthesis,
fermentation, and plastid protein translocation common to plants and
algae but lacks typical eukaryotic light-harvesting complex proteins
(Price et al., 2012). The eukaryotic algal genomes were mainly shaped
by the forces of endosymbiotic gene transfer and lateral gene transfer.
Eukaryotic photoautotrophs appeared at least 1.2 billion years ago
when a nonphotosynthetic unicellular eukaryote successfully took in a
cyanobacterium that resulted in a two-membrane photosynthetic
plastid containing a cyanobacterial-derived genome (Reyes-Prieto and
Bhattacharya, 2007). The analyses according to a plastid multi-gene
phylogeny with Bayesian and maximum likelihood phylogenic
methods supports an ancient origin of photosynthetic eukaryotes with
the primary endosymbiosis sometime before 1.558 billion years ago
(Yoon et al., 2004). This primary endosymbiotic event is hypothesized
to have produced three photosynthetic lineages: a green lineage
(Chlorophyll a and b), and the other red and glaucophyte lineages
(chlorophyll a and phycobilins) (Parker et al., 2008). The split of the
red and green algae is calculated to have occurred about 1.5 billion
years ago (Yoonetal., 2004). Secondary endosymbiotic events generat-
ed further diversity among photosynthetic eukaryotes. Dominant
marine eukaryoticalgae (diatoms,
cryptomonads) were derived from a secondary endosymbiosis
symbiosis is calculated to have occurred about 1.3 billion years ago
(Yoon et al., 2004). Tertiary endosymbiosis created a greater diversity
within the dinoflagellates. Taxa, which were derived from secondary
or tertiary endosymbiosis, obtained the plastid genome by retaining
the engulfed photosynthetic eukaryote while the engulfed nucleus
genome was either lost (including transferring several genes to the
host nuclear) or greatly reduced. The plastid genomes vary broadly in
al peridinin-containing dinoflagellates, the plastid genomes have sub-
stantially decreased to one or two genes or even none at all (Zhang
et al., 1999). Cryptomonads (containing a red algal plastid) and
Chlorarachniophytes (green algal plastid) coordinate four genomes
(two nuclear and one each from the plastid or the mitochondria)
(Gilson et al., 2006, Tanifuji et al., 2011). In the remnant nucleomorph
genome of Cryptomonad Guillardia theta, half of the genes code for
and chlorarachniophyte nucleomorph genomes display high gene
densities, and only a few of the genes are involved in plastid functions.
Lateral gene transfer (LGT) occurs independently of an endosym-
biotic event. The first elegant work describing the full impact of LGT
on photosynthetic eukaryotics
rarachniophyte. The plastid target genes that account for 21% of the
nucleus genome of the chlorarachniophyte, Bigelowiella natans were
acquired by LGT from numerous other sources: streptophyte algae,
red algae (or algae with red algal endosymbionts), as well as bacteria
(Archibald et al., 2003). To date, an increasing number of phylogenet-
ic works found that this mechanism plays a key role in the photosyn-
thetic eukaryotic gene and in genomic evolution (Chan et al., 2011;
Keeling and Palmer, 2008). Adjacent genes transferred from
cyanobateria to eukaryotic dinoflagellates by LGT could acquire a
new fusion plastid-targeting peptide (Waller et al., 2006). In the ge-
nome of Ostreococcus tauri, one chromosome was postulated to be
from a different origin than the remainder of the genome according
to the G+C content, the codon usage, the plentiful amount of trans-
posable elements, and a phylogenetic analysis of peptide-encoding
genes (Derelle et al., 2006). Viruses could mediate the LGT, especially
in a marine environment by transduction. It is acknowledged that the
cyanophages (Sullivan et al., 2006), and between viruses and eukary-
otic algae, such as brown seaweed (Cock et al., 2010; Delaroque et al.,
2001) and diatoms (Montsant et al., 2007).
wascompleted inthe chlo-
3. Methods for transformation in marine algae
To date, more than 20 different strains of marine algae have been
expression of transgenes from either the nucleus or the plastid, but in
many cases, only transient expression was observed.
Trans-conjugation is the transfer of DNA between a cyanobacterial
cell and a bacterial cell (usually Escherichia coli) by direct cell-to-cell
contact or by a bridge-like connection between two cells. Although
this method is more frequently used in the freshwater cyanobacterial
genetic manipulation, the versatility of gene transfer by trans-
conjugation in marine cyanobacteria was first demonstrated in five
strains of marine Synechococcus, one strain of marine Synechocystis
was implemented using a mobilizable transposon and a broad-host-
range vector pKT230 (IncQ). This research confirmed the wide applica-
bility of conjugation in marine cyanobacteria. Three typical marine
Synechococcus, WH7803 isolated from shelf waters and WH8102 and
WH8103 representative of open-ocean environments, were successful-
ly genetically manipulatedbya conjugationmethod tointroducebotha
replicative vector and a suicide vector (Brahamsha, 1996). A plasmid
DNA containing green fluorescent protein (GFP) was transferred into
a Prochlorococcus strain by interspecific conjugation with E. coli, and
the expression of this protein was detected by Western blotting and
cellular fluorescence (Tolonen et al., 2006). This is the first report of
GFP expression in oceanic cyanobacteria.
3.2. Natural transformation and induced transformation
Natural transformation and induced transformation could allow a
competence cells or be artificially induced to competence cells whenev-
er they are undergoing exponential growth. In marine cyanobacteria,
S. Qin et al. / Biotechnology Advances 30 (2012) 1602–1613
natural transformation has been reported only for Synechococcus sp.
PCC7002 (Buzby et al., 1985) to date, while others are mainly found in
freshwater strains. Some marine cyanobacteria could be transformed
by competent cycle determination. By treating with ethidium bromide,
a cured strain marine Synechococcus sp. NKBG042902-YG 1116 success-
and Takeyama, 1995). The mechanism of competence in marine cyano-
bacteria may be similar to that in bacteria (Porter, 1986). However, the
transformation efficiencies of the marine Synechococcus strains were
ten times lower than those for the freshwater Synechococcus strains.
Polysaccharides may prevent DNA uptake, which were found surround-
ing themarineSynechococcus cell. Thus, due to the requisition of simple,
quick and highly efficient transformation methods, these two transfor-
mations were rarely used and gradually replaced with electroporation
Gene transfer by electroporation has been applied in various cells
and a high efficiency with a small amount of DNA (Neumann et al.,
1982, Zimmermann et al., 1975). Electroporation can transfer extrinsic
genes independent of the cell's ability and universally to different gen-
era. The firstusage in marine cyanobacteriumwasperformed inmarine
unicellular Synechococcus sp. NKBG042902-YG 1116 (Matsunaga et al.,
1990). The electric field strength for marine cyanobateria was lower
than that for fresh water strains. The decrease in efficiency due to the
electric field strength can be compensated for by enhancing the CaCl2
pretreatment of marine strains. Efficient electroporation-mediated
transformation was achieved in both wild-type and cell wall-deficient
eukaryotic Chlamydomonas reinhardtti strains (Brown et al., 1991).
The efficiency of electroporation was two orders of magnitude higher
than that obtained with the glass beads method to introduce exog-
enous DNA to algal cells (Shimogawara et al., 1998). To date, the elec-
troporation transformation was established in many marine genera
from prokaryotic cells to eukaryotic red algae, green algae and diatoms.
Recently,the“star” marinealgafor potentialbiofuel production, known
as the Nannochloropsis sp., was successfully genetically transformed by
electroporation, and several genes involved in the nitrogen metabolism
were knockout genes by this homologous recombination method
(Kilian et al., 2011). This technology will rapidly advance subsequent
functional genomic research (Pan et al., 2011) and the application in
biotechnologies, particularly the R&D of marine algal bio-energy. How-
constrained by undeveloped protoplast preparation and regeneration
3.4. Biolistic transformation
Direct gene transfer by the biolistic method (micro-particle bom-
ly reproducible in introducing exogenous DNA into algal cells. This
method has been successfully employed for the transformation of
many microalgal nuclear and chloroplast expression systems, and it is
not surprising that biolistic transformation remains the most useful
tool for transgenic studies of marine macroalgae regardless of their
cell walls and life cycle. The biolistic method has the following advan-
tages: ① exogenous DNA can be introduced into various cells and tis-
sues, including plants, animals, microbes, and even pollen and other
peculiar acceptors. It is the only effective method that can repeatedly
transform chloroplasts, mitochondria and other organelles. ② Diversi-
most algae, it is sometimes difficult to construct useful algal endoge-
nous vectors. The vectors from E. coli were usually used in algal biolistic
transformations. Theoretically speaking, the size of theplasmid froman
the plasmid. Furthermore, the developed and diverse vectors from high
plants provide a repertory for future transgenic manipulation design.
③ Although particle bombardment requires specialized and high cost
equipment (gene gun), the manipulation procedure of biolistic trans-
formation is controllable and mature. Nearly all of the physical and
chemical parameters (rupture pressure, DNA concentration, particle
travel distances, and vacuum degree) in the gene gun can be adjusted
to different algal materials and acceptors.
Marine cyanobacterium Synechococcus sp. NKBG15041c was trans-
formed successfully using particle bombardment with bacterial mag-
netic particles (BMPs) purified from one magnetic bacterium known
pholipid layer and can bind larger quantities of DNA than gold or tung-
sten particles (Matsunaga et al., 1991). In addition, a more efficient
transformation was obtained with BMPs than with gold particles. This
experiment demonstrates the advantage of BMPs as a DNA carrier
during biolistic processes for marine cyanobacteria.
To date, the available tools for genetically engineering marine dia-
toms remain sparse. Biolistic transformation is the only efficient tool
to genetically manipulate marine diatoms. A stable nuclear transforma-
tion system was established and developed for several diatoms using
particle bombardment in the 1990s (Apt et al., 1996; Dunahay et al.,
1995; Falciatore et al., 1999). Recently, the utility of several selectable
marker and reporter genes for use in Phaeodactylum tricornutum was
examined (Zaslavskaia et al., 2000). Transformation methods with
particle bombardment have been established for several species of dia-
toms, including the centric diatom Thalassiosira pseudonana (Poulsen
sp. (Miyagawa-Yamaguchi et al., 2011), C. cryptica (Dunahay et al.,
1995), and the pinnate diatoms Navicula saprophila (Dunahay et al.,
Main characteristics of transformation methods used in marine algae genetic
Trans-conjugationIt is mainly in cyanobacteria and rarely used
It is mainly in cyanobacteria and rarely used
It has simple procedure, and is used
universally to different genera but
constrained in brown algae.
cells and tissues. Diversified vectors can be
applied to overcome the genetic background
insufficiency of the substances. The manipula-
tion is controllable and mature. But it requires
specialized and high cost equipment.
The procedure is simple and it doesn't need
high cost transgenic equipment. But it is
constrained in macroalgae due to immature
protoplast regeneration technology.
It overcomes the cell wall's obstruction of
exogenous DNA compared to glass beads
method and it has inexpensive cost. But it re-
quires strict safeguard to avoid the inhalation
Whereas it is a highly efficient and low cost
method but it has complicated and delicate
Exogenous gene could be directionally
integrated into receptor's genome.
It has potential application in brown algae
but still needs extensive and comprehensive
The efficiency is highly dependent on many
elements and this method is technically
Natural transformation and
Silicon carbon whiskers method
Artificial transposon method
Recombinant eukaryotic algal
mediated genetic transformation
S. Qin et al. / Biotechnology Advances 30 (2012) 1602–1613
1995), P. tricornutum (Apt et al., 1996, Falciatore et al., 1999, Miyagawa
et al., 2009, Zaslavskaia et al., 2000), Cylindrotheca fusiformis (Fischer et
al., 1999; Poulsen and Kröger, 2005).
Since 1992, biolistic methods have been applied to marine macro-
algal genetic engineering (Cheney and Kurtzman, 1992). A transient
transformation system has been established in the genera Porphyra
(Kuang et al., 1998, Zhang et al., 2010) whose cultivation currently
provides a yearly turnover of approximately 1×109US dollars (Pulz
and Gross, 2004) and forms a mature algal industry for food, cos-
metics, and other high value products. The five types of acceptor
cells (juvenile sporophytes, male and female gametophytes, tissue
pieces from sporophytes, and parthenogenetic sporophytes) from
brown macroalgae can all be transformed by particle bombardment
(Jiang et al., 2003, Qin et al., 1999). Because bombardment does not
affect the growth and development of female gametophytes, an
expression system for L. japonica gametophytes could be successfully
established (Qin et al., 2005) (expression system will be discussed in
detail in Section 6).
3.5. Glass beads
Agitation with glass beads has been used to efficiently introduce for-
reinhardtii (Kindle, 1990). The advantages of glass beads are their sim-
plicity and independence from expensive and specialized equipment
for Dunaliella salina, which lacks a rigid cell wall, by glass beads has been
successfully established, and the glass beads method is more efficient
and repeatable, more easily controlled and less physically destructive
to cells than electroporation and particle bombardment for thetransfor-
pared at the same time (Feng et al., 2009). The red seaweed Porphyra
leased conchospores, which have either thin cell walls or none at all.
The maximum number of transformants was more than six out of the
1 million agitated conchospores (Wang et al., 2010). The main draw-
back of this method is its inability to transfer DNA into cells with thick
cell walls (Coll, 2006). Cells with thick cell walls should be pre-treated
to digest the walls with an enzyme to become protoplasts. Then, they
can be mixed and agitated with the glass beads and the membrane fu-
sion agent polyethylene glycol (PEG) during agitation. However, in
some seaweeds, cell viability decreases when the cell walls are re-
moved, and the development and the differentiation of callus tends to
complicate the isolation of transformants (Polnefuller and Gibor,
1984). For the state of the art in macroalgal protoplast regenerations,
the list of seaweed species capableof regeneratinginto completeplants
from protoplasts remains limited; however, the list is steadily growing
(Baweja et al., 2009, Reddy et al., 2008).
3.6. Silicon carbon whiskers method
The disadvantage of the glass beads method stated above is that it
requires that a specific cellular and genetic trait (e.g., the cell wall
deficient mutation or thin cell wall) in the host cells achieves efficient
transformation or that the host cells form into wall-deficient cells by
the use of an enzyme (e.g. autolysin). In contrast to agitation of the
cells with glass beads, agitating the C. reinhardtii cells with silicon
carbon (SiC) whiskers for up to 10 min results in a minor loss in cell
viability. The SiC whisker method produced transformants at an effi-
ciency of up to 10−5per cell for walled cells and up to 10−4per cell
for cell wall deficient mutant strains (Dunahay, 1993). Because of its
ability to overcome the cell wall's obstruction of exogenous DNA
and its inexpensive cost, this technique has been attempted in the sta-
ble genetic transformation of marine dinoflagellates. The efficiency
range was approximately 5–24 per 107cells (ten Lohuis and Miller,
1998).Cell viabilityfollowing SiC whisker agitation is greatly improved,
but because of the unreliable source of SiC whisker materials that are
available in small quantities and the inhalation hazard involved in
handling the whiskers (Dunahay, 1993), the glass beads method is
generally preferred (Potvin and Zhang, 2010), while the SiC whiskers
method is not widely used in either freshwater or marine algal genetic
As a directphysicalmethod that isable to penetrate intact cellwalls,
the microinjection method does not necessarily require a protoplast
regeneration system. Additionally, microinjection allows the introduc-
tion of DNA (orother substances) under microscopical control intospe-
cific targets (Schnorf et al., 1991). Theoretically speaking, the receptor
cells of microinjection could be defined as either compartments of a
single cell or as defined cells within a multicellular structure, from
plants, animals, or microbes. However, due to the difficulties of the
ogy,andthe lownumbersof microinjected cells ata given timethatcan
be achieved (Neuhaus and Spangenberg, 1990), the microinjection
method is rarely used in marine algal transformation. A high yield and
stable nuclear transformation was achieved in the marine unicellular
green alga Acetabularia mediterranea by microinjecting SV40 DNA and
pSV2neo into the isolated nuclei of algal cells and then implanting the
injected nuclei into anucleate cell fragments of the same species
(Neuhaus etal.,1986).This established and high yieldmethod wassuc-
cessfully used to tackle the problem of the nuclear transport of algal
procedure, microinjection, could be considered to be a highly efficient
and low cost transformation method for marine algae.
3.8. Artificial transposon method
Transposons, which are mobile DNA elements originally discovered
in maize, have been strong genetic tools for both prokaryotic and
eukaryotic lives (Haapa et al., 1999). Artificial transposons that are
posons have been broadly developed for in vitro mutagenesis and ge-
netic transformation. It is worth emphasizing that although stable
genetic transformation methods have been used successfully in various
genera of marine algae, these techniques have the drawback that, in
most cases, the genes are integrated randomly into the genome. Such
integration may lead to the rearrangement and/or truncation of DNA
sequences, thus causing unintentional changes or silencing the expres-
sion of the foreign gene. The high frequency of transposition by the
artificial transposons method, which can integrate intact foreign DNA
into a receptor cell's genome, could partly avoid or compensate for
the above-mentioned unintentional changes or silencing of foreign
gene expression (Paszkowski and Whitham, 2001, Wu et al., 2011). A
modified transformation strategy was applied using a natural Tn5
transposon (Reznikoff, 2008), a transpose, and a cation liposome com-
plex by electroporation to improve the transformation efficiency for
Spirulina platensis (Kawata et al., 2004), which is one of the most com-
mercially important species of microalgae and can be mass cultured in
a seawater medium by domestication.
3.9. Recombinant eukaryotic algal viruses as transformation vectors
The large dsDNA viruses that are known to infect eukaryotic algae
show promise as genetic vectors in algal biotechnology. The two main
groups of eukaryotic algal viruses are the Chlorella system, which dis-
plays high levels of infectivity and complete pathogenesis (complete
lysis of the unicellular host), and the brown algal virus system by
which only specialized reproductive cells of a multi-cellular free-living
organism are infected and lysed. The viral effect on the entire marine
brown algae, where the genome of the virus is likely integrated, is not
S. Qin et al. / Biotechnology Advances 30 (2012) 1602–1613
pathogenic. The broad host range including other brown algae permits
(Henry and Meints, 1994). There is an early report of transformation by
the microinjection of the marine unicellular green alga Acetabularia
mediterranea (Langridge et al., 1985). More extensive and comprehen-
sive fundamental studies on eukaryotic algal viruses are necessary,
including its genome, infection mechanism, etc., before this exciting
approach can become a reality in the future (Leon and Fernandez,
2007; Van Etten and Meints, 1999).
3.10. Agrobacterium tumefaciens-mediated genetic transformation
A. tumefaciens-mediated genetic transformation genetically trans-
forms plants by transferring and integrating a portion of the resident
Ti-plasmid with the large segment DNA up to 150 kb to a plant nuclear
genome with the assistance of several virulence (Vir) proteins for
T-DNA transfer, nuclear targeting, and integration into the plant ge-
nome (Gelvin, 2000; Tzfira and Citovsky, 2006). The first report of a
stable genetic transformation by A. tumefaciens in algae was conducted
in themarine red seaweed Porphyra yezoensis (Cheney et al.,2001). The
transformation frequency of gene transfer to the nuclear genome of the
freshwater greenalga C. reinhardtii by A.tumefaciens was50-fold higher
than that of the glass bead transformation (Kumar et al., 2004). The
transformation systems in the marine microalgae Nannochloropsis sp.
(Anila etal.,2011; Cha et al., 2011).IntheDunaliellastudy,thetransfor-
mation frequency obtained (41.0±4 cfu per 106cells) was no higher
thanthose reportedfor glass beads transformation and electroporation,
but the transformants obtained were found to be stable for 18 months
(Anila et al., 2011). It was found that cinnamic acid, vanillin and
coumarin produced higher percentages of GUS positive cells in
Nannochloropsis sp. transformations compared to acetosyringone,
providing possible alternative Agrobacterium vir gene inducers that
are more potent than the commonly used acetosyringone (Cha et al.,
The reliable Agrobacterium-mediated transgenic system for trans-
forming large DNA segments (>100 kb) into marine algae would
make it feasible to introduce a natural gene cluster or a set of formerly
separate foreign genes into a single locus of the nuclear genome
(Hamilton et al., 1996). This strategy could provide a sufficient num-
ber of genes for crop protection (crop protection will be discussed in
Section 6) when marine algae are in the outdoor mass-culture and
constitutes an entirely new metabolic pathway for novel bio-molecule
production, metabolic engineering and synthetic biology in algal cells.
However, the efficiency of this method is highly dependent on many
elements, such as the Agrobacterium strain being used, the plasmid
vectors, and the extent of virulence (vir) gene induction. (Opabode,
2006). This method is also technically challenging because of the large
size and low copy number of Ti plasmids, which leads to difficulties in
oped and advanced binary vector systems are attempting to mitigate
these problems and help integrate multiple foreign genes into the
same T-DNA (Lee and Gelvin, 2008, Tzfira et al., 2005). The limited
applicable genera of marine algae indicate the importance of under-
standing the molecular mechanisms of A. tumefaciens-mediated
marine algal (or possibly including freshwater algae) nuclear genome
4. Vector construction and gene selection strategies
4.1. Vector construction: promoter selection and codon usage
Vector element construction is one of the crucial parts in deter-
mining the stability and frequency of exogenous DNA expression in
algal expression systems. In marine cyanobacteria, shuttle vectors,
which are vectors constructed from a cyanobacterial chromosome
segment, and cyanophages are the main possible sources of the vector
backbone. In eukaryotic algae, vectors are typically constructed based
on their own chromosome segment. Additionally, several vectors
from E. coli and a few constructed vectors from high plants are some-
times used in marine algal transformations.
Promoter availability and selection is a critical factor in genetic
transformation. The CaMV35S and SV40 promoters from viruses are
broadly used, particularly when lacking the necessary genetic informa-
tion of the endogenesis promoters in some marine algae (Anila et al.,
2011, Liu et al., 2003, Qin et al., 2004, Wang et al., 2010). Apart from
the universal promoters from viruses, the endogenesis promoters
from specific marine algae are considered to be the most efficient
when constructing a vector. The diatom fucoxanthin-chlorophyll a/c
binding protein gene (fcp) promoter is effective in marine diatoms
and other marine algae (Apt et al., 1996, Li et al., 2009, Miyagawa-
Yamaguchi et al., 2011, Qin et al., 2004, Zaslavskaia et al., 2000). The
duplicated carbonic anhydrase 1 (DCY1) promoter was identified and
used for stable nuclear transformation in D. salina (Li et al., 2010, Lu
et al., 2011). The endogenesis PyAct1 (5′ upstream region of the
actin1 gene from P. yezoensis) promoter was also confirmed to be effec-
tive in the transient gene expression of 12 red seaweed species (Hirata
et al., 2011, Takahashi et al., 2010). In Nannochloropsis sp. transforma-
tion, the endogenous promoters were developed from two unlinked
violaxanthin/chlorophyll a-binding protein (VCP) genes, VCP1 and
VCP2. The VCP1 promoter is a unidirectional promoter, while the
VCP2 promoter is bidirectional (Kilian et al., 2011). The endogenous
promoter of a nuclear encoded plastid-targeted protein Rubisco SSU
and applied to transgenic research (Hirakawa et al., 2008).
Proteins are often difficult to express outside of their original con-
text. They might contain codons that are rarely used in the desired
host, originate from organisms that use non-canonical code or contain
expression-limiting regulatory elements within their coding sequence
(Gustafsson et al., 2004). These problems are typically observed when
transferring exogenesis genes to several genomes of marine algae. As
is the case for most heterologous genes, optimizing the codon usage
of algae-destined transgenes to reflect this bias increases their expres-
sion efficiency by increasing their translation rates and may decrease
their susceptibility to silencing (Heitzer et al., 2007, Potvin and Zhang,
2010). In prokaryotic or prokaryotic-derived genomes, such as chloro-
plasts from eukaryotic algae, codon bias is one of the most critical ele-
ments for protein expression (Surzycki et al., 2009). The importance
of codon optimization in marine algal genetic transformation applica-
tions is increasingly acknowledged in recent transgenic research
(Lerche and Hallmann, 2009, Takahashi et al., 2010). Several free soft-
ware and web applications have recently been developed to estimate
and optimize the codon usage of sequences (Potvin and Zhang, 2010).
Today, the rapidly developing field of synthetic biology is endowing
codon optimization as a necessity, and it is becoming increasingly sig-
nificant for de novo DNA synthesis with other gene design principles
(McArthur and Fong, 2010, Welch et al., 2009).
4.2. Reporter and marker genes
The protein expressed by a reporter gene is sensitive, intuitionistic
and easy to detect; thus,itcan beused to demarcate thetransformation
foreign gene, as well as to determine the protein locality in the trans-
formed cells. The widely used reporter genes in marine algal transfor-
mation are GUS and lacZ. The GUS gene encoding the β-glucuronidase
is typically selected as an effective reporter for transient and stable
sp., Symbiodinium microadriaticum (ten Lohuis and Miller, 1998),
T. weissflogii (Falciatore et al., 1999), Ectocarpus sp. (Cheney and
Kurtzman, 1992), Porphyra yezoesis (Hirata et al., 2011, Kuang et al.,
1998, Liu et al., 2003), Ulva lactuca (Huang et al., 1996), L. japonica
S. Qin et al. / Biotechnology Advances 30 (2012) 1602–1613
(Li et al., 2009, Qin et al., 1994), Undaria pinnatifida (Qin et al., 1994).
However, because the substrate of this genehas an impermeable mem-
brane, it is toxic to the transformed cell and damages the cellular ultra-
structure during the dyeing process. In addition, in higher plants and
marine seaweeds, it has been detected that there was a weak back-
ground of the GUS gene and, thus the negative control must be set to
and is similar to the GUS gene because it is more effective in conducting
anti-bacteria processes and requires a negative and blank control to
eliminate the background. The lacZ essay is applied in many marine
algae, such as red alga P. haitanensis (Zhang et al., 2010) and brown
alga L. japonica (Jiang et al., 2003, Qin et al., 1998). The Luc gene
encoding luciferase is another reporter gene usually applied in a fresh-
water microalgae and the marine diatom P. tricornutum (Falciatore
et al., 1999). The green fluorescent protein (GFP) of the jellyfish
Aequorea victoria has been used as a universal reporter of gene expres-
sion and in subcellular localization analyses in various marine algae
(Hirakawa et al., 2008, Miyagawa-Yamaguchi et al., 2011, Poulsen
et al., 2006, Takahashi et al., 2010, Wang et al., 2010, Watanabe et al.,
2011, Zaslavskaia et al., 2000). However, because algae typically have
endogenous photosynthetic pigments and other fluorescent sub-
stances, the expression of GFP requires a strong promoter to prevent
the interference from the interior fluorescence background.
Transformation protocols require effective selection markers to dis-
criminate successful transformants from transformed cells. The majori-
ty of the selectable markers contain two types: one type includes genes
otics of high plants or anti-herbicides, are most commonly used for the
selection of marine algal transformants. In contrast to high plants,
marine algae are not sensitive to neomycin and kanamycin but are
typically sensitive to chloromycetin, hygromycin, and herbicide
glufosinate. The other type of marker is the homologous complementa-
tion of metabolic mutants. This method may be particularly useful for
chloroplast transformations. Although lists of selectable markers in
microalgae have been compiled in past reviews (Griesbeck et al.,
2006, León-Bañares et al., 2004, Walker et al., 2005), novel markers
have since been developed, such as Phytoene desaturase (PDS)
(Steinbrenner and Sandmann, 2006) and ARG9 genes (Remacle et al.,
2009). There are concerns regarding the biosafety of antibiotic- and
herbicide-resistant genes when releasing the transgenic plants to the
environment, and efforts are being made to develop alternative marker
systems and standardize marker-free selection systems (Manimaran
et al., 2011). The selection marker removal may significantly reduce
the public acceptance of genetically modified plants (Miki and
McHugh, 2004). Several marker elimination methods in higher plants
have been developed during these years including co-transformation,
which is usually applied in Agrobacterium-mediated transformation
(Sripriya et al., 2008) and various site-specific recombination methods
that eliminate the selection marker by deleting or inverting the marker
gene with the help of an enzyme recombinase (Cotsaftis et al., 2002;
Darbani et al., 2007, Kopertekh et al., 2004, Ow, 2002). The marine
algal genetic transformation will certainly meet these biosafety issues
microalgae in an open pond of a coastal area or transgenic macroalgal
sporophytes in the open sea. Because of the differences in the genetic
type between high plants and marine algae, as well as the particularity
of marine environments, the marker elimination methodology applied
in marine algal genetic transformation needs to be considered synthet-
ically, and somechangesneedtobemade basedonthe changesapplied
in high plants.
4.3. Gene copy number and homology-dependent gene silencing
The expression levels of transgenic genes in marine algae are incon-
sistent and difficult to predict. The significant reasons for unpredictable
variation arise from inconsistencies in the number of integrated trans-
gene copies and the subsequent homology-dependent gene silencing.
Single-copy transformants are preferable and desirable because of
their higher expression level and they are much more predictable,
while, to some extent, this expectation is ideal in marine algal transfor-
gene copies, occurs at the transcriptional or post-transcriptional level
and is believed to have originated as a defense mechanism of plants
against viruses and as a means of regulating gene expression
(Baulcombe, 2004; Depicker and VanMontagu, 1997; Marenkova and
Deineko, 2010; Potvin and Zhang, 2010). Electroporation commonly
results in highly variable integrated transgene copy numbers and low-
copy transformants. Agrobacterium-mediated transformation typically
leads to low copy numbers and higher single-copy transformants, as
discussed previously in Section 3. 10. The direct DNA-transfer methods
such as glass beads and biolistic bombardment usually lead to a large
number of integrated gene copies in the receptor algal genome, which
may increase silencing effects. Single or low number copy transgenic
lines can be obtained by transformation cassettes and control the
amount of cassette DNA when using the biolistic method (Lowe et al.,
2009, Yao et al., 2006).
4.4. RNA interference technology
Gene silencing can occur either through repression of transcription,
termed transcriptional gene silencing, or through mRNA degradation,
of its high specificity and efficiency, RNA interference has been proven
to be an invaluable tool for analyzing the biological function of the
target gene and adjusting the metabolism process considerably by
sequence-specific knockdown (Cerutti et al., 2011; Schramke and
Allshire, 2005, Waterhouse et al., 1998). A wide range of core RNAi
machinery components, which promotes the transient gene silencing
and stable gene repression experiments, was identified in the marine
algae, red alga P. yezoensis (Liang et al., 2010), green alga D. salina (Jia
et al., 2009), diatom P. tricornutum (De Riso et al., 2009), T. pseudonana
(Armbrust et al., 2004), and brown alga Ectocarpus siliculosus (Cock et
al., 2010). To investigate the potential of double-stranded RNA interfer-
ence with gene expression in D. salina, a plasmid was constructed to
express hairpin RNA containing sequences homologous to phytoene
desaturase genes that were transformed into D. salina by electropora-
tion for the transient suppression of gene expression (Sun et al.,
2008). RNA interference was applied in P. tricornutum to ensure that
the genes were related to the uridine-5′-monophosphate synthase
process and revealed the potential usage of this gene silencing and
complementation system as a powerful genetic tool for this marine
alga (Sakaguchi et al., 2011).
5. Current progress of photobioreactor technologies for marine
5.1. Marine algal photobioreactor: co-development of the ‘open’ and the
Marine algae is presently the best choice for cell factories of recom-
binant protein productions and the source of future biofuel because
they have simple and inexpensive growth requirements (free seawater,
inexpensive nitrogen, phosphorus, and carbon sources), rapid growth
rates withsufficientlight,andcanbeused inthemarginallandofcoast-
al area or can even be used in the sea. The key components to promote
the algal growth in photobioreactors are nutrients, light, turbulence,
tion for marine algae is the only path for the development and applica-
tion of these bioresources. The photobioreactor design is a key element
for this process. To some extent, the development of transgenic marine
S. Qin et al. / Biotechnology Advances 30 (2012) 1602–1613
algae (or even algal biotechnology) is extremely dependenton the R&D
of the photobioreactor.
Applied phycologists have generally distinguished between open
ponds and closed photobioreactors, where the latter implies that
light does not impinge directly on the culture's surface and that
there is no direct contact between the culture and the atmosphere
(Tredici, 2007) for growing microalgae and the micro-generation or
cells of macroalgae (Rorrer and Cheney, 2004). Open ponds, com-
monly used for ultra thin-layered cascade systems, are extensively
used in algal biotechnology applications, including the raceways,
sloping and cascade systems (Grobbelaar et al., 1995). However,
closed photobioreactors have several advantages over medium
light-path open ponds, including higher light utilization efficiencies,
nutrient uptake, volumetric biomass concentrations, lower compen-
sation light/dark ratios or respiratory losses, less contamination and
competition with alien algae and less water losses during the culture
system (Grobbelaar, 2009).
5.2. Photoioreactor for marine microalgae
A 200-liter unit flat glass reactor was designed to optimize the
outdoor mass production of Nannochloropsis sp. The highest areal
productivity was obtained in a 10 cm light-path reactor. The cost-
effectiveness of this photobioreactor was compared carefully with the
open pond (raceway) for factors including volume, ground area, and
harvested cell density. The optimal population density in the 10 cm
plate reactor was obtained with a daily harvest of 10% of the culture
volume yielding an annual average of ca. 12.1 g dry wt. m−2day−1or
0.24 g m−3day−1(Richmond and Cheng-Wu, 2001). The performance
of the D. salina cultures outdoors in a closed tubular photobioreactor
was assessed. The maximal biomass productivity (over 2 g dry
wt. m−2day−1or 80 g−3day−1) was achieved (Garcia-Gonzalez
et al., 2005). This productivity value is higher than what the same re-
search group carried out in an open pond system demonstrating that
the closed tubular photobioreactor has significant advantages over the
open ponds in the marine algal yield rate (Garcia-Gonzalez et al.,
2003). The efficiency and reliability of closed photobioreactors for
culturing coccolithophorid algae were also evaluated in open raceway
ponds and several types of closed photobioreactors (Moheimani and
Borowitzka, 2006, Moheimani et al., 2011). Although the yield rate or
biomass concentration at harvesting is typically high when using a
closed photobioreactor, the very low cost for construction and mainte-
nance and the fewer overheating problems experienced and super
tive option when considering the cost control and economic income for
large-scale marine microalgal culture.
5.3. Photobioreactor for marine macroalgae
Photobioreactor design is also one of the key elements and can be a
barrier to bioprocess technology for marine macroalgae (Rorrer and
Cheney, 2004, Rorrer et al., 1998). Gametophytic expression systems
for transgenic L. japonica and U. pinnatifida were established using
bubble-column and airlift closed photobioreactors. Two functional
genes (human acidic fibroblast growth factor gene and tachyplesin
gene from Tachypleus tridentatus) were successfully integrated into
algal nuclear genomes and expressed in this system (Deng et al., 2008,
2009). Although the expression efficiency of tachyplesin in L. japonica
gametophtes is lower than the expression efficiency in E. coli and
Bacillaceae expression systems, the expressed recombinant proteins
possess their natural biological structure without a renaturation
process. This trait gives gametophytic expression systems a remarkable
advantage in the cost reduction in the post-translational medication
process, which constitutes a large portion of the cost for transgenic
6.1. Key elements for evaluating marine algal expression systems
The crucial and fundamental issue of marine algal biotechnology is
the ‘seed’ problem. The ultimate aim of marine algal genetic transfor-
mation is to provide future industrialized large-scale cultivation with
seeds that display powerful and attractive traits to produce valuable
products for algal economic feasibility and remove CO2, heavy metal
as bioremediation for their ecological contribution. Several genera of
tions, such as Dunaliella, Porphyridium, Nannochloropsis, Laminaria,
Undaria, Porphyra and Gracilaria. To establish an effective expression
system for marine algae, several factors need to be taken into account:
① economic target, which includes the cost and profit calculations;
tion, selection of transformants; ③ engineering design, which includes
the bioreactor's design, running and maintenance; ④ the safety issues
of transgenic marine algae (Lin et al., 2011).
6.2. Crop protection
Like terrestrial monocultures, large marine algal monocultures will
be invaded by pests and pathogens; thus, crop protection is a major
challenge in large-scale algal cultivation sustainability (Hannon et al.,
2010), which will be increasingly significant with the development
and scale-up of marine algal cultivation. The open photobioreactors
will have more significant challenges regarding contamination than
the closed photobioreactors, which have the potential to minimize
contamination due to their nearly axenic filtrate system for water and
air.However, this comes at a highcapital expense. There are alternative
solutions for crop protection. Antimicrobial peptides, which are
expressed from only a single heterologous gene, provide a solution for
a broad-spectrum ranging from anti-bacteria, -fungi and -protozoan
(Farrokhi et al., 2008; Rahnamaeian and Vilcinskas, 2012). These
peptides have been successfully integrated and transformed into the
nuclei and chloroplasts of higher plants (DeGray et al., 2001, Jan et al.,
2010, Lee et al., 2011). To prevent the prey from invertebrates, several
allelochemicals from cyanobacteria can be introduced and expressed
to defend against potential predators and grazers (Berry et al., 2008;
Rastogi and Sinha, 2009). An ecological method could also be consid-
ered according to the relationship in the ecosystem (Hansen et al.,
1993, Koski et al., 2005).
6.3. Biosafety assessment
The issue of the biosafety of transgenic marine algae contains two
components: one component is to eliminate harm to human health,
and the other component is to be environmentally friendly. For the
human health concerns, the biosafety evaluation could refer to the
methods applied in higher plants (Kikuchi et al., 2008, Maliga and
Maliga, 2006, Oh et al., 2011, Ramessar et al., 2007) to guarantee that
there are no poisonous substances and allergens. However, with the
aim to remove the potential threats to human health from the geneti-
cally transformed marine algae, it is necessary to develop ‘completely
marine algae-derived’ vectors, which includes searching for endoge-
nous promoters, establishing a directional foreign gene integration
platform according to algal homologous recombination, substituting
mutation, and replacing animal virus DNA sequences with algal endog-
enous vectors or algal virus DNA sequences. In addition, it is worth
paying greater attention to the issue in which the transformed gene es-
capes from the marine algal transformation and the expression system
because the cultivation environment of marine algae is an open and
flowing body of seawater. It is critical to assess the transgenic algae
crossbreed with the domestic species/strains by a set of comprehensive
S. Qin et al. / Biotechnology Advances 30 (2012) 1602–1613
observations and detection systems on the status and changes of the
domestic algalgenetic background.L.japonicaistheonlyspeciesof sea-
species endemic to the Sea of Japan and imported to the northern part
relative species. Thus, the biosafety evaluation on transgenic male and
female sporophytes was conducted with safe containers on the sea
and harvested before maturation of the sporangia (Qin et al., 2005).
logical applications. However, the functional complexity of synthetic
devices has been limited by the available design tools (Muers, 2012;
Purnick and Weiss, 2009). The prospect of being able to design and
build novel algal biomolecules and components, metabolic networks,
and even to rewire and reprogram photoautotrophs is extremely excit-
ing. The pilot study was conducted by using the model freshwater cya-
nobacteria Synechococcus elongates for designing two-species synthetic
2011). Cyanobacteria and eukaryotic algae exhibit potential in their
abundant material pool and programmable photoautotroph cells for
the aforementioned design and construction. It is now essential to de-
velop effective strategies for assembling various and complex devices
and modules in marine algal cells into intricate, customizable larger
scale systems, such as an artificial and recombinant photosynthetic
human health, as well as for worldwide energy shortage and global
The authors thank SOA Public Science and Technology Research
Funds Projects of Ocean (Grant No. 20120527 and 200905021-3),
NSFC general program (Grant No. 41176144), CAS/SAFEA International
Partnership Program for Creative Research Teams ‘Typical Process in
Coastal Zone Area and Its Effects on Bio-resources’ (Grant No. KZCX2-
YW-T14), ‘973’ programof China(GrantNo. 2011CB200902), ‘863’ Pro-
Excellent Young and Middle-aged Scientists of Shandong Province
(Grant No. 2010BSB02009), and NSF of Shandong Province (Grant No.
JQ200914 and 2009ZRB02542), who provided the financial support
over the course of this work.
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