REVIEW

Genetic Engineering of Microalgae www.biotechnology-journal.com

Recent Developments on Genetic Engineering of

Microalgae for Biofuels and Bio-Based Chemicals
I-Son Ng,* Shih-I Tan, Pei-Hsun Kao, Yu-Kaung Chang, and Jo-Shu Chang

creating alternative energy, exploring

Microalgae serve as a promising source for the production of biofuels and sustainable agriculture, supporting food
bio-based chemicals. They are superior to terrestrial plants as feedstock in security, preventing disease transmis-
many aspects and their biomass is naturally rich in lipids, carbohydrates, sion, and providing new concept in
education.[1] However, some conflict
proteins, pigments, and other valuable compounds. Due to the relatively slow
exists between continuous economic
growth rate and high cultivation cost of microalgae, to screen efficient and growth and sustainable development.
robust microalgal strains as well as genetic modifications of the available Therefore, the crucial problem is green-
strains for further improvement are of urgent demand in the development of house effect caused by carbon dioxide
microalgae-based biorefinery. In genetic engineering of microalgae, transfor- from combustion of fossil oil, which had
led to the anthropogenic climate change
mation and selection methods are the key steps to accomplish the target
over the past 20 years. Inaction under
gene modification. However, determination of the preferable type and dosage such circumstances will lead to severe
of antibiotics used for transformant selection is usually time-consuming and consequences, including the abrupt rise
microalgal-strain-dependent. Therefore, more powerful and efficient techni- of CO2 by 15%.[2] In order to decrease
ques should be developed to meet this need. In this review, the conventional carbon dioxide emissions and reduce
and emerging genome-editing tools (e.g., CRISPR-Cas9, TALEN, and ZFN) fossil fuels consumption, many efforts
have been devoted to the search of
used in editing the genomes of nuclear, mitochondria, and chloroplast of
alternative energy.[3] Among the existing
microalgae are thoroughly surveyed. Although all the techniques mentioned fossil fuel alternatives, biomass energy
above demonstrate their abilities to perform gene editing and desired (in a recognized life cycle assessment,
phenotype screening, there still need to overcome higher production cost and LCA) seems to have superior advantages
lower biomass productivity, to achieve efficient production of the desired from the perspective of environmental
protection as biomass absorbs carbon
products in microalgal biorefineries.
dioxide during growth, thereby reducing
the atmospheric concentration of carbon
dioxide.[4,5] In addition, biofuel is porta-
1. Introduction ble and can be used in the transportation sector, which is not
possible with other renewable energies like solar energy and
The global population is expected to increase from 6.3 billion wind power.[6] Microalgal biomass and the energy rich
at 2015 to more than 9 billion at 2050. The humanity’s top ten compounds derived from microalgae, such as carbohydrates
problems for next 50 years will be energy, water, food, and lipids have emerged as the most popular feedstock for the
environment, poverty, terrorism and war, disease, education, production of biofuels.[7] Compared with other biofuels
democracy, and population issues. To solve such kinds of feedstock, microalgae do not compete with food crops for
problems, many policies have been developed focusing on arable land or water resources, and they can grow in seawater
or industrial/domestic wastewaters with a relatively fast
Dr. I-S. Ng, S.-I Tan, P.-H. Kao, Dr. J.-S. Chang growth rate than other plants.[8,9] Therefore, microalgae as the
Department of Chemical Engineering non-food biofuel feedstocks are environment friendly resour-
National Cheng Kung University, Tainan 70101,
ces of biomass energy and they could possible reduce
Taiwan
E-mail: yswu@mail.ncku.edu.tw greenhouse gas effect as they account for about 40% of
Dr. I-S. Ng, Dr. J.-S. Chang global carbon fixation. Furthermore, compared to terrestrial
Research Center for Energy Technology plants, microalgae are more appropriate for biofuel produc-
and Strategy tion due to their relatively higher growth rate and higher lipid
National Cheng Kung University, Tainan 70101, content compared to terrestrial plants. The oil content of
Taiwan
microalgae by dry weight is typically in the range of 20–50%,
Prof. Y.-K. Chang while it can reach up to 70% in some microalgal strains.[10] It
Graduate School of Biochemical Engineering
Ming Chi University of Technology, is also known that an enormous proportion of the crude oil is
New Taipei City 24301, Taiwan of microalgal origin, especially from the diatoms.[11] Several
startup companies have been developed in the past few years
DOI: 10.1002/biot.201600644
trying to commercialize microalgae-derived fuels.[12]

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Moreover, microalgae have metabolic pathways for the

production of high value-added compounds, such as carote- I-Son Ng is an assistant professor in
noids,[13] polyunsaturated fatty acid (PUFA),[14] astaxanthin the Department of Chemical
and lutein,[15] which can be applied in a biorefinery concept to Engineering at National Cheng Kung
make the production economically feasible. University (NCKU), Taiwan since
Despite plenty of advantages for microalgae to be an excellent 2014. She received her Ph.D. from
resource for biorefinery, there are still some technical difficulties NCKU in 2005 and then worked for
that need to be overcome before commercializing biofuels and Academia Sinica in 2006; and
biologically active compound(s) from microalgae.[16] Several Xiamen University in 2010. She is
constraints for microalgae biorefinery are as follows: 1) cell devoted to the study of synthetic
stoichiometry or mass balance to shift composition toward the biology, genetic and enzymatic
desired product; 2) higher cost of cultivation and conditions; and engineering, biorefinery, and bioremediation. She has
3) suitable marketing and sale price.[17] Till now, it is estimated developed a robust and efficient platform to explore the
that the cost of a barrel of algae-based fuel using current novel and functional enzymes by genetic and proteomics
technology is greater than US$300, compared with petroleum approach. She has published more than 40 SCI papers
which is available at US$40 to 60 per barrel. The most cost and also created the first iGEM team in NCKU.
incurring part in microalgal biofuel production is cultivation and
the energy intensive harvesting of microalgae, and the strains Jo-Shu Chang is a university chair
used for biofuel production must have high biomass productivity professor in the Department of
and high carbohydrate/lipid productivity. One of the solutions is Chemical Engineering at National
to cultivate microalgae in heterotrophic mode which offers a Cheng Kung University (NCKU),
promising approach to obtain higher biomass and economically Taiwan. He received his Ph.D.
useful metabolites or high-value compounds.[18] On the other degree from University of California,
hand, increasing the biomass productivity by process engineer- Irvine in 1993. His research interests
ing strategies led to the design of expensive photobioreactors for cover biochemical engineering,
stable outdoor cultivation.[19] Flotation systems for harvesting environmental biotechnology, and
and new reactor designs for high biomass productivity have been applied microbiology focus on
considered to overcome these barriers[20] while different microalgae-based CO2 reutilization, biofuels production,
mediums were tested and optimized for the production of the and biorefineries. He plays an important role in Taiwan’s
target products.[21,22] However, the intrinsic metabolic capability biomass energy R&D and policy making. He also serves
of microalgae to accumulate lipids could not be improved by as executive committee member of Asia Federation of
such strategies and lipid productivity is still limited in many Biotechnology (AFOB). He recently became fellow of
microalgal strains.[14] Lipid accumulation is triggered in micro- American Institute of Medical and Biological Engineering
algae when cell division is blocked by the depletion of certain (AIMBE) in 2015 and has received numerous prestigious
nutrients like sulfur or nitrogen, whereas carbon is continuously domestic and international academic awards.
fixed by cells leading to lipid accumulation. As a result, high lipid
accumulation usually cannot be achieved during rapid micro-
algal growth, thereby strongly limiting the lipid productivity of
microalgae. It was also observed that some microalgal species modification.[31–33] There are four main methods being used for
with fast growth rate possess very lower lipid content.[23] Thus, it transformation of microalgae: 1) agitation with glass beads; 2)
seems very difficult to achieve high cell growth rate and high electroporation; 3) particle bombardment; and 4) Agrobacte-
cellular lipid content simultaneously. To overcome the intrinsic rium-construction. Each method has its own advantages and
limitations associated with production of lipids or other function disadvantages based on efficiency, integration, or stability of the
compounds from microalgae or cyanobacteria, a series of studies transgene. Alternatively, the selection system can be based on
have been focused on genetic modification of microalgae to antibiotic resistance or reporter gene selection. Different host
enhance carbon fixation,[24] lipid accumulation[25–27] and high and promoter strength would affect the selection efficiency.[34]
value-added chemical formation.[28,29] Besides, in metabolic engineering, genome-editing and gene-
Genetic engineering recently gained more attentions because interfering tools are important for efficiently targeting the gene.
new and powerful genetic tools are increasingly available, and Recently, Clustered Regularly Interspaced Short Palindromic
the genome can be edited to our need more accurately than Repeats – CRISPR associated protein 9 (CRISPR-Cas9) and
before.[30] The metabolic approach or synthetic biology has Transcription Activator-Like (TAL) Effector Nucleases (TALEN)
received the most attention, as the design of a metabolic pathway which are the new genome-editing tools, as well as Zinc-Finger
in a system where it does not exist opens up new possibilities for Nucleases (ZFN) are used in gene modification.[35–37] Desired
industrialization of microalgae.[31] In synthetic biology, the basic microalgae phenotypes without gene modification could be
gene manipulation process remains the same, including host achieved by gene-interfering tools, such as CRISPR-dCas9,
selection, gene target, plasmid construction, transformation micro RNA (miRNA), and silence RNA (siRNA), to repress or
tools, selection system, and DNA editing tools. activate the gene expression.[38–40] The Omics approach is also an
In microalgae genetic engineering, the transformation and integrative technology for microalgal research in sustainable
selection methods are key steps to accomplish the target gene development.[41]

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In this review, we summarize the basic gene manipulation genes in algal cells, or by using genetic knockout and knockdown
tools in microalgal genetic engineering, including the transfor- strategies to change the metabolic flow to a specific path. In the
mation methods, selection systems, and tools for genome genetic engineering of microalgae, two important steps are
engineering as well as gene expression levels. In particular, the involved: 1) the genetic delivery tools and 2) selectable and
CRISPR-Cas9 gene manipulation in microalgae will be screenable markers. Some successful examples have been
discussed in detail. Moreover, we will delineate the metabolic compiled in the following sections.
engineering of microalgae with an emphasis on biofuel
production, carbon fixation, and lycopene production, where
the targeted function of cells could be enhanced by single gene 2.1. Transformation Techniques
enhancement, metabolic flux redirection, or new pathway
construction. Finally, future perspectives on genetic engineering Gene delivery by transformation to host is an important
of microalgae are also discussed. technique for molecular biotechnological applications, especially
in the production of foreign proteins or modifications of specific
metabolic pathway. A prerequisite for genetic engineering of any
algal species is the formation of reliable and reproducible
2. Microalgal Genome Editing
transformation systems, ideal for both nuclear and chloroplast
Metabolic engineering has generally become a central strategy genomes. Nuclear transformation usually occurs as a random
for optimizing genetic and biosynthetic pathways within cells to insertion of the transgene in the nuclear genome (Figure 1A) by
increase the yield and rate of any metabolite. More than 30 years agitation with glass beads, where the algal cells and the DNA are
ago, the earliest successful DNA modification of C. reinhardtii agitated in the presence of 0.5 mm beads and this is the oldest
was accomplished by Rochaix and van Dillewijn.[42] From then method reported for microalgal transformation.[46,47] The
on, a number of genetic techniques for improving efficiency[43] transgenes would be inserted at random in the nuclear genome
and approaches have been developed for Chlamydomonas.[24] But and selected by antibiotic resistance or phenotypic variation.
very few has been developed for other microalgae, where genetic This method is quite simple and a high ratio of transformed cells
tools are not available or not yet developed. Most successful can be achieved, but using a cell wall-less strain is required.
biosynthetic results are reported for the model eukaryotic algal Electroporation requires the use of electrical impulses to deliver
systems Chlamydomonas and Chlorella[44] or prokaryotic micro- exogenous DNA into cells. It involves specific instrumentation
algae, cyanobacteria,[45] which produced high value and optimization steps, but has the highest rate of nuclear
biocompound(s) or proteins through the expression of certain transformation for microalgae compared with the other

Figure 1. Genetic transformation strategies of microalgae. A) Nuclear transformation by glass beads agitation, electroporation and agrobacterium
transfection, and random insertion. B) Chloroplasts transformation by biolistic and homologous recombination.

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transformation systems.[48–52] In addition, other strategies that transformation of microalgae.[59,66] The benefit of electroporation
have been deemed suitable for nuclear transformation of transformation is well-documented for different electric field
microalgae involve agitation with silicon carbide whiskers,[53,54] intensity on different alga. For example, voltage ranging from 1000
sonication in the presence of polyethylene glycol[55] or using to 2000 (cm 1) for pulse duration around 5 ms is acceptable for
Agrobacterium tumefaciens-mediated transfection to accomplish Chlamydomonas and Chlorella, while voltage at 6000 (cm 1) for
transformation.[56,57] Currently, the most efficient strategy for pulse 50 ms is preferred for Dunaliella salina. All the genetic
delivering foreign DNA fragments into chloroplast genome of modification was random insertion or deletion. Therefore, the
algal cells, where the DNA must traverse multiple membranes, long-term stability of transformants is a great concern and is
is microparticle bombardment with a biolistic particle gun.[58,59] discussed in the next section.
The chloroplast genome is altered by homologous recombina-
tion between the chloroplast DNA (cpDNA) and the foreign DNA
delivered by transformation (Figure 1B). Therefore, the 2.2. Transgenic Microalgal Strains, Stability, and Antibiotic
transgene would integrate into the chloroplast chromosome Resistant
within left border (LB) and right border (RB).
The four major transformation techniques, including bom- Following the successful and diversified genetic transformation
bardment, glass beads, electroporation, and Agrobacterium of numerous algal species, the genomes of microalgae could be
transfection for microalgae are used differently and summarized more easily manipulated. However, the genetic stability after
in Table 1. Bombardment technique is the only successful method transformation and the effect of continuation were a great
to transform chloroplast DNA,[60] but the numbers of trans- concern. As shown in Table S1, Supporting Information,
formants obtained are relatively low (i.e., 100 clones per 106 cells). Chlamydomonas, Dunaliella, Chlorella, and Nannochloropsis,
Bombardment, as a choice for chloroplast transformation, which are demonstrative microalgal species, show high stability
requires a biolistic device for DNA delivery like PDS-1000/He after transformation.[46,67–70] However, other special algae, such
apparatus (Biorad, CA, USA) to accomplish the foreign gene as Thalassiosira weissflogii, Ulva lactuca, Poryhyra miniata,
transformation in chloroplast, where pressure control is the critical Kappaphycus alvarezii, and Gracilaria changii are unstable after
point.[59] Glass beads method is accomplished by vortexing the nuclear transformation.[71–75] The transgenic DNA in chloro-
DNA and bead, which is the simplest method among four of them plast of Chlamydomonas,[58] Chlorella,[76] Porphyridium,[77] and
but the cell wall must be removed or cell wall less strain is Euglena gracilis[78] are quite stable. Till now, the stability is
required.[61] The efficiency is similar to bombardment and highly uncertain in genetic engineering of microalgae. But
operates in very low cost for C. reinhardtii. However, it is not more stable transformants have been reported in nuclear
directly applied in many kinds of microalgae (i.e., Chlorella, transformation.
Nannochloropsis, or Phaeodactylum) because their cell-walls are Two mechanisms are primarily used to screen microalgal
rigid. Agrobacterium assisted transfection is widely used in many nuclear transformants: 1) generating auxotrophic defective
plants but it is still technically challenging for usage in microalgae, mutants and then transforming them with the wild-type authentic
and only have been reported for C. reinhardtii,[56] Haematococcus gene,[79] or 2) integrating a gene that induces resistance to an
pluvialis,[57] Chlorella vulgaris,[62] and oil-bearing marine algae antibiotic or herbicide. Antibiotic screening is the most frequently
Parachlorella kessleri.[63] Except for eukaryotic microalgae, prokary- used approach. For a powerful genetic selection, the resistance
otic microalgae such as cyanobacteria are amenable to genetic genes require high efficiency and stability. However, the type and
manipulation by conjugation or electroporation.[64] It produced the concentration of selectable marker or antibiotic in different
important natural products, such as protein and vitamin or species plays a crucial role, thus these parts of conditional test are
chemical compounds of 3-hydroxypropionic acid[65] and succinate usually time consuming. The success of antibiotic marker for
acid[42] through electroporation. The electroporation, as an genetic transformation in various species of microalgae and
operation friendly equipment and well established protocol, is specific concentration of antibiotics used are summarized in
widely, effectively and dominantly applied for nuclear Table S2, Supporting Information. For commercial vectors (i.e.,

Table 1. Comparison of bombardment, glass beads, electroporation, and Agrobacterium transfection for transforming microalgae.

Glass
Criteria Bombardment beads Electroporation Agrobacterium
Required equipment Complex PDS-1000/He apparatus Simple Complex Gene Pulser Xcell (Biorad) or Gemini Complex
(Biorad) Systems (BTX)
Equipment cost High Low High High
Difficulty of usage Need of specialized Quite easy Easy Technically
challenging
Predominant type of transformation Chloroplast Nucleus Nucleus Nucleus
Removal of cell wall required No Yes No No
Demonstrated presence of exogenous Yes Yes Yes Yes
DNA

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pChlamy), hygromycin is the commonly used antibiotic for single nucleotide would be much easier for design and
selection. Zeocin, chloramphenicol, erythromycin, spectinomy- operation.[82]
cin, and paromomycin are also used successfully for selection in Over the past 20 years, the dominant genetic editing tools
Chlamydomonas reinhardtii, Chlorella sp., Synechococcus, and were zinc finger nucleases (ZFNs)[83] and transcription
Nannochloropsis sp. As shown in Table S2, Supporting Informa- activator-like effector nucleases (TALENs).[84] They are artificial
tion, the concentration of hygromycin and zeocin resistance enzymes made by fusing an engineered DNA-binding domain
ranging from 10 to 20 mg L 1 in different kinds of algae. to the FokI DNA-cleavage domain for targeting specific DNA
Kanamycin, spectiomycin, and chloramphenicol are usually used sequences. Zinc finger domains or transcription activator-like
at 100 mg L 1. It is worth mentioning that the model diatom effectors (TALEs) can be engineered to bind any desired DNA
P. tricornutum, is resistant to higher concentration of antibiotics sequence that then ZFNs or TALENs perform specific cleavage
like zeocin at 100 mg L 1[80] and chloramphenicol at 300 mg at specific locations (Figure S1A, Supporting Information).
L 1,[81] respectively. The promoters CAMV35S and RbcS2 are used Both ZFNs and TALENs have lots of successful academic
frequently for all species. Table S2, Supporting Information, reports on genome editing in plants, insects and mam-
provided an index for screening of genetically modified microalgae mals[85,86] but seldom in the diatom P. tricornutum.[87]
from the proper antibiotics with referred promoter at the Discovered in 2013, the CRISPR-Cas9 system belonging to
beginning. the bacterial adaptive immune system is receiving extensive
attention. A simplified variant of the type-II CRISPR-Cas9
system from Streptococcus pyogenes rely on CRISPR RNA
2.3. CRISPR Technology for Genome Editing in Microalgae (crRNA) and trans-activating crRNA (tracrRNA) or single
synthetic guide RNA (sgRNA) before the protospacer adjacent
Over the past decade, genetic engineering of microalgae motif (PAM) to lead the Cas9 nuclease for triggering double-
developed from delivery of DNA, focusing on transformation, strand breaks (DSBs) in genomic DNA[88] (Figure S1A,
selection, and currently till the most recent CRISPR/Cas9 Supporting Information). The schematic diagram and time-
technique. The timeline of genetic roadmap is shown in scale of development and applicability of the three gene editing
Figure 2. Since 1988, genetic engineering of microalgae has tools including ZFNs, TALENs, and CRISPR/Cas9 system are
been developing from a variety of gene delivery techniques, summarized in Figure S1, Supporting Information. When
including the chloroplast genome and the nuclear genome, compared with ZFNs and TALEN, CRISPR/Cas9 showed
even some improved methods are used to increase the higher applicability but need more whole genomic data to
efficiency of transformation. A breakthrough came in the prevent off-target sgRNA design. The CRISPR interference
2000s, the regulation of microalgae metabolism began to be (CRISPRi) system uses the same design of guide RNA but with
controlled by the developing gene editing strategies such as nuclease-deficient Cas9 (or dead Cas9) which lack the ability to
RNA interference, ZFNs, and TALENs. However ZFNs is more cleave DNA and only function as a DNA binding complex for
challenging to be programmed, as the finger domain is 3–6 gene interference instead of gene modification for gene
nucleotide triplets and the nucleases to which they are attached regulation.[89] The CRISPRi is a new approach, but the concept
function only as dimers, thus the pairs of ZFNs are required to is similar to traditional RNA interference (RNAi) such as siRNA
target any specific locus. TALENs is similar to ZFNs, but and miRNA (Figure S1B, Supporting Information). RNA
instead of recognizing DNA triplets, each domain recognizes a interference is initiated by the enzyme Dicer, which cleaves

Figure 2. Timeline of genetic engineering development in microalgae.

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molecules into short double-stranded fragments called siRNAs 3. Case Studies for Metabolic Engineering of
and split out miRNA. Inside the cell, protein Argonaute 2 (Ago) Microalgae
would bind with RNA-induced silencing complex (RISC) and
induce RNA regulation, causing interference. As RNAi in 3.1. Biofuels Production
microalgae is challenged by low efficiency, non-specific
targeting and silencing of the RNA constraints, the new Biofuels can be produced via thermal conversion, chemical
technology CRISPRi has great potential as it is much conversion, and biochemical conversion of the biomass or its
controllable. From 2014, demonstrations of CRISPR/Cas9- metabolic products. In the case of microalgae, the entire biomass
mediated genome editing in C. reinhardtii cells marked the and extracts could be converted into different forms of biofuels,
beginning of a new age of genome editing in microalgae. Of such as biogasoline, bioethanol, biodiesel, and even jet fuel by
course, the challenge of CRISPR/Cas9 genetic editing for the biorefinery processes.[98] In addition, with higher growth rate
microalgae is the toxicity of Cas9 nuclease, thus the mutation and lipid productivity, microalgae are more appropriate to serve
rate is within 10%.[89] To use Cas9 protein-gRNA ribonucleo- as the biodiesel feedstock with a genetically modified lipid
proteins (RNPs) is an alternative approach to overcome the pathway, including enhancement of single or multiple genes
toxicity of Cas9.[90] The successful clones are quite low and need involved in the production pathway or blocking the competing
further optimization in the future. pathway.[99] There are three main steps in the lipid production
The first assays to demonstrate the CRISPR-Cas9 based gene pathway: namely, malonyl-CoA synthesis, acyl chain elongation,
modification in Chlamydomonas reinhardtii, the model micro- and triacylglycerol (TAG) formation in the sequential order.[100]
algae, showed clear evidence that Cas9 and sgRNA can Also, three existing competitive pathway to lipid production are
successfully express functions in algae, but lack efficiency and the b-oxidation,[101] phospholipid biosynthesis,[102] and the
have low surviving ratio due to the toxicity of vector-driven conversion of phosphoenolpyruvate to oxaloacetate.
Cas9.[89] This extreme problem was solved by directly delivering In the first step of the lipid biosynthesis, acetyl-CoA
Cas9 protein-gRNA ribonucleoproteins (RNPs) into C. rein- carboxylase (ACC) plays a crucial role in metabolic flux to lipid
hardtii to induce mutations at three loci and improved up to 100- biosynthesis since ACC catalyzes the carboxylation of acetyl-CoA
fold compared to the earlier study.[35] In addition, some simple to form malonyl-CoA, which is the first intermediate product to
features of the application have been reported using the same the fatty acid elongation pathway.[103,104] ACC possesses three
method for the knockout of CpFTSY and ZEP two-gene.[90] There activity subunits, including biotin carboxylase activity, biotin
are still other demonstrations of CRISPR/Cas9-based genome carboxyl carrier protein, and carboxyl transferase activity, which
editing of microalgae species. The genome of the marine diatom are composed of several polypeptides encoded by distinct
Phaeodactylum tricornutum can be efficiently edited by using genes.[105] A fully functional ACC is assembled by these three
optimized CRISPR/Cas9 vector.[91] CRISPR technology has also activity subunits with some specific difference amongst
been successfully implemented on Nannochloropsis spp., which organisms.[105,106] So far, many reports on the overexpression
became new model microalgae of carbon sequestration and oil- of ACC from different species in diverse organisms, such as
producing variety.[92] However, the practical use of this bacteria, microalgae, plants, etc., has concluded that single ACC
technology for the production of metabolites from microalgae overexpression indeed increased the activity of ACC and fatty
is not yet demonstrated. Till 2017, CRISPRi is first applied for acid biosynthesis rate because of the increased malonyl-CoA
C. reinhardtii CC400 to enhance lipid production through pool, while the lipid content was not enhanced signifi-
repression of CrPEPC1 gene effectively.[93] For the study of cantly.[103,107] It is suggested that the committed step of lipid
photosynthetic mechanism, cyanobacteria are the evolutionary production catalyzed by ACC is not the rate-determining step in
ancestors of plastids and serve as the conceptual model. some special species or there are secondary rate-determining
Important chemical compounds, such as 1-butanol, ethylene, steps in the lipid production pathway as the expression of ACC
and limonene are reported to be produced by genetically exceeded in some level.[108]
modified prokaryotic microalgae, Synechococcus sp.[94] In recent After malonyl-CoA is synthesized by acetyl-CoA carboxylase
years, very few studies reported application of a CRISPR/Cas9 from acetyl-CoA, a series of reactions for fatty acid production is
genome editing system in the fast-growing cyanobacterium catalyzed by fatty acid synthase (FAS).[109] The FAS is a cascade
S. elongatus UTEX 2973, as a proof of concept for the ability to protein and classified into two types. Type I FAS existing in
produce a marker-free deletion mutant to target the nblA gene.[95] fungi, mammalian, and CMN group of bacteria is a multi-
The CRISPRi applied in Synechocystis sp. PCC 6803 has been subunit protein, while Type II FAS composed of independent
reported for multiple genes repression recently.[38] Moreover, polypeptides encoded by separate genes are found in the archaea,
several studies employed both CRISPR and CRISPRi system bacteria, and plants.[110,111] Regardless of the classification of
not only to trigger the gene regulation, but also achieved FAS, it belongs to the multi-enzymatic family, which including
significantly improved succinate production in S. elongatus PCC malonyl/acetyltransferase (MAT), acyl carrier protein (ACP),
7942 for the first time.[39,96] For enhancement of lactate ketoacyl synthase (KS), ketoacyl reductase (KR), dehydrase (DH),
production, glutamine synthetase (glnA) repression strains enoyl reductase (ER), and thioesterase (TE).[112] First, malonyl-
were obtained from cyanobacterium Synechococcus sp. PCC CoA:ACP transacetylase catalyzes the reaction of adding acryl
7002 via CRISPRi technology without reducing autotrophic carrier protein (ACP) to malonyl-CoA and produces the
growth rates and mutation on chromosome.[97] The successful intermediate product, which the malonyl-CoA-ACP would influx
applications of CRISPR/Cas9 gene editing in algae are into the fatty acid elongation cycle.[109] In the fatty acid
summarized in Table 2. elongation cycle, a series of enzyme-based Claisen condensation

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Table 2. Overview of published studies using CRISPR technology in microalgae.

Microalgae Methods for verifying

species Editing method Target gene the editing Positive contributions Ref.
[89]
C. reinhardtii Vector-driven CRISPR/Cas9 Three exogenous gene: mGFP, PCR/restriction enzyme First study of successful expression
(CC-530) system (C. reinhardtii codon- mGluc, Hygror analysis CRISPR/Cas9 system in microalgae
optimized Cas9)
A endogenous gene: FKB12
[90]
C. reinhardtii Cas9 ribonucleoproteins (RNPs) MAA7, CpSRP43, ChlM Phenotypic characterization Improve the toxicity problem of producing
(CC-124) Cas9 in algal cells
[35]
C. reinhardtii Cas9 ribonucleoproteins (RNPs) CpFTSY, ZEP Phenotypic characterization Knockout two-gene to constitutively produce
(CC-4349) zeaxanthin and improve the photosynthetic
productivity
[91]
P. tricornutum Vector-driven CRISPR/Cas9 CpSRP54 Phenotypic characterization Showed a demonstration of CRISPR editing
(CCMP2561) system (P. tricornutum codon- in the diatom
optimized Cas9)
[92]
N. oceanica Vector-driven CRISPR/Cas9 NR (nitrate reductase) PCR/restriction enzyme Showed a demonstration of CRISPR editing
(IMET1) system (codon-optimized Cas9) analysis and phenotypic in the emerging oil-producing model
characterization microalgae
[93]
C. reinhardtii Vector-driven CRISPR/Cas9 CrPEPC1 PCR/restriction enzyme First successful enhanced lipid by CRISPRi/
(CC-400) system (C. reinhardtii codon- analysis and phenotypic dCas9 in microalgae
optimized Cas9) characterization
[95]
S. elongatus Vector-driven CRISPR/Cas9 nblA deletion Conjugation efficiency Toxicity effect in Cyanobacteria, markless
UTEX 2973 system demonstration
[38]
Synechcocystic Vector-driven CRISPR/dCas9 phaE, glgC PCR/restriction enzyme Showed a multiple genes repression by
sp. PCC6803 system analysis and phenotypic CRISPRi
characterization
[96]
S. elongatus Vector-driven CRISPR/Cas9 ppc, glgc, gltA PCR/restriction enzyme Succinate production enhanced by passages
PCC 7942 system analysis and phenotypic
characterization
[39]
S. elongatus Vector-driven CRISPRi/dCas9 glycogen accumulation (glgc), PCR/restriction enzyme Succinate production in cyanobacterium
PCC 7942 system succinate dehydrogenase (sdhA analysis and phenotypic
and sdhB) characterization
[97]
Synechococcus Vector-driven CRISPRi/dCas9 Yfp, cpc cluster, ccm, glnA PCR/restriction enzyme Regulation of carboxysome to fix CO2 and
sp. PCC 7002 system analysis and fluorescent produced chemical

will be taken place by KS, KR, DH, and ER to form the final condensed into phosphatidate by AGPAT afterward.[102,122,123]
product palmitic-ACP or stearic-ACP.[113,114] Then, palmitic- Subsequently, dephosphorylation of PA would be catalyzed by
ACP or stearic-ACP thioester bonds are hydrolyzed by the TE, phosphatidic acid phosphatase (PAP) to produce diacylglycerol.
which produces palmitic acid or stearic acid.[115] In order to Finally, the diacylglycerol would be esterified into triacyl-
increase the accumulation of fatty acid, KS was stimulated glycerol by diacylglycerol acyl-transferase (DGAT) which is
resulting in changes in cell physiology and lipid profile, regarded as one of the most important enzyme in TAG
decreases in cell growth rate and lipid synthesis rate.[111] On synthesis pathway.[124–126] Due to the importance of DGAT,
the other hand, it is difficult to enhance the fatty acid production overexpression of DGAT has been reported in many plants and
by modification of the pathway owing to the intrinsic properties microorganisms, which reveals that it could increase TAG
of fatty acid synthase (FAS), as the enzyme is composed of many accumulation.[114,127–130] The successful expression of diacylgly-
subunits, thus the modification on any subunit would affect the cerol acyl-transferase gene in Scenedesmus obliquus were
activity of whole FAS.[99] Besides, for the purpose of obtaining enhanced 128% of lipid content.[131] Therefore, the esterification
longer or unsaturated fatty acid, desaturases and elongases were of diacylglycerol catalyzed by DGAT is the rate-determining step
introduced into the fatty acid synthesis, which uses palmitic acid in the lipid synthesis pathway.
or stearic acid as substrates.[116–121] Aside from the overexpression of gene in the lipid synthesis
Final step of lipid production in microalgae is the pathway, another approach is to block the competitive pathways
triacylglyceride (TAG) formation. In glycerol phosphate-based for lipid synthesis. As mentioned earlier, there are three
pathway, glycerol-3-phosphate would be transformed into competitive pathways. One is the b-oxidation pathway which
phosphatidate (PA) by glycerol phosphate acyltransferase is the most straightforward competitive pathway and breaks
(GPAT) and acylglycerolphosphate acyltransferase (AGPAT) down the fatty acid in cytosol or mitochondria and peroxisome
sequentially. GPAT catalyzes the conversion of glycerol-3- for prokaryotes or eukaryotes separately.[101,132] Studies show
phosphate into lysophosphatidate (LPA) which would be that direct knock out of b-oxidation pathway related genes or

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indirect inhibition of b-oxidation pathway by decreasing acyl- 3-phosphoglycerate, but also a side reaction of ribilose-1,5-
CoA transportation system could also efficiently enhance the bisphosphate and oxygen into 2-phosphoglycolate, a toxic
lipid production pathway.[122,133] Another one is the phospho- metabolite for cell growth.[153] Consequently, the phosphogly-
lipid biosynthesis pathway where the phosphatidate would be colate related metabolic product would be recycled by photo-
converted into CDP-diacylglycerol, and influxes into the respiratory pathway in mitochondria and peroxisomes and
phospholipid biosynthesis pathway to form the cell membrane carbon dioxide would be released in the process of photorespi-
instead of TAG formation.[102,134] However, the inhibition of ration.[154] In order to decrease the futile side reaction and
phospholipid biosynthesis pathway leads to abnormally long enhance RuBisCo activity, a series of research on RuBisCo was
fatty acid production because the inhibition of this pathway focused on genetic modification of RuBisCo to enhance its
affects the cell physiology by lack of phospholipids for cell selectivity and velocity.[155] However, limited success was
membrane formation.[135] Another case is the reaction that reported owing to the intrinsic problems of RuBisCo as
converts phosphoenolpyruvate to oxaloacetate. In bio-lipid selectivity and velocity could not be enhanced simulta-
synthesis pathway, phosphoenolpyruvate (PEP) is the important neously.[156] Another approach to enhance carbon fixation is
metabolite that would be converted into pyruvate or oxaloacetate. to heterologously overexpress some natural variants of RuBisCo,
As a general rule, PEP is converted into pyruvate and the especially from red-algae, as it is slightly more effective than
metabolic flux follows TCA cycle or lipid production pathway; others.[157] To identify an efficient RuBisCo from the diverse pool
however, through genetic engineering for the phosphoenolpyr- is more effective than mutation of RuBisCo gene.[156,158]
uvate to be converted into oxaloacetate, catalyzed by phospho- Additionally, assembling different RuBisCo with high selectivity
enolpyruvate carboxylase (PEPC), the metabolic flux would only or catalytic velocity has been reported to enhance carbon
flow into the TCA cycle.[136] Consequently, many reports have fixation.[159] Overall, genetic modification of RuBisCo to increase
exhibited that inhibition of the PEPC activity would enhance the catalytic velocity is preferable than that of improving selectivity,
lipid content by knock down of PEPC gene.[137–141] CRISPRi since the selectivity problem could be overcome by bioreactor
enhanced lipid production up to 94% by successful knock-down design with high concentration of carbon dioxide.[160]
of PEPC in C. reinhardtii.[93] On the other hand, expression of Actually, RuBisCo is not the sole enzyme to enhance carbon
transgenic malic enzyme in P. tricornutum enhanced lipid fixation. Metabolic flux control and regulation of metabolic
productivity by 2.5-fold compared to wild-type, while retaining pathway from Calvin cycle are also important to enhance carbon
the same growth rate.[142] fixation.[153] As for the neighboring pathway of Calvin cycle,
photorespiratory pathway could be bypassed by introducing the
phosphoglycolate rerouting enzyme, which results in a higher
3.2. Carbon Fixation carbon fixation rate and biomass accumulation owing to less
energy consumption in the process of rerouting phosphoglyco-
Carbon fixation is the process by which the autotrophic late.[161] As for the metabolic flux control of Calvin cycle, some of
organisms convert inorganic carbon available from the atmo- Calvin cycle enzymes have been tested, such as sedoheptulose-
sphere to organic compounds. The metabolic pathway for carbon 1,7-bisphosphatase, transketolase, aldolase and so on, in order to
fixation in carbon-fixing organisms is crucial, because the enhance carbon fixation. A successful metabolic flux control for
atmospheric carbon dioxide must be mobilized as energy-rich enhancing carbon fixation by overexpression of sedoheptulose-
organic forms for life on earth. Till now, six autotrophic carbon 1,7-bisphosphatase indeed increased photosynthetic effi-
fixation pathways have been reported. The photoautotrophic ciency.[144,162,163] However, it is not always the enhancement
organisms, such as the plants, algae, and cyanobacteria, absorb in carbon fixation by strengthening all the enzymes involved in
sunlight to convert water and carbon dioxide into organic carbon Calvin cycle.[162] Therefore, the flux balance in Calvin cycle plays
like glucose in chlorophyll by Calvin cycle.[143,144] Other special a critical role in carbon fixation. Through computer-based
photoautotrophic organisms such as purple sulfur bacteria, metabolic pathway construction/analysis by modeling the
convert hydrogen sulfide, instead of water, and carbon dioxide photosynthesis in algae and cyanobacteria or dynamic tracking
into organic carbon while releasing solid sulfurs.[145,146] Except of isotopes, Calvin cycle regulation could be more precisely
for the Calvin cycle, the other five of six carbon fixation pathways controlled to enhance carbon fixation.[144,164] On the other hand,
are the reductive citric acid cycle, reductive acetyl CoA pathway except RuBisCo, thioredoxin regulated enzymes such as
and three relative cycles of 3-hydroxypropionate production sedoheptulose-bisphosphatase (SBPase), fructose-1,6-bisphos-
discovered in some kind of bacterium, Chlorobium, Clostridium, phatase (FBPase), and ribulose-5-phosphate kinase (PRKase) are
Chloroflexus, enabling survival in harsh environment.[147–150] key enzymes in the Calvin cycle which involved in plant
RuBisCo (ribulose-1,5-bisphosphate carboxylase/oxygenase) growth.[165] These enzymes are also supposed to accelerate
is involved in the first step of carbon fixation in Calvin cycle and carbon fixation.
directs the carbon dioxide into Calvin cycle.[151] RuBisCo usually Abiotic factors for carbon fixation also affect the efficiency of
consists of two subunits. One of the subunit is encoded by rbcL carbon fixation. For example, photoautotrophic organisms use
gene or large-chain gene present in chloroplast DNA. Another light as energy to perform photosynthesis. However, excess light
subunit is encoded by small-chain genes, including several would induce photoinhibition, which results in inefficient
related genes in the nuclear DNA. A fully functional RuBisCo is utilization of light and decreased photosynthetic efficiency.[160]
assembled by eight large-chains and eight small-chains into a To reduce photoinhibition, Beckmann et al.,[166] Mussgnug
complex of about 54 000 kDa.[152] RuBisCo catalyzes not only the et al.,[167] and Masuda et al.,[168] have reported that shrinking
conversion of ribilose-1,5-bisphosphate and carbon dioxide to chlorophyll antenna size allows more light transmission and

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higher absorptive capacity of light resulting in higher biomass carotenoids play a crucial role in effective photosynthesis.[177] As
productivity. Another abiotic factor is the concentration of for humans, carotenoids have been reported to prevent some
inorganic carbon. As mentioned above, the concentration of diseases and lung cancer.[178–180] Therefore, carotenoids are
carbon dioxides affects the efficiency of Calvin cycle. Photoauto- becoming attractive in the field of health-promoting foods.
trophic organisms have developed their own carbon concentrat- Nowadays, carotenoids are produced in large quantities and
ing strategies. In algae, carboxysomes and pyrenoids are used to extracted especially from microalgae due to their high
concentrate carbon dioxide by diverse carbonic anhy- productivity and high growth rate.[33,181] In order to enhance
drase.[169,170] Enhancement of carbonic anhydrase (CA) or other the mass production of carotenoids, aside from screening a high
enzymes involved in carbon concentrating pathway may increase carotenoid producing microalgae, metabolic engineering
the carbon fixation rate.[171] One of the example is genetic approaches have been also applied because the pathway of the
S. elongates PCC7942 with CA which accelerates the CO2 fixation carotenoids biosynthesis has been extensively studied and most
as well as biomass productivity.[172] Many environmental factors of the genes have been identified.[182–184]
significantly influence the efficiency of Calvin cycle, especially In the carotenoid synthesis pathway, phytoene synthase (PSY)
metal ions and temperature.[173–176] Therefore, the abiotic factors catalyzes the first step of carbon flux toward the production of
are also critical for carbon fixation. phytoene, which is considered as the rate determining step in
this pathway.[185,186] Afterward, the phytoene would be converted
into lycopene by a cascade of enzyme, phytoene desaturase
3.3. Other Biocompond(s) Production (PDS), z-carotene desaturase (ZDS), and carotene cis-trans
isomerase (CRISCO).[187–189] Lycopene is the most significant
First at all, carotenoids are a vast group of pigments widely found intermediate which would turn on the production of high-value
and synthesized in higher plants and green algae. In carotenoids. Therefore, enhancement of the expression of
photosynthesis, carotenoids serve as light energy absorber and specific genes upstream of lycopene synthesis would indeed
also protect chlorophyll from photodamage, which means that increase carotenoids production. The PSY and PDS were

Table 3. Summary of the bio-chemicals production by transgenic microalgae.

Transformation
Host Target gene Genetic method method Result Ref.
Biofuel production
[107]
Navicula saprophila Acc1 gene from Cyclotella cryptica Over-expression Bombardment 2–3 ACC activity, no change in lipid content
[127]
C. reinhardtii DGAT2 gene from Brassica napus Over-expression Electroporation 1.5 times increase in the lipid content and
change the lipid profile
[130]
P. tricornutum DGAT2 gene from Phaeodactylum Over-expression Electroporation Increase the neutral lipid content by 35%
tricornutum
[139]
C. reinhardtii PEPC1 gene RNA interfere Glass bead Increase the TAG level by 20%
[141]
P. tricornutum PEPCK gene RNA interfere Electroporation 1.5 times increase in the lipid content and
increase the TAG accumulation about 1.1 times
Carbon fixation
[166]
C. reinhardtii Modified NAB1 gene from Over-expression Glass bead Reduction of LHC antenna size by 10–17%
Chlamydomonas reinhardtii (T541A, T676A) and about 50% increase of photosynthetic
efficiency
[172]
S. elongates Carbonic anhydrase (CA) Over-expression Electroporation Carbon dioxide fixation increased 41%
PCC7942
Other chemical compounds
[185]
C. reinhardtii PSY gene from Chlorella zofingiensis Over-expression Glass bead Content of the carotenoids were 2.0- and
2.2-fold increase
[191]
C. reinhardtii PSY gene from D. salina Over-expression Glass bead Increase content of carotenoids by 25–160%
[194]
H. pluvialis Modified PDS gene from Over-expression Bombardment 43-fold higher resistance to the bleaching
Haematococcus pluvialis (L504R) herbicide norflurazon and increase the
astaxanthin production by about 35%
[195]
C. reinhardtii Modified PDS gene from Over-expression Glass bead 27.7-fold higher resistance to the herbicide
Chlamydomonas reinhardtii (L505F) norflurazon and increase the carotenoids
production by about 20%
[198]
C. zofingiensis Modified PDS gene from Chlorella Over-expression Bombardment Produce 32.1% more total carotenoids (TCs)
zofingiensis and 54.1% more astaxanthin
1 [208]
C. vulgaris Human growth hormone Over-expression PEG-mediated 200–600 ng mL of protein in the culture

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highlighted to be the candidate genes for carotenoids produc- challenged as non-GMO products are more favorable for human
tion.[190] Regulation of the PSY gene was investigated in some use, although over 100 Nobel Prize winners claimed that the uses
microalgae, such as C. reinhardtii, H. pluvialis, and of GMO products have no security issues in 2016. This review
P. tricornutum, and the results indeed show an increase in describes the essential strategies of genetic modification of
carotenoids production on PSY gene over-expression.[185,191–193] microalgae, the tools used for gene editing, along with detailed
On the other hand, manipulation of PDS gene expression has discussion of genetically modified microalgae used for various
also been reported to enhance carotenoids production in purposes. In particular, the development of using CRISPR
C. reinhardtii, H. pluvialis, and C. zofingiensis.[194–197] technology for targeted genome editing in certain species of
High-value carotenoids will be synthesized in vivo just after the microalgae and cyanobacteria are summarized. More successful
production of lycopene. Lycopene is first cyclized to form a- or b- and efficient studies need more whole genomic data of microalgae.
type carotene by different lycopene cyclase, which would influence Omics approaches are also in high priority among all the
the carotenoid to be produced. Next, hydrolysis of carotene by techniques provided. As CRISPR technology can further evolve to
different carotene hydrolase would produce lutein and zeaxanthin seamless or scarless cloning without antibiotic marker, it is
from a- and b-type carotene, respectively.[198–200] It has been expected that the CRISPR-Cas9 system holds the potential to
reported that the deprivation of nitrogen also enhanced expression revolutionize future solutions for efficient and precise genetic
of cyclase.[198] However, reports regarding the regulation of cyclase engineering to produce biofuels or other valuable products from
and hydrolase by genetic engineering for microalgae are very few. microalgae in a more efficient and commercially viable way.
Additionally, b-carotene and zeaxanthin could be oxidized to form
more high-value biocompound(s), such as canthaxanthin, viola- Abbreviations
xanthin, or astaxanthin in specific microalgae, H. pluvialis and
C. zofingiensis.[201–204] In this pathway, b-carotene oxygenase (BKT) ACC, acetyl-CoA carboxylase; ACP, acyl carrier protein; AGPAT,
is the key enzyme to be enhanced or introduced into other model acylglycerolphosphate acyltransferase; BKT, b-carotene oxygenase;
CRILSO, carotene cis-trans isomerase; CRISPR, clustered regularly
microbes to produce astaxanthin. BKTwas successfully introduced interspaced short palindromic repeats; Cas9, CRISPR associated nuclease
in C. reinhardtii and astaxanthin was produced as a result.[205] In 9; CRISPRi, CRISPR interference; dCas9, dead Cas9; DH, dehydrase;
this field, most researchers focus on enhancement of carotenoids DGAT, diacylglycerol acyl-transferase; ER, enoyl reductase; FAS, fatty acid
production by changing culture conditions, by introducing some synthase; GPAT, glycerol phosphate acyltransferase; KR, ketoacyl
nutritional or abiotic stress. Enhancement of carotenoid produc- reductase; KS, ketoacyl synthase; LPA, lysophosphatidate; MAT,
tion by genetic engineering is still limited and needs more malonyl/acetyltransferase; miRNA, micro RNA; PAP, phosphatase; PA,
phosphatidate; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate
attention. Moreover, the codon optimization for all genes
carboxylase; PDS, phytoene desaturase; PSY, phytoene synthase; PUFA,
expressed in different microalgae remains significant. polyunsaturated fatty acid; siRNA, small interfering RNA; TE, thioester-
Fast growth rate, high protein content, and FDA-approval for ase; TALENs, transcription activator-like effector nucleases; TAG,
human use are the advantage of Chlorella species. For example, triacylglyceride; ZFNs, zinc finger nucleases; ZDS, z-carotene desaturase.
lutein content is up to 42.0 mg L 1 in C. sorokiniana.[206] C.
pyrenoidosa and C. vulgaris consisted 57 and 58% of protein while Supporting Information
the average of protein content in Chlorella was near 50%.[17,207]
Most successful genetic modification of C. zofingiensis has used in Supporting Information is available from the Wiley Online or from the
author.
stimulating carotenoids,[197–199] other cases are dominating in
heterologous expressed protein in Chlorella species.[44] For
example, the human growth hormone was the first report for
genetically modified C. vulgaris in pharmaceutical use.[208] Acknowledgement
However, the yield was quite low and genetic Chlorella are still The authors are grateful to the financial support for this study provided by
difficult to adapt in industry at the current stage. Some scientific the Ministry of Science and Technology (MOST 105-2221-E-006-225-MY3,
reports on transgenic microalgae to produce versatile biocom- MOST-105-2621-M-006-012-MY3 and MOST-105-2218-E-006-021) in
pound(s) are summarized in Table 3. Taiwan.

4. Conclusion and Prospects Conflict of Interest

The authors declare no commercial or financial conflict of interest.
Currently, microalgae are becoming an ideal biomass feedstock
due to their ability of increased CO2 sequestration, rapid growth
rate, enhanced lipid content, and production of other high-value
compounds like fatty acids and pigments. To reduce the Keywords
production cost of high-value biocompounds from microalgae, CRISPR-Cas9, genetic engineering, microalgae, selection, transformation
new extraction methods, expansion of the use of microalgae and
aquaculture, and the utilization of whole cells need to be Received: February 23, 2017
explored further. Development of high performance Revised: July 24, 2017
microalgal strains by metabolic engineering is significant for Published online: September 18, 2017
making microalgae-derived products economically competitive.
However, the use of genetically modified microalga is still

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