Bioresource Technology 291 (2019) 121932

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Challenges and opportunity of recent genome editing and multi-omics in T

cyanobacteria and microalgae for biorefinery
⁎
Way-Rong Lin, Shih-I Tan, Chuan-Chieh Hsiang, Po-Kuei Sung, I-Son Ng
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan, ROC

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: Microalgae and cyanobacteria are easy to culture, with higher growth rates and photosynthetic efficiencies
Microalgae compared to terrestrial plants, and thus generating higher productivity. The concept of microalgal biorefinery is
Cyanobacteria to assimilate carbon dioxide and convert it to chemical energy/value-added products, such as vitamins, car-
Biorefinery otenoids, fatty acids, proteins and nucleic acids, to be applied in bioenergy, health foods, aquaculture feed,
Gene editing
pharmaceutical and medical fields. Therefore, microalgae are annotated as the third generation feedstock in
CRISPR
bioenergy and biorefinery. In past decades, many studies thrived to improve the carbon sequestration efficiency
as well as enhance value-added compounds from different algae, especially via genetic engineering, synthetic
biology, metabolic design and regulation. From the traditional Agrobacterium-mediated transformation DNA to
novel CRISPR (clustered regularly interspaced short palindromic repeats) technology applied in microalgae and
cyanobacteria, this review has highlighted the genome editing technology for biorefinery that is a highly en-
vironmental friendly trend to sustainable and renewable development.

1. Introduction and harvesting solar power. Microalgae and cyanobacteria have many
promising potentials as they are highly diverse in ecology, metabolism,
Microalgae and cyanobacteria are oxygenic photosynthetic micro- chemical and biological applications. Carbon fixation by microalgae
organisms that have pivotal roles in global biological carbon mitigation, and cyanobacteria accounts for 40% among all photosynthetic plants
oxygen production and nitrogen cycle. A preeminent microbial manu- which implied that they played a key role in energy conversion and
factory can be constructed with cyanobacteria and microalgae which carbon cycling (Pierobon et al., 2018). The prokaryotic cyanobacteria
could generate useful products via capturing CO2 from the atmosphere and the eukaryotic microalgae are quite similar in certain

⁎
Corresponding author.
E-mail address: yswu@mail.ncku.edu.tw (I.-S. Ng).

https://doi.org/10.1016/j.biortech.2019.121932
Received 6 June 2019; Received in revised form 26 July 2019; Accepted 27 July 2019
Available online 30 July 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
W.-R. Lin, et al. Bioresource Technology 291 (2019) 121932

Fig. 1. Conceptual development of biorefinery in microalgae and cyanobacteria through genetic approach.

characteristics. Both of them have photosynthetic organs such as is a green and sustainable alternative for biofuel, health care chemicals,
chlorophyll and chloroplast, which can effectively use photosynthesis nutrients, medical and pharmaceutical materials (Li et al., 2014).
to convert solar energy, water, carbon dioxide and inorganic salts into In the past, the focus was on improvement in integrative process and
organic matter, while fixing carbon dioxide and reducing greenhouse cultural techniques, such as effective photo-bioreactor designs (Shin
impact; the propagation of microalgae and cyanobacteria is usually a et al., 2018), reduce energy consumption for harvesting microalgae in
simple division with a short cell cycle (no complicated sexual re- downstream processes (Cheng et al., 2019a), as well as extraction
production cycle), and the ease in scale-up make the whole biomass techniques for high-value compounds (Chew et al., 2017). The concept
easy to harvest and utilize. Furthermore, both capable to culture in of microalgal biorefinery is used to develop a low-cost, high efficiency
seawater which could be replaced by brine or semi-salt and important and thoughtful use of the whole microbial cell to produce valuable
to obtain effective biomass in the absence of freshwater resources (Chen products. However, the goal of achieving a thriving algal biorefinery
et al., 2017a; Singh et al., 2016). Microalgae are rich in protein, lipids with high efficiency is still a challenge. Due to the rapid development of
and carbohydrates while cyanobacteria contain high amounts of phy- molecular biotechnology in recent years, algal gene technology have
cocyanin. Some species are also rich in pigments, trace elements and expanded from the traditional process to genetic, systematic and syn-
minerals that are the essential food and oil resources for human beings thetic engineering, as well as regulations in the metabolic pathway to
(Li et al., 2018a). Especially for marine one, due to its unique growth attain high rate, high titer and better productivity in a biorefinery (Ng
environment, it synthesizes different kinds of biological materials with et al., 2017).
unique structural and physiological functions. After a certain induction More than 30 years ago, Rochaix and van Dillewijn successfully
method, including physical stresses like pH, photo-intensity, and tem- transformed the yeast DNA into Chlamydomonas reinhardtii (C. re-
perature, or chemical stresses like nitrogen starvation, carbon dioxide inhardtii), completing the earliest microalgae genetic engineering
concentration, and salinity concentration, microalgae and cyano- (Rochaix and van Dillewijn, 1982). However, the development of ge-
bacteria produce much more commercially valuable compounds, which netic technology is limited due to unsophisticated transformation

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Table 1
General genetic components used for Cyanobacteria, Chlamydomonas, Chlorella and other eukaryotic microalgae.
ORI and host Selectable Resistance (mg/L) Promoter RBS for cyanobacter or References
marker reporter gene in
microalgae

Cyanobacteria
pRSF1010 aadA Spc (50–100) Ptrc B0034 Elhai (1993), Englund et al. (2016), Heidorn et al. (2011),
pDC1 nptII Kan (25–100) Ptac B0032 Huang et al. (2010), Ng et al. (2000), Ruffing et al. (2016),
pDU1 cat Cm (10–20) Plac B0030 Shen et al. (2002), Summers et al. (1995), Zhu et al. (2010)
pUH24 J series RBS*
pCC5.2 Tet Synthetic RBS**
rbcL native
Locus A2520
Locus A2579

Microalgae
Chlamydomonas aph7 Hyg (10–25) TUB CaMV35S Ars, gfp, crluc, yfp Baier et al. (2018), Fuhrmann et al. (1999), Kumar et al.
reinhardtii aphH Para (5–30) hsp70A (2004), Lumbreras et al. (1998), Shao and Bock (2008)
aadA Spc (25–100) rbcS2
Sh bla Zeocin (10–20) psbA
U6-26
Chlorella vulgaris cat Cm (100–300) CaMV35S, U6- GUS, gfp, cfp Chow and Tung (1999), Kumar et al. (2018), Niu et al. (2012),
Chlorella neo G418 (30–100) 26, Yang et al. (2016)
sorokiniana aph7 Hyg (20–75) NR
Sh bla Zeocin (10–100)
Nannochloropsis species Sh bla Zeocin (1–10) VCP GUS, egfp Ajjawi et al. (2017), Kilian et al. (2011), Simionato et al.
aph7 Hyg (5–300) TUB, hsp, (2013)
NR, TEF1
Phaeodactylum Sh bla, nat, neo, Zeocin (5–50) FcpA, FcpB, NR, uidA, gfp, CAT, GUS Niu et al. (2012), Zaslavskaia et al. (2000)
tricornutum NTC (50–300)
G418 (30–100)
Thalassiosira pseudonana Sh bla, nat Zeocin (5–50) NTC Fcp, NR gfp Poulsen et al. (2006)
(50–200)

TUB: β-tublin gene, NR: nitrate reductase gene, Fcp: fucoxanthin chlorophyll a/c-binding protein gene, CaMV35S: cauliflower mosaic virus 35S, hsp: heat shock
promoter, VCP: violaxanthin-chlorophyll binding proteins, TEF1: elongation factor 1 alpha-encoding gene, Nourseothricin: NTC.

methods and lack of genetic information. The first completely se- pigments and high-value compounds produced from cyanobacteria and
quenced and annotated genome was the unicellular green alga, C. re- microalgae as a promising resource in biorefinery. Then, genetic en-
inhardtii that was regarded as the model organism. Particle bombard- gineering and genome editing by novel CRISPR system, multi-omics
ment technique has been demonstrated to deliver the exogenous DNA technology including genomics, transcriptomics, proteomics, and me-
into its chloroplast (Boynton et al., 1988). Prior to 2000, many studies tabolomics as well as the biorefinery concept for bio-products in nu-
focused on the model microalgae C. reinhardtii (Harris, 2001). Cur- merous cyanobacteria and microalgae strains would also be particularly
rently, genome sequencing of some microalgae has been completed, mentioned and discussed.
such as C. reinhardtii CC503 (Merchant et al., 2007), and the remaining
genomes of Chlorella variabilis NC64 (Blanc et al., 2010), Chlorella sor- 2. From genetic engineering to gene editing era
okiniana UTEX1602 (Arriola et al., 2018), which laid the foundation for
the genetic modification of microalgae. In the last decade, the rapid Fig. 1 shows the process flow of genetic modifications in microalgae
development of clustered regularly interspaced short palindromic re- and cyanobacteria. Four critical steps are to be considered. Four critical
peats with associated protein 9 (CRISPR/Cas9) appeared to be the most steps are to be considered. First, select a suitable microalgal strain for
efficient and novel technology (Cong et al., 2013; Doudna and the desired product and then search for a gene of interest from a me-
Charpentier, 2013). CRISPR technology has been widely used to opti- tabolic pathway via bio-informative platform like KEGG, NCBI, Biocyc.
mize cellular metabolism, regulate biosynthetic pathways, and increase Afterwards, decide and set up the gene editing technique including
the rate and yield of metabolites. The first CRISPR-Cas9 technology RNAi, ZFN, TALEN and CRISPR system. Finally, analyze and char-
used for gene editing in the model microalgae C. reinhardtii was re- acterize the productivity and properties of chemicals such as biogas,
ported in 2014 (Jiang et al., 2014). bioethanol, biodiesel, pigment, protein or polysaccharides after genetic
On the other hand, prokaryotic cyanobacteria including Nostoc sp. manipulation in the microalgae or cyanobacteria of interest. The
PCC 7120 Synechocystis strains PCC 6803 and PCC 7002, Synechococcus paradigm of genetic approach in cyanobacteria and microalgae applied
strains UTEX 2973 PCC 6301 and PCC7942, with a considerably high in the field of biorefinery is discussed below.
amount of lipid content and a higher growth rate possess a relatively
small genome and have been completely sequenced (http://genome. 2.1. Cyanobacteria
microbedb.jp/cyanobase). Thus, it is a much simpler operating system
for genetic engineering compared to eukaryotic microalgae and several Genetic engineering of cyanobacteria relies on homologous re-
attempts have been made to increase the fatty acid production and combination and neutral sites (Berla et al., 2013). In the model cya-
other green chemicals (Santos-Merino et al., 2018; Eungrasamee et al., nobacteria Synechocystis sp. PCC6803, several loci of neutral sites in-
2019). cluding slr0646 (Xue et al., 2014), slr0168, slr2030-slr2031, psbA1
Scientists have developed a variety of approaches to enhance bio- (Yao et al., 2015a,b), and slr1495-sll1397 had not been shown (Wang
chemical production rate by either overexpression of protein or genetic et al., 2017), while Synechococcus elongatus PCC7942 has three reg-
modification of microorganisms’ genome in the past decades. The ob- ularly used sites as NSI, NSII, and NSIII (Kim et al., 2017). The plasmids
jective of this review is to summarize the potential biofuels (i.e., hy- for engineering of cyanobacteria consisted of a replication origin (ORI
drogen, ethanol, butanol, biodiesel, and lipid), bulk chemicals, in pRSF1010 as broad-host replicons, pDC1 and pDU1 from Nostoc spp.,

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Fig. 2. Genetic elements for (A) prokaryotic cyanobacteria and (B) eukaryotic microalgae. The plasmid of interest was cloned in E. coli and transformed into
cyanobacteria through electroporation, conjugation or nature uptake while the transformation of eukaryotic microalgae was used electroporation, gene gun or glass
bead.

pUH24 and pCC5.2 from Synechocystis spp.) for replication, a selection in the loss of natural transformability in the highly nature-transform-
marker gene (usually an antibiotic resistance gene), and ribosome able Synechocystis PCC6803 (Yoshihara et al., 2001). Li et al. further
binding site (RBS) or synthetic RBS (Gordon et al., 2016) expression transform the pilN gene in novel cyanobacteria which originally lost its
cassettes for protein expression. The general components for genetic natural transformation ability and could recover the ability (Li et al.,
engineering in cyanobacteria are shown in Table 1 and Fig. 2a. 2018a). In other words, transformation of pilN gene could complement
Three main approaches are used to transform the genetic element the lost native pilN component and restore transformability. Among the
into cyanobacteria; natural uptake transformation, electroporation and different methods, electroporation is the common method to introduce
conjugation (Huang et al., 2010; Jin et al., 2018). Natural transfor- foreign DNA into the host. The electroporation condition of cyano-
mation of cyanobacteria has been described for more than 30 years as it bacteria (Koksharova and Wolk, 2002) was similar to E. coli in which
easily introduced the DNA into host. However, natural transformation voltage, capacity, resistance and normal time constant are 12–18 kV/
was restricted to a small number of unicellular strains. Genes encoding cm, 25 μF 200–600 Ω, and 5–15 ms, respectively. Conjugation is the
pili or porin proteins were considered as important proteins for natural most complex method for introducing the DNA into cyanobacteria.
transformation. Yoshihara et al. (2001) has demonstrated that the gene However, due to limited information on natural uptake transformation
pilN encoding pilus assembly protein or competence protein was es- and difficulties of electroporation in the novel cyanobacteria strain,
sential for natural transformability and the knockout of pilN resulated conjugation was developed for cyanobacteria. Conjugation of target

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gene cluster in plasmids from E. coli to cyanobacteria required three expression level (Lumbreras et al., 1998). Apart from this, as the first
plasmids: a conjugal plasmid (i.e. pRL443), a helper plasmid with mob intron was located in the upstream of rbcS2 promoter, the level of an-
gene (i.e. pPMQAK) and a cargo plasmid with a lethal gene (i.e., sacB) tibiotic resistance rises 6-folds dramatically, which suggested the ex-
to be incorporated into the genome. When the conjugal and helper istence of an enhancer element in this particular intron. They also im-
plasmids in E. coli strain HB101 were mixed with cyanobacteria, the plied that the position of the intron influences the expression of ble, and
cyanobacteria would be able to receive a plasmid transfer from the E. that insertion of two copies of the intron has an additive effect.
coli cells (Elhai et al., 1997). The detail processing of natural uptake Recent study observed that the optimal insertion motif of intron was
transformation, electroporation and conjugation of foreign DNA frag- between two guanine nucleotides (NG/GN) (Baier et al., 2018), which
ment to cyanobacteria are shown in Fig. 2a. was consistent with the positive result from Lumbreras et al., as they
Traditional transformations with selectable markers connected to inserted the intron between two guanines. Due to the rich GC content in
any sequence of interest and flanked by homologous arm to neutral Chlamydomonas (∼64%) (Merchant et al., 2007), it was easy to locate
sites on the chromosome are limited by the antibiotic resistance of proper insertion points for the intron. Specht et al. (2010) further de-
cyanobacteria. Berla et al. (2013) had mentioned several strategies monstrated the influence of different intron sequences from rbcS2,
without adding selective pressure from antibiotic resistance (Berla namely intron 1, 2 and 3, and their position upon the expression of
et al., 2013). First, a cassette consists homologous arm, resistance mark recombinant peptides and proteins (Specht et al., 2010). The result of
and a conditionally toxic gene would be introduced into cyanobacteria. intron_1 was in compliance with Lumbreras et al., while intron_3 lo-
Besides, the inducement of pernicious gene magnifies the selectivity on cated in the downstream of rbcS2 promoter led to a dramatic increase of
the plate. Or, by using the empty homologous cassette to replace the gene expression level higher than that of intron_1. However, no further
detrimental gene results in completing the deletion of the original test was performed to clarify the exact function of this intron. In ad-
segments on the chromosome. dition, integration of the three rbcS2 introns in their physiological
Conventional genome editing in cyanobacteria was solely depen- number and order resulted in a 4-folds increment in expression among
dent on homologous recombination. The homologous arm was at least all transformants, for the arrangement was similar to the natural pat-
500 bp and highly recommended to be 700 bp to 1000 bp to get more tern in the genome of wild type.
successful transgenic strains (Ruffing et al., 2016). In genome editing, Despite the wide use of rbcS2-intron system in Chlamydomonas,
targeting size is recognized as the neutral site in which the any mod- there were still some limitations to be overcome, such as how the first
ification (insertion or deletion) would not affect cell growth. The intron effectively enhanced transgene expression in the nuclear genome
marker-less modification could be achieved using the counter-selection, or elucidating the precise activity of intron_3. Moreover, although the
sacB (Lagarde et al., 2000) or recombinase of FRT (Tan et al., 2013) and combination of intron and transgene indeed stimulated its expression
Cre-loxP (Zhang et al., 2007). However, the counter-selection is time level, further enhancement is required to reach the standard of com-
consuming due to a second transformation procedure while in the re- mercial interest. Nevertheless, this system provides a simple strategy in
combinase-based approach; it remained as a scar on the modified the transformation system of Chlamydomonas, while similar technolo-
genome. Finally, due to the polyploidy nature of cyanobacteria, it was gies may be adapted for other microalgae as well as higher plants.
not easy to confirm by colony PCR and would lead to an almost 0%
successful rate. Thus, three patches are needed in order to get a highly 2.3. Chlorella and other species
aggregated strain, which contribute to about 50% successful rate rather
than that by colony PCR (Lagarde et al., 2000). Belonging to the division of Chlorophyta, unicellular microalgae
Chlorella is in the shape of a sphere with sizes ranging from 2 to 10 μm.
2.2. Chlamydomonas reinhardtii With the advantage of rapid growth rate, easily cultivation and scale
up, Chlorella has elicited much interest recently. However, the lack of
Being a model organism, microalgae Chlamydomonas reinhardtii, is genetic resource center and low efficacy of transformation methods has
one of the most well-studied photosynthetic eukaryotes. The thorough hindered the development of genetic engineering of Chlorella species.
and comprehensive understanding of its structure and genomic se- To enhance the transformation efficiency, several reports have been
quence provides researchers a well-established genetic manipulation utilizing plasmid containing left (LB) and right border (RB). Originally,
platform for C. reinhardtii (Harris 2001). However, its characteristic low LB and RB served as homologous flanks in the transfer DNA (T-DNA)
expression of foreign DNA hampered the development of genetic and binary system used in Agrobacterium-mediated transformation which
metabolic engineering in the nucleus of C. reinhardtii (Shao and Bock, permits specific gene exchange into the chromosome of the host
2008). Factors responsible for this may be the epigenetic transcriptional (Fig. 2b). It is widely applied to generate transgenic plants with a short
inactivation of a foreign gene in the nuclear genome, the lack of ap- segment of T-DNA in a tumor-inducing plasmid (Ti-plasmid) that is
propriate promoter/enhancer or an improper codon usage. Previously, transferred from Agrobacterium into the nuclear genome of infected cells
studies have been conducted to improve the poor transgene expression (Gelvin 2000). Nevertheless, Agrobacterium-mediated transformation
rate, including designing synthetic promoter (Scranton et al., 2016), was not a preferred method for microalgae Chlorella due to its unclear
and codon optimization of transgenes (Fuhrmann et al., 1999; Weiner molecular mechanism and was rarely reported in previous genetic
et al., 2018). Nevertheless, the random integration of foreign DNA often studies of Chlorella (Yang et al., 2016). Instead, electroporation was the
resulted in undetectable or unstable expression and the integration sites most commonly used method in Chlorella transformation due to its re-
in nuclear genome also affected the expression level (Weiner et al., latively lower cost, high efficiency and ease of manipulation. It creates
2018). The genetic components of eukaryotic microalgae for random micropores in cell membranes by applying electrical pulses, allowing
insertion of gene are shown in Table 1, while the schematic illustration plasmid with foreign DNA to enter the cell and integrate in the chro-
is shown in Fig. 2b. mosome (Azencott et al., 2007). However, the transformation efficiency
Researchers have found that the insertion of endogenous intron was relatively low in C. reinhardtii (Shimogawara et al., 1998) and C.
sequences in transgene greatly improved the transformation rate in C. vulgaris (Chow and Tung 1999). Kumar et al. (2018) demonstrated
reinhardtii (Kumar et al., 2004). Furthermore, the transcription level successful transformation with plasmid pCAMBIA by electroporation in
increased as the enhancer or repressor element was located within the C. vulgaris, and that green fluorescence can be observed by laser con-
intron (Brooks et al., 1994). Lumbreras et al. (1998) fused the bacterial focal microscope (Kumar et al., 2018). On the other hand, recent report
derived selection marker gene ble with the non-coding regions of rbcS2 showed the plasmid pMDC (which is based on pCAMBIA) assisted
gene of Chlamydomonas. The endogenous introns were introduced into transformation of Scy-Hepc fusion protein into Chlorella for protection
the ble gene, which resulted in at least two-fold increase of transgene against the infection of Aeromonas hydrophila (He et al., 2018).

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Moreover, with the aid of LB and RB in the binary vector pBI121 har- product (i.e. Cas9-RNP) could be applied in any host without additional
boring heterologous genes (GPAT, LPAAT, PAP, and DGAT) related to modification. Finally, to ensure the successful functional genome
the Kennedy pathway, lipid accumulation was enhanced up to 60% (w/ editing in the host, a screening platform was often set up based on
w) in Chlorella sp. (Chien et al., 2015). To sum up, the T-DNA in- phenotypes and selectable markers carried in the plasmid (Fig. 3). For
tegration associated with other transformation methods turned out to further confirmation, the clones would be verified by DNA sequencing.
be a flexible and efficient platform for the genetic engineering of Recently, genome editing by Cas9 or Cpf1 across diverse species of
Chlorella. cyanobacteria has been successfully applied, but the Cas9 possessed
On the other hand, diatoms are referred as a diverse group of uni- more toxicity than Cpf1 (Ungerer and Pakrasi, 2016). Alternatively,
cellular, photosynthetic microalgae, which can be found in both fresh Cas9 could be provided in the non-replicable plasmid without homo-
water and marine system. They are responsible for nearly 20% of global logous region. Within cyanobacteria, the cell doubling time was in the
carbon fixation (Tréguer et al., 1995). Over the years, diatom Nanno- range of 6–12 hr, where Cas9 expression was sufficient enough to ac-
chloropsis species, Phaeodactylum tricornutum and Thalassiosira pseudo- complish genome editing and obtain the marker-free mutant strains
nana had attracted considerable attention in the field of biorefinery (Ma (Behler et al., 2018). With the assistance of Cpf1 nuclease, the homo-
et al., 2014a,b; 4–20; Radakovits et al., 2011), due to their inherent logous arm for cyanobacteria could be shortened to 400 bp (Li et al.,
large amount of lipid accumulation. For example, triacylglycerol ac- 2016). Finally, the marker-less and scar-less modification could be
cumulation of Nannochloropsis can reach 38% of its total biomass on a achieved by serial inoculation of cells in medium without the selective
dry weight basis under nitrogen depletion (Simionato et al., 2013). condition for 10 generation, in which the plasmid curing rate was about
Furthermore, they are also rich in pigment production and high-quality 75% (Wendt et al., 2016). Compared with Cas9, Cpf1 is more favorable
protein (Eilers et al., 2016). All these features and ample genomic in- for application in cyanobacteria for synthetic regulation, large DNA
formation (Hildebrand, 2008) makes them a suitable host for genetic fragment deletion or editing and also it is feasible in broader hosts (Niu
engineering. et al., 2018).
Till now, several transformation methods have been developed for
diatoms, especially for the model organism P. tricornutum. One of the 3. Understanding microalgae and cyanobacteria by multi-omics
commonly used strategy is electroporation, with several successful re-
ports in diatoms and the highest transformation efficiency (∼2500 The rapid development of biotechnology relied on advanced gene
transformants/μg of DNA) by applying a very high electric field manipulation and the multi-omics analysis, leading to the emergence of
strength (∼11,000 V/cm field strength, 50 μF capacitance, and 500 Ω new terms such as synthetic biology, metabolic engineering, and sys-
shunt resistance) (Kilian et al., 2011). Likewise, P. tricornutum was able tems biology. Synthetic biology seeks for the standard biopart in order
to express chloramphenicol acetyltransferase (CAT) gene (Niu et al., to develop a rapid and novel application of biology. With the genomic
2012). The other method is by particle bombardment, which mainly sequence and analysis, the biopart of functional protein has been mined
targeted the chloroplast of microalgae. This technique has been widely from different organisms. Furthermore, transcriptome analysis coupled
used in diatoms for years and was well developed. Zaslavskaia et al. with the genome sequence could predict the strength and inducibility of
(2000) successfully demonstrated the possibility of expressing a variety promoters. Metabolic engineering aims to construct a robust host for
of selectable markers and reporter genes in P. tricornutum by the aid of high-level production of the value-added chemical through genome
particle bombardment (Zaslavskaia et al., 2000). In another diatom, T. editing and genetic circuits design for redirecting the carbon flux, and
pseudonana, it enabled the development of stable introduced gene ex- also involve proteomics and metabolomics at certain conditions. System
pression both in a constitutive and an inducible manner (Poulsen et al., biology utilizes at least two omics analysis to describe a complex bio-
2006). logical system (Choi et al., 2019b). Here, recent omics-contributions on
the research of microalgae and cyanobacteria are summarized below.
2.4. Gene editing era by CRISPR technology
3.1. Genomics analysis
Because the traditional homologous recombination displayed low
efficiency, the CRISPR-assisted method was introduced to increase the Most efforts on genomics analysis have been made with cyano-
efficiency of genome editing. The toolkit based on CRISPR/Cas9, bacteria and Nanochloropsis sp (Lockhart and Winzeler, 2000), because
CRISPR/deactivated Cas9 (dCas9), CRISRP/Cpf1 and Cas9- ribonu- high-gene-density (i.e., less intron) provided the direct gene annotation.
cleoprotein (RNP) are illustrated in Fig. 3. First of all, a 2–6 bp DNA A recent genome-wide comparative analysis to determine codon usage
sequence in terms of protospacer adjacent motifs (PAM) was selected bias and patterns in 41 genomes of cyanobacteria indicated that T-
with a 20-nucleotide complementary guide RNA (gRNA) target se- terminal codons are predominantly located in the genome regardless of
quence adjacent to it. In the case of Cas9-related system, the canonical GC content, and there is no codon usage bias among the genes (Prabha
PAM site was in the form of 5′ –NGG- 3′, while in the Cpf1 system, it et al., 2017). Beside the DNA-aspect comparison, the genome-wide
recognized a T-rich PAM (5′ -TTTTN- 3′). After that, a CRISPR/Cas9 or comparative analysis supported the prediction of proteomes and me-
Cpf1 component was generated and delivered into cells. For Cas9 tabolomics. Transcription factor is an essential protein because it con-
system, sgRNA (tracrRNA::crRNA hybrid) would guide the Cas9 for trols the phenotype of microalgae and cyanobacteria. Hu et al. (2014)
binding and cleavage; on the other hand, the Cpf1 only required a identified the transcription factor (TF) and binding site (TFBS) in the
crRNA. Moreover, the CRISPR/Cas9 system has also been adapted to oleaginous microalgae Nannochlorpsis to demonstrate an in-depth-in-
generate technologies like CRISPRi (CRISPR interference) and CRISPRa terrogation of regulatory links in TAG biosynthesis (Hu et al., 2014). A
(CRISPR activation). Thus, a nuclease-deficient Cas9 (dCas9) was uti- novel pipeline analysis of TF was developed by integrating the big data
lized for binding to the DNA targets rather than creating double and showed almost 99% precision to identify the TF family in several
stranded break (DSB). Combined with repression or activation subunit, eukaryotic microalgae, including Pavlova sp., P. tricornutum, N. gadi-
it could result in transcriptional repression or upregulate the expression tana, P. purpureum and C. reinhardtii (Thiriet-Rupert et al., 2016). Ex-
of the targeted gene. Following the DSB are two general repair path- cept for TF as the target, prediction of protein–protein interaction (PPI)
ways, non-homologous end joining (NHEJ) and homology directed re- based on the genome-based comparison also proofed the biochemical
pair (HDR). For Cas9-RNP, theplasmids containing the Cas9 and gRNA, mechanism. For example, photosynthesis and DNA repair were suc-
each driven by their own promoter were co-transformed into E. coli cessfully studied by prediction of protein–protein interaction (PPI) in
first. After purification, the Cas9-RNP was then delivered into the host. Synechocystis sp. PCC6803. Besides, the protein with unknown function
The importance and advantage of this strategy was that the purified in photosynthesis (ssl3451), organic ion trans-membrane transporter

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Fig. 3. CRISPR technology applications in microalgae and cyanobacteria. Plasmid driven CRISPR system would construct the plasmid of interest in three different
forms as CRISPR-Cas9, CRISPRi-dCas9 and CRISPR-Cpf1 for genome editing or interference. The ribonucleoprotein based method was cloned the Cas9 protein and
sgRNA in E. coli at first. The Cas9-RNP complex would delivery into microalgae by electroporation to obtain the mutants without antibiotic marker.

(sll1252) and sigma factor (ssl0822) have been successfully character- cyanobacteria with doubling time 6.95 h. Ungerer et al. (2018) first
ized (Lv et al., 2015). By the comparative genome analysis, cyano- performed a comprehensive mutational analysis of S. elongatus UTEX
bacteria have been explored to accomodate multiple natural synthetic 2973 and identified three genes, atpA, ppnK, and rpaA with SNP con-
pathways for secondary metabolites, in which the intermediate or final ferring rapid growth. With the allele gene replacing in S. elongatus PCC
products are promising therapeutic potentials, such as for anti-cancer, 7942, the cell growth of S. elongatus PCC 7942 was enhanced, and the
multidrug-reserving, antifungal, antibacterial, anti-flammatory, anti- doubling time of mutated strain was 2.25 h (Ungerer et al., 2018).
viral and potent enzyme-inhibiting bioactivities (Dittmann et al., 2015). Besides, Li et al. (2018b) performed comprehensive gene information
Leao et al. (2017) utilized the comparative genomics approach to figure analysis and found out that the pilN gene with the SNP leading to the
out the potential of cyanobacterial genus Moorea, showing the prolific early translational termination maybe the main reason for the natural
and distinctive gene cluster for novel secondary metabolites while the non-transformable nature. With the allele gene complementation from
compound was still unknown (Leao et al., 2017). S. elongatus PCC 7942, S. elongatus UTEX 2973 recovered its natural
Realizing the difference based on the genomics analysis is the first transformability with transformation efficiency of around 4 × 10−8
step, while the experiment-based characterization would pragmatically colonies/plated cells (Li et al., 2018b). Therefore, enough genomic in-
accelerate the progress of synthetic biology. Two successful examples formation accelerates the application of cyanobacteria in biorefinery.
have been reported based on the comparative genome analysis between
S. elongatus UTEX 2973 and the model cyanobacterium S. elongatus PCC 3.2. Transcriptomics
7942 and it was observed that the identical gene set differ by only 55
single nucleotide polymorphism (SNP), 7.5 kb deletion and 188 kb in- Transcriptomics mainly studies the type, structure, and function of
version, leading to vast difference of phenotype (Yu et al., 2015). Fi- transcripts produced by a cell under certain conditions. For prokaryotic
nally, S. elongatus UTEX 2973 is a naturally non-transformable and fast- cyanobacteria, genomics data could provide enough information for
growing cyanobacterium with doubling time of 2.13 h, but the S. design. However, due to the complex intron and exon system in eu-
elongatus PCC 7942 is naturally transformable and slow-growing karyotic microalgae, genomics data coupled with the transcriptomics

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W.-R. Lin, et al. Bioresource Technology 291 (2019) 121932

data is critical to obtain comprehensive genome information. An olea- microalgae have been reported to evaluate the alterations in transla-
ginous green microalgae Chlorella protothecoides sp. 0710 utilized glu- tional level that provided valuable information for synthetic biology
cose as sole carbon source effectively to accumulate lipid intracellularly and metabolic engineering in microalgae and cyanobacteria. A salinity-
than other well-sequenced microalgae, such as Chlorella variabilis tolerant C. reinhardtii was analyzed by comparative proteomics. Results
NC64A and Coccomyxa subellipsoidea C-169 (Gao et al., 2014). More- showed that high salinity tolerance was contributed from the increased
over, transcriptome coupling with genomic data could serve as a sti- protein abundance involved in membrane transport and trafficking,
muli-response and transcription start site (TSS) characterization. The stress and defense, iron uptake and metabolism, as well as protein de-
notable event was that the light-response mechanism, which was gradation. Notably, the proteomics approach uncovered that several
commonly applied in synthetic biology to program the cell behavior by house-keeping proteins were modified through the putative salinity-
light based on the transcriptome data (Liu et al., 2018). TSS analysis specific post-translational modification process (PTM) in this study
provided the precision design of targeted RNA in length, especially for (Sithtisarn et al., 2017). A lactate-producing Synchocystis sp. PCC6803
the sgRNA design in CRISPR technology to get high on-target efficiency, was reported to show substantial growth retardation, which was in-
as well as prediction of RBS location (Tan et al., 2018). itially attributed to the lactate toxicity. However, through the pro-
Besides understanding of the genome information, the comparative teomics, another reason was proposed as the imbalance of redox couple
transcriptome served as an essential approach to identify mutant strains due to an up-regulated protein involved in dehydrogenation (Borirak
with extraordinary ability. Random insertion of plasmid pGreenI1000 et al., 2015). Coupling the proteomics, transcriptomes and genomics, a
in the non-model green microalgae Dunaliella tertiolecta increased the comprehensive understanding could be obtained, especially for the
lipid content, which was contributing to the gene-upregulation in the difference between transcription and translation (Carrieri et al., 2017).
photosynthesis pathway and inositol phosphate metabolism (Yao et al., Recently, it has been addressed that different changing fold between
2015a,b). Apart from random insertion, a mutant C. reinhardii produ- transcriptomes and proteomics reveal the different translational me-
cing 5.2-fold enhancement on H2 production than wild type was ob- chanism in a CA-deficient Nannochloropsis sp (Wei et al., 2019).
tained by exposure of microalgae to the atmospheric and room tem-
perature plasma (ARTP). A recent successful example was demonstrated 3.4. Metabolomics
in C. reinhardii, in which H2 production was increased up to 5.2 times
than wild type. Further, comparative transcriptome revealed that the Metabolomics is the quantitative and qualitative analysis of all
gene-upregulation of the photosynthesis, including photosystem I, II, metabolites in the organism. The known metabolite concentrations are
cytochrome b6/f complex, electron transporter and ATPase, contributes measured after separation by various chromatographic methods, i.e.,
to improved H2 production (Ban et al., 2019). The alternative approach liquid chromatography, gas chromatography. A glucose-tolerant green
was based on adaptive laboratory evolution (ALE). Two Chlorella strains algae Crypthecodinium cohnii was produced by adaptive laboratory
were mutated and adapted to high phenol tolerance and high flu-gas evolution ALE; through the comparative metabolomics, the glucose
tolerance (Zhou et al., 2017; Cheng et al., 2019b). Chlorella strain with tolerance was attributed to the higher production of glutamate, gly-
high phenol tolerance was examined by comparative transcriptome, cerol, malonate and succinate, which could protect the cells against the
and the results showed that the photosynthesis pathway, antioxidant substrate inhibition, as well as lower production of fructose and xylose,
enzyme and biosynthesis of carotenoids were up-regulated, while the which would cause the osmotic stress (Li et al., 2017). In addition to
flu-gas tolerant Chlorella showed upregulation of photosynthesis, oxi- identifying the known metabolite, nuclear magnetic resonance (NMR)
dative phosphorylation and extracellular sulfur transporter to confer was used to identify unknown metabolites. Through the metabolomics
tolerance to 10% CO2, 200 ppm NOX and 100 ppm SOX (Cheng et al., coupled to NMR, a novel columbamide A was found as a secondary
2019b). metabolite in the distinctive and prolific cyanobacteria Moorea
A carbonic-anhydrase-deficient Nannochloropsis sp. has elevated (Kleigrewe et al., 2015). Furthermore, mass spectrometry can also be
biomass at 5% CO2. Through comprehensive transcriptome analysis, used for isotope analysis to confirm the distribution of metabolic fluxes.
the mechanism of high CO2 tolerance was suggested as the “in-activa- Kanno et al. (2017) engineered Synechococcus elongatus PCC7942 to
tion of carbon concentration machinery” that could generate the hyper- improve glucose utilization, enhance CO2 fixation and increase the 2,3-
CO2 assimilating strain and autonomously containable industrial mi- butadoil production. By metabolomics analysis coupled with the C13
croalgae for flue-gas-based oil production in Nannochloropsis sp. (Wei isotope supply, they demonstrated the importance of the availability of
et al., 2019). Another case was performed in a NobZIP1-overexpressing RuBP for CO2 fixation and chemical production in cyanobacteria
Nannochloropsis sp, which possessed a remarkable elevation in lipid (Kanno et al., 2017). A strategy coupling the genomics, proteomics, and
production and secretion. Comparative analysis on transcriptome in- metabolomics have been applied to systematically understand the cel-
dicated that the overexpression of NobZIP1 conferred the up-regulation lular behavior of 3-hydroxypropionate-producing Synechocystis sp.
of 4 genes located on the Acyl-CoA-derived TGA biosynthesis pathway PCC6803 (Wang et al., 2016b), and ethanol-producing Synechococcus
and down-regulation of UDP-glucose dehydrogenase (UGDH). Re- sp. PCC7002 (Kopka et al., 2017). Recently, in silico modeling of Sy-
markably, the silencing of UGDH altered the cell wall composition, nechocystis sp. PCC6803 for ethanol production was established based
which resulted in secretion of lipid (Li et al., 2019). on trans-omics data (combine genomics, transcriptomics, proteomics
and metabolomics), where based on the modeling, a highly productive
3.3. Proteomics ethanol-producing Synechocystis sp. PCC6803 was constructed
(Nishiguchi et al., 2019).
Proteomics is a systematic study of all proteins expressed by or-
ganisms or cells in a particularly physiological condition for protein 4. Trends in genetic microalgae for biorefinery
sequence identity and quantity. It involves mass spectrometry and li-
quid chromatography to identify the protein via 2-dimensional gel 4.1. Biogas: Hydrogen and ethylene
electrophoresis (2-DE) or gel-free isobaric tags for relative and absolute
quantification (iTRAQ) (D'agostino et al., 2016). In 2014, Yang et al. Hydrogen is one of the most promising clean fuels, since it only
(2014) reported the importance of proteomics in the validation of releases water and produces fairly high heat per mole after combustion.
predicted genes, correlation of initiation and stop-codon positions as Moreover, microalgae could be directly used for combustion for clean
well as revealing the novel protein that was missing in the genome energy (Choi et al., 2019a). The eukaryotic Scenedesmus obliquus, C.
annotation (Yang et al., 2014). Besides, several successful applications reinhardii, and Chlorella vulgaris have the ability to produce hydrogen
of proteome analysis in the bioengineering of cyanobacteria and but only C. reinhardii generated approximately 5 mL H2/L/h via genetic

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W.-R. Lin, et al. Bioresource Technology 291 (2019) 121932

engineering of hydrogen producing enzymes (Khetkorn et al., 2017), photosynthetic microbial-based n-butanol production (Fathima et al.,
but much less than that was produced by cyanobacteria. In the cya- 2018).
nobacteria Anabaena sp. PCC 7120, three types of enzymes partici- Isobutanol has similar prominent properties of butanol. Among the
pating in hydrogen metabolism have been reported: a nitrogenase, a next-generation biofuels synthesized from pyruvate, isobutanol pos-
reversible bidirectional hydrogenase (Hox), and an uptake hydrogenase sesses fewer reaction steps from pyruvate to product than the synthesis
(Hup), where both △hupL and △hupL/△hoxH mutants produced H2 at of n-butanol or biodiesel. Producing isobutanol in Synechocystis sp. PCC
a rate of 3–6 times high than that in wild-type cells (Masukawa et al., 6803 required overexpression of two heterologous genes of the Ehrlich
2002). In Synechocystis sp. PCC 6803, after redirecting the electron pathway and a plasmid harboring the genes kivd and adhA from
supply from the nitrate assimilation pathway by mutating △narB:△- Lactococcus lactis under the control of an IPTG-inducible promoter, Ptac
nirA, a high hydrogen production rate was observed (Baebprasert et al., was constructed (Varman et al., 2013). Since isobutanol is toxic to the
2011). Under anoxic conditions, HydA activity is capable of supporting cells and may also be degraded photochemically by hydroxyl radicals
light-dependent hydrogen production in S. elongatus PCC7942, which during the cultivation process, researchers exploited in situ removal of
contributed to a maximum production rate at 2.8 μmol/h/mg-Chl-a the isobutanol using oleyl alcohol as a solvent. This resulted in a final
(Ducat et al., 2011). For hydrogen production, the outstanding trans- net concentration of 298 mg/L of isobutanol under mixotrophic culture
formation efficiency and clear genomic information from cyanobacteria conditions. However, the expression level of Kivd was significantly af-
supported the gene regulation promoting hydrogen production suc- fected when co-expressed with another gene downstream in a single
cessfully, but rare reports indicated the possibility of hydrogen pro- operon and in a convergent oriented operon. Therefore, the expression
duction in genetically engineered eukaryotic microalgae (Khetkorn of the ADH encoded by codon-optimized slr1192 and co-expression of
et al., 2017). IlvC and IlvD were identified as potential approaches to further enhance
Recently, genetic ethylene producing cyanobacterium has been ex- isobutanol production (Miao et al., 2018).
plored by the xylA and xylB genes from E. coli, which was required to
regulate xylose utilization (Lee et al., 2015). By introduction of both 4.3. Lipids
xylAB and xylose transporter xylFGH into an ethylene-producing strain
Synechocystis sp. PCC 6803, the rate of ethylene production was much The diverse definition of microalgal lipids includes cholesterol, fatty
improved in the presence of xylose. On the other hand, introducing a acids, triglycerides and phospholipids. Fatty acids and phospholipids
codon-optimized ethylene-forming enzyme (EFE) from Pseudomonas are more valuable and attractive. In cyanobacteria, the metabolic
syringae in Synechocystis sp. PCC 6803 achieved higher ethylene pro- pathway for fatty acid and phospholipid synthesis involved the trans-
duction with a volumetric production rate of 9.7 mL/L/h (Zhu et al., formation of acetyl-CoA (acc) to Malonyl-CoA by acetyl-CoA carbox-
2015). Another example was ethylene forming enzyme from Pseudo- ylase (accABCD), followed by the 2-step initiation reaction of forming
monas syringae (sy-efe), that was introduced in two Synechococcus β-ketoacetyl-ACP by fabD and fadH and the elongation cycle. In the
strains, the ethylene productivity remained stable and reached the elongation cycle, a series of reduction (catalyzed by FabG), dehydration
highest recorded yields of 140 µL/L/h/OD750 (Carbonell et al., 2019). (FabZ and FabA), reduction (FabI) and elongation by condensing ad-
ditional malonyl-ACP molecules (FabB and FabF) would take place. It
4.2. Biofuel: Ethanol and butanol has been reported that the reaction catalyzed by 2,4-dienoyl-CoA re-
ductase (fadH) is the rate-limiting step in the fatty acid synthesis by
With respect to bioethanol produced by engineered cyanobacteria, Synechococcus sp. PCC 7002 (Kuo and Khosla, 2014). Eungrasamee
S. elongates PCC7942 co-expressing ictB, ecaA, and acsAB and co-fer- et al. (2019) recently engineered Synechocystis sp. PCC 6803 with
mentation with Z. mobilis enables the cyanobacteria to reach the highest overexpression of acyl-ACP synthetase to recycle the free fatty acid, and
ethanol production of 7.2 g/L (Chow et al., 2015). In another case, by the engineered strain resulted to maximal lipid content and production
introducing the exogenous pyruvate decarboxylase from Z. mobilis and rate of 34.5% in DCW and 41.4 mg/L/day in the engineered strain
over-expression of an endogenous alcohol dehydrogenase (slr1192) in (Eungrasamee et al., 2019). Besides, in order to get long-chained un-
Synechocystis sp. PCC6803, the production of poly-β-hydroxybutyrate saturated fatty acid, Santos-Merino et al. (2018) reported that the β-
was alleviated and a final ethanol concentration of 5.5 g/L over 26-days ketoacyl-ACP synthase II (fabF) overexpression, deletion of fadD, en-
was acheived (Gao et al., 2012). The similar concept was adapted in coding acyl-CoA synthetase that would compete the flux from the
Synechococcus sp. PCC7002 with integration of the pdc-slr1192 in elongation cycle to β -oxidation, and introduction of delta(12)-fatty-
pathway on a neutral site NS0027 and ethanol productivity was suc- acid desaturase (desA and desB) into Synechococcus sp. PCC 7002 which
cessfully endowed (Wang et al., 2019). Among the different biofuel enhanced the levels of omega-3 fatty acid (Santos-Merino et al., 2018).
targets, n-butanol has received significant attention for its suitability in Most eukaryotic microalgae grow faster and have higher biomass;
the current infrastructures as a chemical feedstock. However, en- thus, a growing number of studies have focused on the genetic en-
gineering cyanobacteria to produce n-butanol has been challenging as gineering of microalgae (Jeon et al., 2017). For example, the exogenous
the metabolic pathway for synthesizing n-butanol comes from strict Acyl-ACP thio-esterase gene was transformed into Phaeodactylum tri-
anaerobes and the metabolism is very different from cyanobacteria. cornutum to increase the production of shorter chain length fatty acids,
Since competing ethanol synthesis would occur after introducing the which are more desirable for biofuel application (Radakovits et al.,
individual polyhedral-body-associated CoA-acylating aldehyde dehy- 2011). P. tricornutum was genetically manipulated by antisense
drogenase important for 1,2-propanediol degradation by S. enterica knockdown of a key gene, pdk involved in lipid metabolism without
(PduP) enzymes with NADPH-dependent alcohol dehydrogenase compromising the biomass (Ma et al., 2014b). Pyruvate dehydrogenase
(YqhD) into S. elongates PCC 7942. A synthetic operon was expressing kinase (PDK) deactivated the pyruvate dehydrogenase complex which
acetoacetyl-CoA synthase (NphT7), acetoacetyl-CoA Reductase (PhaB), catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. The
(R)-specific enoyl-CoA hydratase (PhaJ), and YqhD in the malonyl-CoA neutral lipid content in dry cell weight of transformants was obtained at
dependent pathway, and a PduP homologue from S. enterica under the 42.1%, while there was no much impact on the fatty acid composition
control of an IPTG inducible PLlacO1 promoter was constructed (Lan and and growth rate compared to wild type. A multifunctional lipase/
Liao, 2012). In addition, the accumulation of acetyl-CoA suggested that phospholipase/acyltransferase was knocked down in the diatom Tha-
ACCase from Yarrowia lipolytica was the rate-limiting step in the n-bu- lassiosira pseudonana to enhance lipid content without affecting its
tanol pathway. Thus, the DC11 strain additionally integrated ACCase growth (Trentacoste et al., 2013). The transgenic strains revealed a 2.4-
and reached a production titer of 418.7 mg/L ethanol at 6-days culture. to 3.3-fold increase in lipid content during exponential growth and up
The resulting strain showed promise for future application in to 4.1-fold compared to wild type after 40 h of silicon starvation. By

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introducing a disrupted ω-3 fatty acid desaturase (fad3) into Chlorella backbone structures and cyclic groups, which can be divided into two
vulgaris, it was able to accumulate over 30% of total lipid content and broad categories including carotenes, such as α-carotene, β-carotene
increase the proportion of C16:0, saturated fatty acid (SFA) without and lycopene, and the other was xanthophylla containing of lutein,
affecting its growth (Lau et al., 2017). zeaxanthin and violaxanthin (Zhang et al., 2014). The main car-
The first report for applying CRISPRi system in cell wall deficient C. otenoids, such as α-carotene, β-carotene and lutein, are mainly directly
reinhardtii CC400 down regulated the phosphoenolpyruvate carbox- involved in photosynthesis reactions, which are the key to cell survival.
ylase (PEPC) gene related to the tricarboxylic acid (TCA) cycle (Kao and Secondary carotenoids such as astaxanthin, canthaxanthin would be
Ng, 2017). This approach has effectively driven the carbon flux from expressed as a response to specific environmental stimuli (Orosa et al.,
TCA cycle of protein production towards the formation of fatty acid. 2000). For example, a phytoene synthase (PSY) gene from Dunaliella
Results suggested that the lipid content and productivity of transfor- salina was cloned into C. reinhardtii. PSY is a key enzyme in the car-
mants was enhanced up to 28.5% DCW and 34.9 mg/L/day. Attention otenoids pathway and can regulate the carbon flux to the synthesis of
for the industrial alga Nannochloropsis spp arised from their ability to carotenoids (Couso et al., 2011). The expression of exogenous PSY in C.
accumulate high amount of triacylglycerol (TAG) and poly-unsaturated reinhardtii led to a significant increase in the content of carotenoids with
fatty acids (PUFAs) (Ma et al., 2014a). However, lipid production in a level of 1.8- and 2.6-fold higher than untransformed cells. On the
microalgae Nannochloropsis gaditana could be maximized under nu- other hand, insertion of mutant norflurazon-resistant PDS gene into
trient deprivation condition, but this often led to poor growth rate. Chlorella zofingiensis greatly improved the desaturation activity, hence
CRISPR/Cas9 system was to accomplish the genome editing in the increased the total carotenoid content and the accumulation of astax-
model oleaginous microalga, Nannochloropsis sp., recently (Wang et al., anthin (Liu et al., 2014). Recently, high-intensity light exposure to
2016a). A CRISPR-Cas9 based reverse-genetics pipeline was developed Chlamydomonas sp. JSC4 caused a higher lutein accumulation which
and was used to identify a lipid regulator named ZnCys in N. gaditana was due to the up-regulation of 2 in the lutein pathway, which implied
(Ajjawi et al., 2017). The lipid accumulation in gene knock-out trans- that physical stress affects biological functions in microalgae (Ma et al.,
formants was improved to 40–55% in nutrient-replete condition, while 2019).
lipid productivity was doubled to 5.0 g/m2/day under semi-continuous Limonene is a 10-carbon isoprenoid produced by plants which
growth conditions without affecting the growth capabilities (Ajjawi mainly found in citrus fruits which gives the characteristic scent of
et al., 2017). orange or lemon. In fact, limonene is being evaluated for chemo-pre-
ventative or anticancer effects. A new use for limonene is as a third-
4.4. Bulk chemicals generation biofuel, especially in jet fuel and diesel applications due to
its immiscibility in water, combustibility, high energy density, and low
Bulk chemicals from microalgae consist of acids, esters, and freezing point. Regarding to previous studies, limonene synthase (lims)
polymer-related compounds. The maximal lactic acid production rate is from Citrus limon and Mentha spicata produce limonene of high purity.
achieved in Synechocystis sp. PCC6803 with SAA023 (Ptrc::ldhco), that The codon-optimized lims was introduced into Synechocystis sp. PCC
is, a production rate of 0.0175 mmol lactic acid/g-DCW/h with LDH 6803 which up-regulated ribose 5-phosphate isomerase (rpi) and ribu-
under control of the trc promoter (Angermayr and Hellingwerf, 2013). lose 5-phosphate 3-epimerase (rpe) genes in the PP pathway, and a
Afterwards, a lactate dehydrogenase (LDH) strain SAW039 based on the geranyl diphosphate synthase (gpps) optimize the limonene biosynthetic
design with SAA023 plus ldh, the production rate of L-lactic acid re- pathway and limonene concentration reached 6.7 mg/L (Lin et al.,
sulted to 5.6-fold increase in Synechocystis sp. PCC6803 (Angermayr 2017). Insertion of the mutant sequences (Ptrc-ls) at neutral site of S.
et al., 2014). In the step prior to the LDH-catalyzed reaction, pyruvate elongatus PCC 7942 enhanced limonene content of up to 92.2 μg/g-
kinase (PK) converted phosphoenol pyruvate (PEP) into pyruvate, DCW/h. But photosynthesis limitations were shown that the strong li-
making PK an attractive target for overexpression. Finally, the lactic monene sink led to NADPH accumulation and slowed down photo-
acid concentration increased from 5.2 in SAW039 to 9.3 mmol/L in synthesis electron flow (Wang et al., 2016a,b).
SAW041 (Angermayr et al., 2014). By using CRISPRi down-regulation β-Phellandrene is a monoterpene with commercial value as a key
of the ccm operon in Synechocystis sp. PCC 7002, a 2-fold more lactate ingredient in synthetic chemistry, medical, cosmetic and cleaning pro-
was produced than the original strain (Gordon et al., 2016). Recently, ducts, and potentially as a fuel. The CpcB-PHLS fusion protein (cya-
genes essential for glycogen accumulation (glgC), succinate conversion nobacteria phycocyanin β-subunit following β-phellandrene synthase)
to fumarate (sdhA and sdhB) targeted by CRISRPi in S. elongatus PCC retained the activity of the PHLS enzyme and catalyzed β-phellandrene
7942 reduced the expression of the concerned genes and glycogen ac- synthesis (Formighieri and Melis 2015). However, catalysis of hetero-
cumulation and significantly enhanced the succinate titer up to logous terpene synthesis in cyanobacteria was in the past compromised
0.63 mg/L (Huang et al., 2016). In the model eukaryotic C. reinhardtii, by low levels of transgenic terpene synthase expression. A recent study
synthesis of poly‐3‐hydroxybutyrate (PHB) required three key enzymes mentioned that fusion of the transgene to a highly expressed gene in
which are encoded as phbA, phbB and phbC. The transgenic strain of C. cyanobacteria could be a means to substantially enhance transgene
reinhardtii revealed that the amount of polyhydroxybutyrate (PHB) can translation and recombinant β-phellandrene accumulation as accom-
be produced up to 6 μg/g-DCW (Wang et al., 2010). However, the re- plished by geranyl diphosphate synthase (gpps) fusion with the kana-
duction of growth rate could be observed, since PHB cannot be used by mycin (nptI) and chloramphenicol (Cm) resistance (Betterle and Melis,
plant cells. Interestingly, gene silencing of stearoyl-ACP desaturase 2018). It is concluded that both homologous (cpcB) and heterologous
(SAD) enhanced the stearic acid content in C. reinhardtii (de Jaeger (nptI and Cm) genes highly expressed in cyanobacteria leads to the
et al., 2017). Besides, C. reinhardtii was engineered to produce the (E)- overexpression of a fusion construct. This method was proposed to
α-bisabolene which is a biodiesel precursor with the productivity up to overexpress is difficult, or low level expression genes in cyanobacteria
10.3 mg/g-DCW by tailored carbon partitioning (Wichmann et al., (Betterle and Melis, 2018).
2018). Isoprene (C5H8) is a volatile C5 hydrocarbon that is preferentially
used as feedstock in the rubber industry. The Synechocystis sp. PCC 6803
4.5. Pigment and high-value compounds strain, in which the isoprene synthase (ispS) is under the control of the
strong rbcL promoter, resulted in the highest productivity of 1.16 ng/
Carotenoids are the most popular, and carotenoids derived from mL/h/OD (Pade et al., 2016). After the attempt to regulate 2-C-methyl-
microalgae have high antioxidant and anti-inflammatory properties, d-erythritol 4-phosphate (MEP) pathway for increasing isoprene pro-
and their content affects the cell tones of organisms (Gong and Bassi duction, a better understanding of the regulation and optimized flux of
2016). Carotenoids are isotonic polyene chains derived from 40 carbon carbon to the precursors would be required to further increasing the

10
 Table 2
Biogas, biofuel, lipid, pigment and high-value compounds production by genetic cyanobacteria and eukaryotic microalgae.
Species Target genes Strategy Product, productivity and titer References
W.-R. Lin, et al.

Biogas: Hydrogen and ethylene

Anabaena sp. 7120 hupL Deletion of hupL and induction of hupH by NH4+ Hydrogen, 45 μmol H2/mg chla/h Masukawa et al. (2002)
Synechocystis sp. PCC 6803 narB and nirA Deletion of narB and nirA Hydrogen, 300 nmol H2 /mg chla/h Baebprasert et al. (2011)
Synechococcus elongatus PCC7942 hydA Overexpression of gydA from Clostridium acetobutylicum Hydrogen, 2.8 μmol H2 /mg chla/h Ducat et al. (2011)
C. reinhardtii mutant strain L159I- psbA Chromosome psbA was intron-less and mutated in the L158I and N230Y Hydrogen, 5.77 mL H2/L/h Khetkorn et al. (2017)
N230Y
Synechocystis sp. PCC 6803 Ele, xylAB and glgC Overexpression of efe from P. syringae pv and xylAB from E. coli. Deletion Ethelene, 945 μL/L/h Lee et al. (2015)
of glgC
Synechocystis sp. PCC 6803 Efe and kgtP Overexpression of efe from P. syringae pv with triplicate copy and kgtP Ethylene, 9.7 mL/L/h Zhu et al. (2015)
Synechococcus elongatus PCC 7942 efe Overexpression of efe from P. syringae pv Ethylene, 140 μL/L/h/OD750 Carbonell et al. (2019)

Biofuels: ethanol, butanol and isobutanol

Synechococcus elongatus PCC 7942 ictB, ecaA, and acsAB Overexpression of ictB, ecaA, and acsAB to increase the hydrocarbon Ethanol, 7.2 g/L Chow et al. (2015)
production.
Synechocystis sp. PCC 6803 Pdc and adh Overexpression of Pdc from Z. mobilis and adh Ethanol, 5.5 g/L Gao et al. (2012)
Synechocystis sp. PCC 7002 Pdc and adh Overexpression of Pdc from Z. mobilis and adh integrated on a novel- Ethanol, 0.6 g/L Wang et al. (2019)
identified NS site
Synechococcus elongatus PCC 7942 ter, nphT7, bldH, yqhD, phaJ and phaB Overexpression of ter from T. denticola, nphT7, bldH, yqhD, phaJ and phaB Butanol, 30 mg/L Lan and Liao, (2012)
Synechococcus elongatus PCC 7942 Ter, pduP, CAAase, nphT7, yqhD, phaj and Overexpression of ter from T. denticola, pduP from S. enterica, CAAase Butanol, 481.7 mg/L Fathima et al. (2018)
phaB from Yarrow lipolytica, nphT7, yqhD, phaJ and phaB
Synechocystis sp. PCC 6803 Kivd and adhA Overexpression of kivD and adhA from Lactococcus lactis Isobutanol, 0.298 g/L Varman et al. (2013)
Synechocystis sp. PCC 6803 Mutant kivDS286T, adhA, ilvC and ilvD Overexpression of mutant kivDS286T, adhA from Lactococcus lactis and Isobutanol, 0.911 g/L Miao et al. (2018)
ilvCD

Lipid
Synechocystis sp. PCC 6803 acyl-ACP synthetase (aas) Overexpression of aas Lipid, content 34.5% in DCW, production rate Eungrasamee et al. (2019)

11
41.4 mg/L/day
Synechococcus elongatus PCC 7942 fabF, fadD, desAB Overexpression of fadF and desAB as well as deletion of desAB omega-3 fatty acid, content of total lipid Santos-Merino et al. (2018)
reached 22.6%
Phaeodactylum tricornutum Acyl-ACP thioesterase Overexpressing Acyl-ACP thioesterase in cell Lauric and myristic acid Radakovits et al. (2011)
Phaeodactylum tricornutum PDK Antisense gene knockdown Lipid content (42.1% DCW) Ma et al. (2014a,b)
Thalassiosira pseudonana Thaps3_264297 Antisense gene knockdown Lipid content increased 2.4- ∼ 3.3-fold Trentacoste et al. (2013)
Chlorella vulgaris (UMT-M1) ω-3 FAD Introduced a copy of disrupted gene into the host Lipid (> 30% of total lipid content) Lau et al. (2017)
Chlamydomonas reinhardtii CC400 PEPC1 Gene down regulation by CRISPRi/dzCas9 Lipid (content and productivity of 28.5% DCW Kao and Ng, (2017)
and 34.9 mg/L/day)
Nannochloropsis gaditana Zn(II)2Cys6-encoding genes Gene knock out by CRISPR-Cas9 reverse-genetics pipeline Lipid (content and productivity of 40–55% Ajjawi et al. (2017)
DCW and 5.0 g/m2/day)

Bulk chemicals
Synechocystis sp. PCC 6803 Ldh Overexpression of ldh from Lactococcus lactis Lactate, 1.84 g/L Angermayr and Hellingwerf
(2013)
Synechocystis sp. PCC 6803 Ldh, Pyruvate kinase (pk) and PEPC Overexpression of ldh from Lactococcus lactis and pk from E. faecalis. Lactate, 0.36 g/L Angermayr et al. (2014)
Partially knockout of pepc
Synechocystis sp. PCC 7002 Ldh and glnA Overexpression of mutant LdhV39R and CRISPR-dCas9 repression of glnA Lactate, 0.8 g/L Gordon et al. (2016)
Synechococcus elongatus PCC 7942 glgc, sdhA and sdhB CRISPRi down regulation Succinate acid, increased to 12.5-fold and Huang et al. (2016)
reached 0.63 mg/L
Chlamydomonas reinhardtii (CC-849) phbB, phbC Introduced foreign gene into cell PHB (3.3–6 μg/g-DCW) Wang et al. (2010)
Chlamydomonas reinhardtii stearoyl-ACP desaturase Silenced by artificial microRNA to knockdown of fab2 gene Stearic acid content in TGA molecules de Jaeger et al. (2017)
increased 1.97 fold.
Chlamydomonas reinhardtii AgBs Introduced foreign gene into cell by glass bead Bisabolene (10.3 mg/g-DCW) Wichmann et al. (2018)

Pigment and high-value compound

Chlamydomonas reinhardtii PSY Overexpression of exogenous PSY from Dunaliella salina Carotenoids content increased up to 2.6-fold Couso et al. (2011)
Chlorella zofingiensis PDS Overexpression of mutated PDSL156F gene in cell Increased 32.1% carotenoids and 54.1% Liu et al. (2014)
astaxanthin
Phaeodactylum tricornutum DXS Introduced foreign gene into cell by particle bombardment Fucoxanthin (24 mg/g-DCW) Eilers et al., 2016
(continued on next page)
Bioresource Technology 291 (2019) 121932
W.-R. Lin, et al. Bioresource Technology 291 (2019) 121932

isoprene synthesis in cyanobacteria. Finally, pinene synthase (PS) is

Formighieri and Melis (2015)

competing with limonene synthase for the usage of geranylpyropho-

Betterle and Melis (2018)

sphate (GPP) in MEP pathway. The performance of pinene synthase

Wang et al. (2016a,b)

Tashiro et al. (2016)

mutant and wild type (PSmut and PSwt) in Synechocystis sp. PCC 6803

Pade et al. (2016)

was compared. PSmut accumulated ∼80 μg/L pinene in the strains for
Lin et al. (2017)
Ma et al. (2019)

168 h is 2-folds over the PSwt in the same condition. However, the
References

amount of pinene accumulated was approximately one third of the

reported value (i.e., 250 μg/L) for limonene synthesis by expressing
LMS in the same strain which reflected the higher catalytic capacity of
LMS over PS in the MEP pathway (Tashiro et al., 2016).
Microalgae biotechnology industry has been established by opti-
mizing the culture environment and other cultivation strategies (eg,
High lutein productivity (5.08 mg/L/d)

light intensity, temperature, pH, dissolved oxygen and nutrients, fed-

mode or semi-continuous), and many algae production systems for
β-phellandrene, 5.95 mg/g-DCW
Limonene, 750 μg/L/OD730/day
β-phellandrene, 3.2 mg/g DCW
Product, productivity and titer

high-priced chemicals have been established, such as Dunaliella salina.

Isoprene, 0.336 mg/g-DCW

Haematococcus pluvialis, Chlorella, and Scenedesmus for carotenoids (β-

carotene, astaxanthin, canthaxanthin, lutein, and isoprene) (Gong and
Limonene, 6.7 mg/L

Bassi 2016; Chen et al., 2017b; Chen et al., 2018). In summary, Table 2
Pinene, 140 mg/L

has displayed the progress of biogas, biofuel, lipid, pigment, and high-
value compounds produced by genetic cyanobacteria and eukaryotic
microalgae. Although the improvement by gene regulations in both
groups has competitive advantages, such as easy to culture and produce
bio-fuels diversely, the productivity and cost are still far away from
Overexpression of Lims from Mentha soicata and overexpression of rpi, rpe

industrial level (Tashiro et al., 2016).

Overexpression of ispS in a replicable plasmid pVZ325 isolated from A.
Overexpression of PHLS with N-terminal fusion of phycocyanin β

Overexpression of PHLS with N-terminal fusion of phycocyanin β

High-intensity light exposure induced repression of lut1 and zep

5. Challenges and prospective

To date, genetic engineering of microalgae and cyanobacteria still

face many constraints. For instance, the lack of sufficient genomic re-
source center has hindered the development of genetic engineering in
microalgae, such as Chlorella (Yang et al., 2016). Another obstacle is the
low efficiency of transformation methods, which may be caused by the
Overexpression of mutant PSH346Y, Q456L

rigidity and robustness of the cell wall (Tanwar et al., 2018). Take
electroporation for example. Although the adjustment of parameters
and gpps in the RSF1010 plasmid

could enhance the permeability of cell, it may lead to low recovery after
the procedure and hence decrease the efficiency (Muñoz et al., 2018).
In the economic aspect, scale-up and industrial application needs tre-
Overexpression of lims

mendous research focus. Although cyanobacteria and microalgae could

-subunit and GPPS

directly convert CO2 into chemicals, the concentration was much lower
than respective value in the current industry setting. For example, the
maximum production yield of ethanol in cyanobacteria is 5.5 g/L, but
Strategy

-subunit

thaliana

in E. coli 10 g/L could be easily achieved (Yazdani and Gonzalez, 2008).

On the other hand, the disruption of glycogen synthesis decreased the
glycogen utilization, thereby enhancing the carbon flux to the targeted
β-phellandrene synthase (PHLS) and GPP-

chemical production, but the cell growth decreased (Gordon et al.,

Liminene synthetase (lims), rpi, rpe and

2016). To improve or maintain a balance between glycogen utilization

and synthesis of higher desired chemical is a critical issue in the future.
β-phellandrene synthase (PHLS)

Is it possible to create a consortium of cyanobacteria and super-pro-

ducer strains to attain high-level targeted chemical and simultaneous
Isoprene synthase (ispS)

Pinene synthetase (PS)

fix CO2?
In the past, cyanobacterium is a predominant host to be engineered
synthase (GPPS)

for simultaneous CO2 fixation and chemical production. Multi-omics

Target genes

lut1 and zep

infers the consistent results that all the engineered cyanobacteria with
outstanding traits enhance the expression of protein in photosynthesis.
Lims
gpps

However, some reports revealed the limitation of native photosynthesis

with chemical production (Kanno et al., 2017; Wang et al., 2016a,b),
Synechococcus elongatus PCC 7942

indicating that depending on sole carbon source from CO2 using native
RuBisCO pathway is not suitable for the production of chemicals and
Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803

results in low productivity (Gao et al., 2012; Fathima et al., 2018; Miao
Chlamydomonas sp. JSC4

et al., 2018; Huang et al., 2016; Lin et al., 2017). Therefore, how to
Table 2 (continued)

overcome the limitation of photosynthesis and increase the CO2 toler-

ance is the critical step to produce the chemicals with high productivity
autotrophically. Introduction of different carbon fixation pathway in
cyanobacteria may be a new approach, but it would remain a challenge
Species

due to the unknown mechanism of photosynthesis limitation. It needs

more multi-omics analysis to uncover the mechanism, which could

12
W.-R. Lin, et al. Bioresource Technology 291 (2019) 121932

accelerate the development of synthetic biology in cyanobacteria ex- simultaneously.

ponentially. Thus, the challenge will be to create a super-producer
strain by multi-omics approach which will metabolize intracellular 6. Conclusion
glycogen and produce the targeted chemical with simultaneous CO2
mitigation. Microalgae and cyanobacteria are both important and promising
It is possible to extrapolate the knowledge base in cyanobacterial alternative feedstocks for biorefinery. Over the past decades, research
genetic engineering to the eukaryotic microalgal genetic engineering, progress has been shifted from gene expression to genome editing and
but it is hindered by the lack of genetic information. For example, metabolic regulation or synthetic biology in microalgae and cyano-
double homologous recombination is a common approach for en- bacteria. Further developments in bioinformatics, genomes, tran-
gineering the cyanobacteria. The same concept could be applied for scriptome, proteome, metabolome and multi-combination of data ana-
eukaryotic microalgae based on the homologous recombination, in- lysis in cyanobacteria and microalgae strains will achieve a robust
cluding the successful construction of rbcS intron in Chlamydomonas as genetic engineering platform, thus, obtaining higher growth rate, better
well as LB and RB in Chlorella. The failure in introducing gene in carbon sequestration capacity, more high-value compounds, high
Chlorella by the rbcS intron suggested a highly difference between the carbon dioxide tolerance and temperature resistance. This could help in
rbcS sequence of two species. Thus, studies in cyanobacteria could ac- attaining designer microalgae proficient in outdoor conditions and apt
celerate the progress of synthetic biology in eukaryotic microalgae as for industrial use in the future.
well.
Despite of these situations, progress of utilizing microalgae and Declaration of Competing Interest
cyanobacteria in the field of genetic engineering is well underway. Take
Chlorella for example, genomes and transcriptome of several Chlorella The authors declare that they have no known competing financial
strains was available, and others are currently being developed. interests or personal relationships that could have appeared to influ-
Meanwhile, more and more newly developed genetic tools and methods ence the work reported in this paper.
have been published to improve the transformation and expression
system, such as some pretreatment of removing the cell-wall to enhance Acknowledgements
the transformation efficiency. Adjustment of cultivation condition may
lead to great influence on the content of some bio-compounds in mi- The authors are grateful for the financial support received from the
croalgae as well. With a proper combination of genetic manipulation Ministry of Science and Technology (MOST 105-2621-M-006-012-MY3,
and culture process, optimal productivity could be achieved. As a MOST 108-2218-E-006-006, and MOST-108-2621-M-006-015) in
summary, there are several important conditions to be overcome in the Taiwan.
genetic editing of microalgae. First was the design and regulation of Ethics approval and consent to participate: All the authors have read
multiple target sites, followed by the establishment of intact metabolic and agreed the ethics for publishing the manuscript.
pathway, stability after translocation and efficiency of screening mar- Consent for publication: The authors approved the consent for
kers. Moreover, the need for different algae, the label-free editing to publishing the manuscript.
achieve non-basic reform issues as well as sufficient genomics and
transcriptomics data and information were also essential factors. References
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