Biotechnology

Biotechnol. J. 2016, 11 DOI 10.1002/biot.201500617

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Review

Chlorella species as hosts for genetic engineering  

and expression of heterologous proteins:  
Progress, challenge and perspective

Bo Yang1,2, Jin Liu1,3, Yue Jiang 4 and Feng Chen1,3

1 Institute
for Food and Bioresource Engineering, College of Engineering, Peking University, Beijing, China
2 Schoolof Light Industry and Food Sciences, South China University of Technology, Guangzhou, China
3 Singapore-Peking University Research Centre for a Sustainable Low-Carbon Future, CREATE Tower, Singapore
4 Runke Bioengineering Co., Ltd., Zhangzhou, China

The species of Chlorella represent a highly specialized group of green microalgae that can produce
Received 11 NOV 2015
high levels of protein. Many Chlorella strains can grow rapidly and achieve high cell density under Revised 30 JUN 2016
controlled conditions and are thus considered to be promising protein sources. Many advances Accepted 05 JUL 2016
in the genetic engineering of Chlorella have occurred in recent years, with significant developments
in successful expression of heterologous proteins for various applications. Nevertheless, a lot of
obstacles remain to be addressed, and a sophisticated and stable Chlorella expression system has
yet to emerge. This review provides a brief summary of current knowledge on Chlorella and an
overview of recent progress in the genetic engineering of Chlorella, and highlights the advances in
the development of a genetic toolbox of Chlorella for heterologous protein expression. Research
directions to further exploit the Chlorella expression system with respect to both challenges and
perspectives are also discussed. This paper serves as a comprehensive literature review for the
Chlorella community and will provide valuable insights into future exploration of Chlorella as a Supporting information 
available online
promising host for heterologous protein expression.

Keywords: Chlorella ∙ Genetic engineering ∙ Heterologous proteins ∙ Microalgae ∙ Transformation

Correspondence: Jin Liu, Institute for Food and Bioresource Engineering,

1    Introduction
College of Engineering, Peking University, No.60 Yannan Yuan, Beijing,
100871 China  Recombinant proteins have broad applications in health-
E-mail: gjinliu@pku.edu.cn  care, scientific research, agriculture, and various indus-
  tries [1]. Recent advances in recombinant proteins have
Additional correspondence: Feng Chen, Institute for Food and Bioresource resulted in the development of several expression sys-
Engineering, College of Engineering, Peking University, Beijing, China  tems based on various living cells and organisms. Estab-
E-mail: sfchencoe@pku.edu.cn
lished expression systems include bacteria [2], yeast [3],
Abbreviations: CAI, codon adaptation index; CaMV35S, cauliflower mosaic- insect cell lines [4], mammalian cell lines [5], animals [6],
virus 35S; cfu, colony forming units; EGFP, enhanced green fluorescent and plants [7]. Each of these expression platforms has
protein; EPA, eicosapentaenoic acid; EST, expressed sequence tag; fGH, distinct strengths and limitations with regard to the type
flounder growth hormone; G418, geneticin; GFP, green fluorescent protein; of protein, ease of manipulation, period and cost of pro-
GRAS, generally recognized as safe; GUS, β-glucuronidase; HDGS, homol- duction, and safety risks [8].
ogy-dependent gene silencing; hGH, human growth hormone; IRs, inverted
Microbial cells, such as bacteria and yeast, are the
repeats; LINE, long interspersed DNA element; LUC, firefly luciferase;
MerA, mercuric reductase; NR, nitrate reductase; PDS, phytoene desatu-
most commonly used platforms to express heterologous
rase; PEG, polyethylene glycol; rbcS, small unit of ribulose-1,5-bisphosphate recombinant proteins. Bacteria, such as Escherichia coli,
carboxylase/oxygenase; siRNA, short interference RNA; Ubi1, maize poly- serve as a powerful and versatile workhorse for large-scale
ubiquitin enzyme production [6]. Although bacterial expression

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systems have the advantages of low cost, rapid growth,
and ease of manipulation, they have several drawbacks,
such as limited type of produced proteins (viz. small pro-
teins with a simple structure), lack of post-translational
modifications, and potential contamination of the product
with bacterial endotoxins [2, 6, 9]. Yeast expression sys-
tems, such as Saccharomyces cerevisiae, can express
secretory proteins and perform post-translational modifi-
cations in addition to having advantages similar to those
of bacterial systems. However, they tend to express het-
erologous proteins with hyperglycosylation and improper
folding [3, 10–12].
Insect and mammalian cells as well as transgenic ani-
mals and plants are employed to overcome these limita-
tions. Insect cell lines, when utilized in combination with
the baculovirus expression system, can express safe and
size-independent proteins with a high expression level,
eukaryotic post-translational modifications, and proper
folding [4, 6]. However, this system suffers from several
limitations, such as proteolysis, high cost, and incorrect
glycosylation [13]. Mammalian cell lines are regarded as
ideal systems for the expression of large complex thera-
peutic proteins requiring mammalian-specific post-trans-
lational modifications, proper folding, and glycosylation
[14, 15]. Nevertheless, the mammalian cell-derived prod-
ucts are expensive because of high nutrient requirements
and long generation time [16]. Entire transgenic animals
can also be engineered to produce complex proteins in
milk, egg white, and blood on an industrial scale, but ani-
mal generation is an expensive and time-consuming
process [17].
The use of transgenic plants to synthesize heterolo-
gous recombinant proteins, which is also known as
“molecular farming,” has elicited interest because of this
system’s advantages over other expression systems in
terms of protein type and complexity, economic feasibil-
ity, scalability, and safety [7]. However, this system has
several practical disadvantages, such as relatively low
levels of protein yield, long cycle time, and lack of precise
control over growth conditions [8, 10]. Therefore, explor-
ing an alternative and desirable system to express heter-
ologous proteins is necessary.
Unicellular microalgae represent a group ofdiverse
photosynthetic organisms present in a wide range of
habitats. Compared with terrestrial plants, they are more
efficient in converting sunlight into chemical energy as
green cell factories; thus, they are particularly attractive
for the production of lipids, proteins, carbohydrates, pig-
ments, and other bioactives [18, 19]. Recently, microalgae
have elicited much interest as simple models for higher
plants and as a natural source of valuable compounds
ranging from therapeutic proteins to biofuels from a bio-
technological perspective [20, 21]. Promising achievements
have been obtained in the genetic engineering of several
microalgae, including Chlamydomonas reinhardtii [22],
Phaeodactylum tricornutum [23], Nannochloropsis sp.
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[24], and Chlorella spp. [25], although microalgae are gen-
erally considered relatively difficult to engineer because of
the lack of a high-efficiency genetic toolbox and a stable
genetic system [26]. Given the advantages of rapid
growth, low cost, ease of culture, and broad industrial
applications ranging from biodiesel feedstock to food
additives, Chlorella has received increasing attention and
is a potential newcomer as a host for the expression of
heterologous proteins through genetic engineering.
This review provides an overview of the progress in
the expression of heterologous proteins in Chlorella and
discusses the advantages of Chlorella cells, Chlorella
genomics, the genetic toolbox of Chlorella, including
transformation methods and vector construction strate-
gies that could affect protein yields, and claims on heter-
ologous proteins expressed in Chlorella. Future challeng-
es and perspectives regarding the expression of
heterologous proteins in Chlorella are also examined.
2    Advantages of Chlorella as an expression
system
Species of the Chlorella genus are unicellular eukaryotic
microalgae in a spherical form, and their diameters typi-
cally vary from 5 to 10 μm. Considering that the systemat-
ics of Chlorella still remains provisional according to the
conflicts among multidisciplinary approaches based on
morphology, chemotaxonomy, and molecular phylogeny
(Fig. 1A) [27–30], the term “Chlorella” utilized in this paper
refers to the spherical cell phenotype of Chlorella sensu
lato within the division Chlorophyta and class Chlorophy-
ceae and Trebouxiophyceae (including Chlorella, Para-
chlorella, Auxenochlorella, and other Chlorella-like strains
that were traditionally designated as Chlorella genus).
Many key physiological and biotechnological traits ena-
ble Chlorella to function as a promising host for the
expression of heterologous proteins and other high-value
compounds.
The competitive advantage of Chlorella is that it
grows fast and is easy to culture and scale up both indoors
and outdoors [31–33]. Fast growth means less time is
required for biomass accumulation and protein produc-
tion. In addition, Chlorella can achieve a relatively high
cell density under various culture modes, including pho-
toautotrophy [34, 35], heterotrophy [36, 37], and mixotro-
phy [38, 39]. In photoautotrophic culture modes, regard-
less of enclosure in photobioreactors or being placed in
outdoor raceway ponds, the media requirements are only
carbon dioxide, water, and certain simple nutrients.
These simple requirements endow this culture mode an
affordable cost and energy input and low risk of contami-
nation. Most Chlorella species can also grow heterotroph-
ically in the dark by using simple carbon sources, such as
glucose, and can therefore be utilized in existing indus-
trial fermenters in accordance with FDA-approved stand-
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ard protocols, such as continuous fermentation [40].
Moreover, several species, such as C. vulgaris, Auxeno-
chlorella protothecoides (C. protothecoides), C. pyrenoi-
dosa, and Chromochloris zofingiensis (Chlorella zofingien-
sis), can utilize low-cost industrial waste products as
carbon and energy sources for biomass production [41–
44], thus showing a great potential for cost-effective
expression of heterologous proteins. Other advantages
include the following:
(i) As a eukaryotic microalga, Chlorella can produce
eukaryotic proteins with post-translational modifi-
cations, although this pattern still remains unclear.
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Figure 1.  (A) Phylogenetic relationship inferred from 18S
ribosomal RNA gene sequences of Chlorella and Chlorella-
like microalgae with the maximum likelihood method
(1000 bootstrap replicates). Taxa indicated with a blue dot
were previously designated as Chlorella species but are now
checked and revised as close relatives of Chlorella within
the classes of Trebouxiophyceae and Chlorophyceae
through modern molecular approaches: Pseudochlorella
pringsheimii (”Chlorella ellipsoidea”), ­Parachlorella kessleri
(“Chlorella” kessleri), Chromochloris zofingiensis (“Chlorella”
zofingiensis), Auxenchlorella protothecoides (”Chlorella” proto-
thecoides), Chloroidium saccharophilum (”Chlorella saccha-
rophila”). The outer circle indicates the corresponding
classes, which are identified by two different colors, to show
the taxonomic position. (B) Electroporation transformation
process in Chlorella cells. It consists of four major steps.
(i) The plasmids containing the gene of interest are mixed
with carrier DNA and added to the cell suspension of Chlo-
rella before applying electrical pulses. (ii) Electrical pulses
are applied to the suspension to induce the formation of
transient micropores in cell membranes, (iii) thus enabling
the ­foreign DNA to enter the cell, (iv) and integrate into the
Chlorella genome. (C) Schematic of particle bombardment
used for Chlorella transformation. The gene of interest is
first inserted into the plasmid. Following coating of gold
particles with plasmids, the DNA-coated particles are bom-
barded into Chlorella cells with a particle gun. Once in the
cell, the DNA disassociates from the gold particles and is
then integrated into the Chlorella genome. (D) Schematic of
PEG-mediated method used for Chlorella transformation.
The protoplasts of Chlorella cells are prepared by enzymatic
cell wall removal and then incubated with plasmids (con-
taining the gene of interest), carrier DNA, and PEG. With
the aid of PEG, foreign DNA penetrates into the protoplasts
and integrates into the Chlorella genome. (E) Putative mod-
el for Agrobacterium-mediated genetic transformation in
Chlorella. The transformation process comprises six major
steps. (i) The gene of interest is inserted into the left (LB)
and right T-DNA border (RB), and this T-DNA-based plas-
mid is delivered to Agrobacterium. (ii) Cell-cell recognition
and attachment of Agrobacterium to the host Chlorella cells
occur. (iii) With the activation of the vir gene region by
response chemicals, such as acetosyringone, a T-strand is
generated by certain Vir proteins. (iv) The T-strand travels
through the Chlorella cell cytoplasm (v) and is directly
imported into its nucleus. (vi) Once in the nucleus, the
T-DNA is stripped of the unnecessary escorting proteins
and integrated into the Chlorella genome.
The possibility of expressing high-value complicat-
ed proteins and even several therapeutic proteins is
thus highlighted [45].
(ii) Chlorella has long been used as a popular functional
food worldwide, especially in Asian countries such as
Japan, Korea, and China [46]. Two Chlorella species,
namely, C. vulgaris (GRAS Notice No. GRN000396)
and A. protothecoides (C. protothecoides) (GRAS
Notice No. GRN000519), have recently been catego-
rized as “generally recognized as safe” (GRAS). This
categorization indicates that Chlorella cells are safe
for human consumption and are thus a potential
3
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source for the oral delivery of therapeutic proteins
with little or no purification [47].
(iii) Chlorella can produce significant amounts of high-
value fine components, such as lipids, proteins, ca-
rotenoids (e.g. lutein and astaxanthin), vitamins,
and minerals. The integration of these high-value
products is expected to significantly expand the use
of Chlorella in industrial, medical, and pharmaceuti-
cal applications (see Section 8 for a detailed discus-
sion) [48–51].
(iv) Chlorella has a solid research basis for the produc-
tion of bio-compounds even at a large scale. Many
Chlorella species have long been utilized for the
production of high-value compounds and bioener-
gy from laboratory to large scale. This solid founda-
tion will, to a large extent, benefit the expression of
heterologous proteins in Chlorella and facilitate the
commercial use of the Chlorella expression plat-
form.
(v) Several Chlorella species, such as C. sorokiniana
and Chlorella sp., have strong adaptability to the
environment, such as high tolerance to tempera-
ture (up to 42°C) and CO2 concentration (up to 40%)
[52].
3    Current state of Chlorella genomics
Genomics offers a foundation for the development of
transformation toolbox and rational genetic engineering.
It provides powerful approaches to elucidate metabolic
networks and identify key regulatory elements that can
promote protein expression [53]. Genetic analysis with
Chlorella began in the early 1990s [54, 55]. To date, sig-
nificant advances have been obtained in Chlorella genom-
ics. Expressed sequence tag (EST) databases from sev-
eral species have been established and are being actively
expanded (available from the Genbank EST database).
The chloroplast genome of C. vulgaris, Chlorella sp.
ArM0029B, and A. protothecoides (C. protothecoides) and
the mitochondrial genome of Chlorella sp. ArM0029B
have been sequenced [56–58]. At present, full nuclear
genomes and/or transcriptomes of three Chlorella spe-
cies, namely, C.  variabilis [59], C.  pyrenoidosa [60], and
A. protothecoides (C. protothecoides) [61, 62], and tran-
scriptomes and proteomics of C.  vulgaris [63, 64] are
­available. Entire genome sequencing of six additional
­Chlorella strains is currently underway; these six strains  revealed that several transformation methods have been
are C. pyrenoidosa (Bioproject accession number
­PRJNA171991), C. vulgaris (Bioproject accession number
PRJNA278897), C. sorokiniana (Bioproject accession
number ­PRJNA218510), C. minutissima (Bioproject acces-
sion numbers PRJNA245012 and PRJNA245966), C. emer-
sonii (Bioproject accession number PRJNA243839), and
C. zofingiensis (in progress in our laboratory). These com-
prehensive genome data, together with the upcoming
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release of considerable transcriptomes, will provide valu-
able insights into the critical factors that determine the
level of heterologous protein expressed in Chlorella and
may have a great potential to break through the inherent
limits on its capacity for the expression of heterologous
proteins through rational manipulation.
4    Transformation methods
The instability and relatively low yields of protein impede
the use of Chlorella for heterologous protein expression.
Efforts have been and/or are being exerted from the per-
spective of genetic engineering to optimize the Chlorella
expression system for stable and improved expression of
heterologous proteins, including transformation method
development and vector construction improvement by
selecting proper promoters, enhancers, marker genes,
and codon usage.
Transformation methods are currently considered the
most severe limitation in Chlorella transformation. This
hampers the use of engineered Chlorella for heterologous
protein expression to a large extent. Therefore, develop-
ing a reliable transformation method for easy DNA deliv-
ery to Chlorella cells is important.
The first attempt for Chlorella transformation was con-
ducted by Jarvis and Brown in 1991; firefly luciferase was
transiently expressed in C. ellipsoidea via electroporation
[65]. Later, Dawson et al. [66] reported the first stable
transformation by introducing a nitrate reductase gene
into the nitrate reductase-deficient C. sorokiniana mutant
through particle bombardment. These two pioneering
studies provided a starting point for the genetic engineer-
ing of Chlorella. Afterward, many reports on Chlorella
transformation have been presented and have led to sig-
nificant advances. To date, more than 12 different Chlo-
rella species are claimed to have been successfully genet-
ically engineered by nuclear transformation with various
transformation methods (Table  1). No transformation of
chloroplasts or mitochondria have been reported so far. In
most cases, transformation leads to the stable expression
of transgenes. However, in several cases, only transient
expression has been achieved. In stable transformants,
the exogenous genes introduced are integrated and
maintained in the genome over generations [67]. By con-
trast, rapid loss of introduced exogenous genes is usually
observed in transient transformants [68]. Reports have
developed for Chlorella. These transformation methods
include electroporation [69–85], particle bombardment
[66, 81, 83–91], polyethylene glycol (PEG)-mediated meth-
od [65, 74, 92–99], and Agrobacterium-mediated methods
[100, 101]. Each method has its advantages and limita-
tions with regard to transformation efficiency, repeatabil-
ity, ease of manipulation, cost and period, and so on (Sup-
porting information, Table S1).
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Table 1.  Chlorella species reported for nuclear transformation and their transformation methods

Chlorella species Transformation methods Stability References

C. ellipsoidea PEG Transient transformation [65, 97]
PEG Stable transformation [92, 95, 96, 98]
Particle bombardment Transient transformation [81, 88]
Electroporation Stable transformation [75, 78, 82]
Transient transformation [79, 81]
C. saccharophila Electroporation Transient transformation [76]
C. sorokiniana Particle bombardment Stable transformation [66]
PEG Transient transformation [94]
P. kessleri(C. kessleri) Particle bombardment Stable transformation [87]
Chlorella sp. MACC/C95 Electroporation Transient transformation [80]
Stable transformation [70]
C. vulgaris PEG Transient transformation [94]
Stable transformation [93]
Electroporation Transient transformation [77]
Stable transformation [71–73]
Agrobacterium Stable transformation [100, 101]
Particle bombardment Stable transformation [86, 89, 91]
A. protothecoides (C. protothecoides) Electroporation Stable transformation [83–85]
Particle bombardment Stable transformation [83–85, 90]
Chlorella sp. DT PEG Stable transformation [74]
Electroporation Stable transformation [74]
C. minutissima Electroporation Stable transformation [69]
Particle bombardment Stable transformation [83, 85]
C. desiccata PEG Transient transformation [99]
C. emersonii Particle bombardment Stable transformation [83, 85]
Chromochloris zofingiensis (C. zofingiensis) Particle bombardment Stable transformation [102]
Electroporation Stable transformation [102]

4.1    Electroporation
Reports show that electroporation is the most commonly
utilized method for Chlorella transformation because of its
simplicity and high efficiency with a small amount of DNA
[69–85, 102, 103]. Jarvis and Brown [65] were the first to
report on the effectiveness of electroporation in Chlorella
transformation. They exposed protoplasts to high-intensi-
ty electric field pulses for easy uptake of exogenous DNA.
The method can transfer exogenous genes independent
of the ability of cells and is applicable for different Chlo-
rella species. As shown in Fig. 1B, this method induces
the formation of transient micropores in cell membranes
by applying short high-intensity electricfield pulses and
thus allows exogenous DNA to enter the cell and integrate
into the nuclear genome [104]. Given that the thick cell
walls of Chlorella hinder macromolecule movement, in
several cases, Chlorella protoplasts or cell-wall-deficient
mutants can be used for electroporation [21, 105].
Several parameters, including electrical field strength,
pulse length, medium composition, temperature, and
foreign DNA concentration, have been found to affect the
transformation efficiency of electroporation in Chlorella
[71, 76, 80, 102]. Currently, reports claim that the maxi-
mum transformation efficiency of electroporation in Chlo-
rella has resulted in 2500–7137 colony forming units (cfu)
per μg DNA [71, 77]. Although this value is much smaller
than that reported for Chlamydomonas reinhardtii
(1.9  ×  105  cfu per μg DNA) [105], it is acceptable for
genetic manipulations and represents an effective trans-
formation method.
4.2    Particle bombardment
Particle bombardment, also referred to as microprojectile
bombardment or biolistics transformation, is a widely
utilized and effective transformation method [106]. It is
suitable for almost all cell types regardless of the rigidity
or thickness of the cell wall [21]. This method employs
DNA-coated tungsten or gold microprojectiles that are
accelerated and propelled into target cells by a particle
gun apparatus [107]. Figure  1C illustrates the typical
transformation process of Chlorella cells through particle
bombardment. Particle bombardment has long been suc-
cessfully applied to the transformation of microbial cells,
plants, and animals [108, 109] as well as several microalgal
species, such as C. reinhardtii, Volvox carteri, and
Dunaliella salina [110–112]. Accordingly, this transforma-
tion method has been reported to adopt for several Chlo-
rella species, including C. ellipsoidea, C. vulgaris, C. zof-
ingiensis, C. sorokiniana, A. protothecoides (C. protothe-
coides), C. minutissima, C. emersonii, and Parachlorella

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kessleri (C. kessleri) [66, 81, 83–91, 102]. Several transfor-
mation parameters, including bombardment pressure,
distance of a sample from the adding screen, topology and
vector sequence, and plasmid DNA concentration, have
been found to affect DNA delivery to the Chlorella genome
[66, 86]. Although this method suffers from several limita-
tions, such as high cost of the required specialized equip-
ment and low repeatability [45], it remains a useful
method, particularly for Chlorella plastid transformation.
However, given that the diameters of typical Chlorella
cells range from 5 to 10 μm, the commonly used micropro-
jectiles may need to be modified for size reduction to
achieve effective nuclear or plastid transformation of this
alga.
4.3    PEG-mediated method
Gene transfer through PEG-mediated method has been
applied in plants [113], bacteria, and yeast [114] for over
30 years because of the method’s distinct advantages,
including no requirement for specialized equipment, sim-
ple manipulation, minimal damage to host cells, and good
reproducibility [115, 116]. This method simply requires
the protoplasts of cells to be exposed to recombinant DNA
with the aid of PEG, which condenses the plasmid DNA
molecule to prevent attack by nucleases and to facilitate
the penetration of DNA into the cells (Fig. 1D) [117]. The
PEG-mediated method has been successfully used to
transform several Chlorella species, including C. ellip-
soidea [65, 92, 95–98], C. sorokiniana [94], C. vulgaris [93,
94], C. desiccata [99], Chlorella sp. [99], and Chlorella sp.
DT [74].
Compared with that of other transformation methods,
the reported transformation efficiency of the PEG-mediat-
ed method is relatively low in Chlorella (356–2250 cfu per
μg DNA) [74, 92–95], although several attempts have been
made prior to transformation to optimize the transforma-
tion efficiency, such as heat shock treatment, co-precipi-
tation of exogenous DNA with Ca2+ and Mg2+, vortex with
glass beads, and addition of chloroquine and 2-deoxy-D-
glucose [92, 94]. The PEG-mediated method cannot be
directly applied to intact Chlorella cells, and cell wall
removal and protoplast preparation are needed.
4.4    Agrobacterium-mediated method
Agrobacterium-mediated transformation, which has
been widely utilized in plant genetic engineering [118] ,is
also claimed to be a feasible transformation method for
Chlorella. Empirically speaking, this method usually
exhibits high proportions of transgenic events with a low
gene copy number in plants [119, 120]. It genetically
transforms cells by means of pathogenic soil bacteria
Agrobacterium, which can introduce part of their Ti-
plasmid DNA (viz. transfer DNA or T-DNA) into the
nuclear genome of infected cells [121–123]. Agrobacteri-
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um has been reported to have a wide host range that
includes plants, fungi, mammalian cells, and microalgae
[124]. It can process and transfer any inserted DNA, even
several large DNA segments (>10 kb), between the right
and left borders that delimit T-DNA [125].
T-DNA transfer in plants is initiated with the aid of
bacterial virulence (Vir) proteins, which are induced by
certain phenolic or sugar compounds released from
wounded plant cells in dicotyledons or added exogenous-
ly (e.g. acetosyringone) in monocotyledons [118, 123].
Although reports have claimed that Agrobacterium can
transform Chlorella cells with completely different cell
wall components from plants [100, 101, 126], the molecular
mechanism of its T-DNA transfer process remains unclear.
However, given the success achieved in plants and other
related microalgal species [100, 101, 124, 126–130], the
T-DNA transfer process in Chlorella may also comprise
several routine steps similar to those in plants, but with-
out injury to the cell (Fig. 1E). Interestingly, studies have
found that C. reinhardtii and Haematococcus pluvialis
can be transformed in the absence of acetosyringone
[127, 128], but C. vulgaris was found to be transformed
only with the inclusion of acetosyringone [100]. This con-
dition suggests that C. vulgaris may be unable to produce
response chemicals to induce the activation of the Vir
system.
With the Agrobacterium-mediated method, studies
have claimed that C. vulgaris has been stably transformed
with several reporters, such as β-glucuronidase (gus),
green fluorescent protein (gfp) [100], and the Vitreoscilla
hemoglobin gene, which can significantly improve cell
growth and lutein production [101]. In addition, a recent
study that utilized short interference RNA (siRNA) strate-
gies claimed to have knocked down PsbO (a subunit
involved in oxygen evolution) in Chlorella sp. DT by
Agrobacterium-mediated transformation method; this
knockdown of PsbO led to a significant decrease in PsbO
expression and about a 10-fold increase in hydrogen pro-
duction compared with the wild type (WT) [126].
Several factors were found to affect the transformation
efficiency of this method in Chlorella; these factors include
the status of starting materials, Agrobacterium density,
co-cultivation conditions, and acetosyringone concentra-
tion [100], which is consistent with previous findings
reported for plants and fungi [131, 132]. However, consid-
ering that this method is rarely reported in Chlorella and
its mechanisms remain to be fully elucidated, it may not
be a preferred method for Chlorella transformation.
5    Vector construction strategies
The expression performance of heterologous proteins in
Chlorella is also influenced by factors pertaining to vector
construction, including promoters, enhancers, reporter
and marker genes, and codon usage.
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5.1    Promoters and enhancers
Promoters are a critical factor for gene expression and
exert a significant effect on transcriptional regulation. The
promoters commonly used for Chlorella transformation
are presented in Supporting information, Table S2. Vari-
ous studies have reported that cauliflower mosaic virus
35S (CaMV35S) is the most widely used constitutive pro-
moter in Chlorella, especially when endogenously strong
promoters are unavailable [65, 69, 70, 76, 83, 85, 87, 90, 93,
94, 99]. Similar to their function in plant genetic engineer-
ing, the constitutive promoters of Ubi1 (from maize poly-
ubiquitin) and Actin1 (from rice actin) are also claimed to
facilitate efficient gene expression in Chlorella [74, 75, 78,
89, 92, 98, 126]. Several studies have reported the use of a
Chlorella virus promoter for constitutive gene expression
in Chlorella and have demonstrated good transgene
expression over time [73, 83–85, 90, 94]. Given that Chlo-
rella viruses can specifically infect certain strains of Chlo-
rella or Chlorella-like species [133], they are likely to be
considered valuable sources of transgene promoters in
Chlorella transformation. Further studies on host transfor-
mation can therefore be conducted by developing high-
efficiency promoters from Chlorella viruses.
Inducible promoters have also been found to facilitate
efficient and tightly controlled gene expression in Chlo-
rella. The rbcS promoter (small unit of ribulose-1,5-bispho-
sphate carboxylase/oxygenase from C. reinhardtii or
C. zofingiensis) is a strong inducible promoter activated
by light. The efficacy of this promoter in the expression of
several heterologous proteins and marker genes in Chlo-
rella has been demonstrated [69, 82, 92, 94, 102]. The
promoter of the NR gene encoding a nitrate reductase is
another inducible promoter. It is negatively regulated by
the presence of ammonium at both transcriptional and
post-transcriptional levels and is actively induced when
ammonium is substituted by nitrate [79]. Hence, the
expression of transgenes is switched on or off when Chlo-
rella cells are grown in the presence of nitrate or ammo-
nium. Studies have reported that several reporter and
marker genes have been successfully expressed in
C.  ellipsoidea, C. vulgaris, and C. zofingiensis by using
this promoter [77, 79, 102]. Given the good controllability
of inducible promoters, they may have a great potential to
express heterologous proteins in a well-regulated manner.
Enhancers are a part of DNA that can activate tran-
scription from a target promoter regardless of its orienta-
tion and location [134]. Studies have reported that the
omega enhancer derived from tobacco mosaic virus can
significantly increase the gene expression in Chlorella
when placed upstream of the rabbit defensin gene [75,
98]. This is similar to the promoting function of the omega
enhancer in gene expression reported in plants and ani-
mals [135, 136]. In addition, some introns or homologous
long interspersed DNA element (LINE)-like retrotranspo-
sons that probably contain several enhancer-like regula-
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tory elements also play an important role in improving the
stability and efficiency of transgene expression. For
example, in C. zofingiensis, the inclusion of the first intron
of the phytoene desaturase (PDS) gene led to a 91%
increase in transformation efficiency [102]. Insertion of
LINE-like retrotransposons from C. vulgaris, viz. Zepp
sequence, was also found to significantly increase hGH
expression in Chlorella cells [94]. These findings demon-
strate the potential importance of enhancer or enhancer-
like regulatory elements in promoting transgene expres-
sion in Chlorella.
5.2    Reporter and marker genes
Reporter and marker genes are vital to the development of
an efficient transformation system. Reporter genes encode
proteins that have a typical enzymatic activity or are eas-
ily recognizable from various cellular proteins [137]. Thus,
they are useful in studying the transformation efficiency,
expression efficiency, protein localization, and stability of
transgenes. By contrast, selectable markers are proteins
that confer resistance to antibiotics or herbicides or oth-
erwise function by a unique complementary experiment
on metabolic mutants for the selection of positive trans-
formants [45].
Currently, various reporter and selectable marker genes
(summarized in Table  2) are claimed to be available for
Chlorella. Reports have shown that the most widely utilized
reporter in Chlorella transformation is the GUS gene, which
encodes β-glucuronidase. Given its stability, the GUS gene
is claimed to be an efficient reporter for transient or stable
transformation in Chlorella. Additionally, studies have
claimed that the green fluorescent protein (GFP), enhanced
green fluorescent protein (EGFP), and firefly luciferase
(LUC) can be used as intuitionistic reporters for Chlorella
transformation [65, 89, 93, 95, 96, 99, 100]. However, con-
sidering that Chlorella cells possess intrinsic photosyn-
thetic pigments and some fluorescent substances, fluores-
cent reporters should be driven by a strong promoter to
achieve a high expression level for distinguishing them-
selves from the background fluorescence.
The commonly used selectable markers in Chlorella
are claimed to be bacterial-derived markers that confer
resistance against antibiotics, including nptII, ble, hpt,
and cat genes (Table 2). Among them, the nptII marker
gene, which confers resistance to geneticin (G418), has
been claimed to be more applicable to the transformation
of most Chlorella species. Endogenously derived novel
markers, as an alternative to antibiotic-resistant genes,
have also been developed recently. Phytoene desaturase
(PDS), a rate-limiting enzyme involved in carotenoid bio-
synthesis in C. zofingiensis, is a target of bleaching herbi-
cides, such as norflurazon. A study has indicated that
introduction of point mutations in the PDS gene can
endow transgenic cells with resistance to norflurazon.
This scenario highlights the potential of utilizing modified
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Table 2.  Marker and reporter genes used in Chlorella transformation

Gene Description Marker/reporter Gene source Expressed in References

GUS β-Glucuronidase Reporter E. coli C. saccharophila [76]
P. kessleri (C. kessleri) [87]
C. ellipsoidea [79, 81, 88, 92,
97]
Chlorella sp. MACC/C95 [80]
C. vulgaris [71, 86, 100]
LUC Firefly luciferase Reporter Firefly C. ellipsoidea [65]
GFP Green fluorescent protein Reporter Aequorea victoria C. vulgaris [89, 95, 100]
C. ellipsoidea [95, 96]
Chlorella sp. [99]
EGFP Enhanced green fluorescent protein Reporter Aequorea victoria C. vulgaris [93]
NR Nitrate reductase (rescue of nitrate Marker C. vulgaris C. sorokiniana [66]
­reductase-deficient mutants)
PDS Modified phytoene desaturase   Marker C. zofingiensis Chromochloris zofingiensis [102]
(resistance to norflurazon) (C. zofingiensis)
SUC2 Sucrose invertase Marker Saccharomyces A. protothecoides   [83–85, 90]
­cerevisiae (C. protothecoides)
C. minutissima [83, 85]
C. emersonii [83, 85]
nptII Neomycin phosphotransferase II   Marker E. coli C. sorokiniana [94] 
(resistance to G418) C. vulgaris [86, 89, 93, 94,
101
P. kessleri (C. kessleri) [87]
C. ellipsoidea [75, 78, 81, 98]
Chlorella sp. MACC/C95 [70, 80]
Chlorella sp. [99]
C. desiccata [99]
C. minutissima [69]
ble Bleomycin resistance protein (resistance   Marker Streptoalloteichus C. ellipsoidea [82, 92, 95–97]
to phleomycin and related antibiotics) hindustanus
hpt Hygromycin B phosphotransferase Marker E. coli Chlorella. sp. DT [74, 126]
C. vulgaris [71–73, 100]
A. protothecoides   [83–85]
(C. protothecoides)
cat Chloramphenicol acetyltransferase Marker E. coli C. vulgaris [77]
­(chloramphenicol resistance)

PDS gene as a dominant selectable marker for C. zof-
ingiensis transformation [102]. Another endogenous
selectable marker is the NR gene encoding nitrate reduc-
tase from Chlorella. It has been reported to be success-
fully used to rescue NR-deficient mutants of C. sorokini-
ana in a medium containing nitrate as the sole nitrogen
source [66]. However, this marker is only applicable to
certain species with available corresponding defective
mutants. Sucrose invertase, which allows positive trans-
formants to grow on sucrose as a sole carbon substrate,
has also been developed recently as a novel complemen-
tary marker for Chlorella [83–85, 90].
5.3    Codon usage
The genomes of different kinds of organisms and the differ-
ent genomes within an individual organism have codon
usage biases for protein expression [138]. Biased codon
usage indicates that a codon that is usually used in certain
genomes is infrequently used in others, resulting in differ-
ences in tRNA abundance and thus affecting translation
efficiency and protein expression levels significantly [22].
Therefore, optimization of the codon-choice patterns of
transgenes is helpful in promoting their expression effi-
ciency by enhancing the translation rates and possibly
reducing the susceptibility to silencing [139].
Similar to C. reinhardtii, several Chlorella genomes
have an extremely high GC content of 63–67% [59, 61,
140]. This high GC content reveals their biased codon
usage in G and C. Given that several codon-optimized
exogenous transgenes have achieved significantly
increased expression levels in C. reinhardtii [140–142],
codon optimization should facilitate protein expression in
Chlorella. However, only a few attempts have been made

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to optimize the codon usage of transgenes in Chlorella
because the genetic engineering of Chlorella is still in the
initial stage. The extracellular secretion signal sequence
of the hGH gene is claimed to be the first report involving
codon optimization in Chlorella genetic engineering [94].
Bovine lactoferrin N-lobe, Saccharomyces cerevisiae
sucrose invertase, and white spot syndrome virus VP28
peptide are claimed to be other fully codon-optimized
heterologous proteins expressed in Chlorella [72, 83–85,
90, 91]. Although these studies did not prove the positive
effect of codon optimization on expression efficiency,
tentative studies show the promising use of codon-opti-
mized strategies in Chlorella genetic engineering.
Several software and databases have been developed
recently for codon optimization. The codon adaptation
index (CAI) is generally employed as a quantitative meth-
od to measure the expression levels of exogenous genes
according to their codon usage [45]. A useful database
containing CAI and codon usage indices for the majority
of sequenced host species, including two Chlorella spe-
cies (P. kessleri (C. kessleri) and C. protothecoides), is
available online at http://www.kazusa.or.jp/codon/ [143].
E-CAI (http://genomes.urv.es/CAIcal/E-CAI), OPTIMIZER
(http://genomes.urv.es/OPTIMIZER), and CAIcal (http://
genomes.urv.es/CAIcal) represent a series of powerful
online tools for the estimation and optimization of codon
usage [144]. These useful databases and tools will consid-
erably facilitate the development of codon optimization
strategies for enhanced expression of heterologous pro-
teins in Chlorella.
6    Current claims of heterologous proteins  
in Chlorella subspecies
Although the commercial production of heterologous pro-
teins by Chlorella is currently unachievable, several stud-
ies that utilized engineered Chlorella to express heterolo-
gous proteins provide much promise (Table  3). Various
studies have reported that the yields of heterologous
proteins by Chlorella vary from 200 ng/L to 11.42 mg/L [75,
94–96, 98, 145, 146], which is relatively lower than that for
other eukaryotic expression hosts, such as plants (0.1 μg/L
to 247  mg/L) [1, 147, 148], mammalian cells (0.55 to
80 mg/L) [16, 149], and insect cells (80 to 300 mg/L) [150,
151]. However, low protein yields can be compensated by
the short cycle time (typically several days) because of the
rapid growth of Chlorella. All heterologous catalytic pro-
teins from Chlorella, as summarized in Table 3, are claimed
to retain the expected bioactivity, which requires proper
protein folding and subunit assembly and/or possible
glycosylation or disulfide bridge formation, thereby under-
scoring the ability of Chlorella to process complicated
eukaryotic proteins.
Hawkins and Nakamura exerted the first attempt to
express heterologous proteins in Chlorella; in their study,
transient expression of the human growth hormone (hGH)
was observed in engineered C. vulgaris and C. sorokini-
ana by using the PEG-mediated method [94]. hGH was
expressed under the control of cauliflower mosaic virus
35S (CaMV35S) promoter and/or Chlamydomonas rein-
hardtii rbcS2 promoter or Chlorella virus promoter with

Table 3.  Current claims of heterologously expressed proteins in Chlorella

Heterologous protein Function/Application Expression host Notable results References

Human growth hormone
Flounder growth hormone
Mercuric reductase from
Bacillus megaterium
Phytase
VP28 peptide from the
White Spot Syndrome
Virus (WSSV)
Trypsin-modulating oostat-
ic factor (TMOF) peptide
Single-chain antibody
against bladder cancer
(anti-bladder scFv)
Bovine lactoferrin N-lobe
Rabbit neutrophil
­peptide-1
Growth-enhancing effect on children C. sorokiniana   Protein accumulated to 200– [94]
and adolescents, pharmaceutical and C. vulgaris 600 ng/mL of algal culture
Growth-enhancing effect on flounder C. ellipsoidea Protein accumulated to 420 μg/L [95, 96]
fry, feed additives of algal culture
Mercury removal, bioremediation Chlorella sp. DT Enhanced mercury removal ability [74]
compared with WT
Increase phytate phosphorus Chlorella sp. About 4.2-fold increase in phytase [70]
­utilization, feed additives MACC/C95 activity compared with WT
Provide protection to penaeid C. vulgaris Soluble protein expression [91]
shrimp against infection caused  
by WSSV
Control mosquito populations C. desiccata and Protein accumulated to about [99]
Chlorella sp. 2–30 μg per 107 algal cells
Anti-bladder cancer therapy C. vulgaris Protein accumulated to 1.1%   [89]
of total soluble protein
Antibacterial activity, pharmaceutical C. vulgaris Soluble protein expression [72, 73]
Antibacterial and antifungal activity, C. ellipsoidea Protein accumulated to [75, 98,
pharmaceutical 11.42 mg/L of algal culture 145, 146]

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the nptII gene as a selectable marker. The amount of hGH
produced was claimed to be approximately 200 to 600 ng/
mL. Although the presence of hGH could not be detected
after a few months probably because of the instability of
the foreign DNA, this pioneering work highlighted the
potential application of Chlorella in the expression of
heterologous proteins.
Moreover, a previous study successfully expressed the
flounder growth hormone (fGH) under the control of
CaMV35S promoter in C. ellipsoidea by using the PEG-
mediated method. The G418 resistance (nptII) gene was
utilized to select positive transformants. The product
level was estimated to be 420 μg/L of algal culture [95, 96].
This was claimed to be the first report on the stable
expression of heterologous proteins in Chlorella species.
Flounder fry fed with zooplankton that was fed with fGH-
producing Chlorella biomass for 30 days exhibited a 25%
increase in total length and width. Another protein
phytase that functions by releasing inorganic phosphate
from phytate as a source of nutrients was also claimed to
be stably expressed in Chlorella sp. MACC/C95 in a previ-
ous study [70]. The phytase gene was driven by a CaM-
V35S promoter and delivered to Chlorella cells through
electroporation. The study reported that the G418-resist-
ant transformed cells showed approximately 4.2-fold
increase in phytase activity compared with the wild type
(WT).
In addition, the VP28 gene derived from the white spot
syndrome virus (WSSV) was claimed to be stably
expressed in C. vulgaris in a previous study [91]. The VP28
antigenic peptide was used to provide protection to
penaeid shrimp or prawn against infection caused by
WSSV. The VP28 gene was under the control of SV40
promoter with ampicillin-resistant gene as a selectable
marker and then introduced into the Chlorella genome
through particle bombardment. Chlorella cells that pro-
duced VP28 peptides were then fed to penaeid shrimp.
The study clearly showed that the engineered VP28-
Chlorella was successful in protecting the challenged
prawns against acute death by WSSV. Altogether, these
studies demonstrate the potential use of engineered Chlo-
rella as feed additives in aquaculture and agriculture.
Engineered Chlorella has also been claimed to be fea-
sible for the control of pests. A previous work claimed to
have stably expressed trypsin-modulating oostatic factor
(TMOF) peptide in Chlorella cells [99]. The TMOF peptide
was found to inhibit the biosynthesis of trypsin and
trypsin-like enzymes in mosquito larvae and thus con-
trolled mosquito populations. The TMOF gene was under
the control of CaMV35S promoter with the nptII gene as a
selectable marker and then introduced to the genomes of
Chlorella sp. and C. desiccata by using the PEG-mediated
method. Chlorella cells that produced TMOF were then
fed to mosquito larvae. The study found that almost all the
larvae fed with TMOF-Chlorella died within several days.
This result demonstrates that the expression of exoge-
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nous pesticidal polypeptide in Chlorella is feasible for pest
control.
Various studies have also claimed that using engi-
neered Chlorella as a bioreactor for bioremediation is
feasible. Mercuric reductase (MerA) mediates the reduc-
tion of Hg2+ to volatile elemental Hg0 and has been intro-
duced into land plants, such as Arabidopsis, tobacco, and
rice for phytoremediation purposes [152–154]. Huang et
al. [74] engineered Chlorella sp. DT for the expression of
MerA derived from Bacillus megaterium. MerA was driv-
en by a rice actin1 promoter with hygromycin phospho-
transferase gene as a selectable marker and then deliv-
ered to Chlorella cells through the PEG-mediated method
or via electroporation. The study reported that the trans-
formed Chlorella cells had higher growth rate and photo-
synthetic activity and better capability to efficiently
remove Hg2+ than WT by both in vivo and in vitro assays.
In the presence of mercuric ion, the transformed Chlorella
cells were claimed to exhibit a lower expression level of
superoxide dismutase and a lower level of oxidative stress
than WT. These results indicate that exogenous MerA
can be functionally expressed in Chlorella for the efficient
removal of Hg2+ from the aquatic environment.
Chlorella has elicited attention as a platform for the
expression of therapeutic proteins as well. The single-
chain antibody against bladder cancer (anti-bladder scFv)
is reported to have been stably expressed in C. vulgaris
through particle bombardment. Anti-bladder scFv has a
good potential for use as an anti-bladder cancer therapeu-
tic agent. In a previous study, it was expressed under the
control of a Ubiquitin promoter, and the NR gene was
used for selection of positive transformants. The results
showed that anti-bladder scFv was highly expressed in C.
vulgaris, and the expression level was claimed to be 1.1%
of the total soluble protein. This pioneering study paved
the way for the expression of therapeutic proteins in engi-
neered Chlorella cells. Bovine lactoferrin N-lobe, a peptide
with antimicrobial properties functioning in the nonim-
munological defense system, was also claimed to be sta-
bly expressed under the control of CaMV35S promoter in
C. vulgaris through electroporation [72, 73]. The hygromy-
cin resistance (hpt) gene was used to select transgenic
cells. A previous research also showed that heterologously
expressed lactoferrin has significant antimicrobial activi-
ty against E. coli [73]. However, the lactoferrin yield was
not reported in detail in this study.
Recently, a series of research reported the expression
of rabbit defensin (rabbit neutrophil peptide-1) in C. ellip-
soidea [75, 145, 146]. Similar to lactoferrin, defensins are a
family of small cationic peptides constituting the first line
of defense against pathogens in the innate immune sys-
tem of animals and humans [146]. They exhibit broad-
spectrum antimicrobial and cytotoxic properties against
many Gram-positive and Gram-negative bacteria, fungi,
and some enveloped viruses [75]. Chen et al. [75] initially
reported the expression of defensinin C. ellipsoidea via
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electroporation (transient transformation). The defensin
gene was driven by an ubiquitin promoter, and the nptII
gene was used as a selectable marker. Then, the biologi-
cally active defensin was examined by means of in vitro
antimicrobial tests. Afterward, the same research group
reported to have successfully obtained stable cell lines of
transformed C. ellipsoidea expressing bioactive defensin
[146]. The recombinant defensin was claimed to be as
high as 11.42 mg/L even after 15 generations. This value
is regarded as the highest yield of heterologous proteins
ever reported in engineered Chlorella.
Altogether, these pioneering efforts demonstrate the
great potential of utilizing Chlorella as an attractive sys-
tem for heterologous protein expression ranging from feed
additives to therapeutic proteins.
7    Future challenges in the Chlorella
expression platform
Chlorella offers manifold advantages as an expression
platform for the synthesis of heterologous proteins. How-
ever, this expression platform is still in its infancy and
cannot fully meet the requirements of commercial use.
Many challenges need to be addressed, and many impor-
tant issues need to be resolved for Chlorella-made
heterologous proteins to move forward.
A major challenge is the relatively low yield of heter-
ologous proteins in Chlorella, although this is a common
issue encountered in almost all existing expression plat-
forms. This situation depends on many factors, including
promoters, enhancer or enhancer-like regulatory ele-
ments, codon usage bias, proteolytic degradation, and
gene silencing. The first three factors have been thor-
oughly discussed in this paper. They are highly important
and exert direct effects on the yields of heterologous pro-
tein in Chlorella. At present, the lack of high-efficiency
endogenous promoters and enhancers is the most com-
monly encountered issue in the Chlorella expression plat-
form. This issue often leads to low yields of heterologous
proteins in Chlorella. However, protein expression is
likely to be enhanced in the future through the develop-
ment of strong endogenous promoters, optimal regulating
elements, and codon-optimized strategies. The last two
factors, namely, proteolytic degradation and gene silenc-
ing, are also vital and exert indirect effects on heterolo-
gous protein expression (to be discussed in detail herein
after). In particular, proteolytic degradation is a serious
concern and has been increasingly acknowledged in
recent genetic research [13, 155].
The amount of heterologous protein produced reflects
balance between protein synthesis and degradation; the
latter plays a vital role in determining the overall yield of
heterologous proteins. Heterologous recombinant pro-
teins are usually recognized by the host cells as foreign
and thus undergo degradation much faster than most
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endogenous proteins [156]. Proteolytic enzymes, which
are vital for the effective processing and quality control of
proteins, may be responsible for the degradation of heter-
ologous proteins after synthesis [155]. Although no stud-
ies focused on proteolytic degradation of heterologous
proteins in Chlorella, this condition should be considered
because of the relatively low yields in Chlorella. Many
relevant studies have been conducted on plants and some
microalgae, and these may provide valuable implications
for the Chlorella expression platform [157–159]. At pre-
sent, several remedial strategies are available to minimize
the proteolytic degradation of heterologous proteins in
plants. These strategies include targeting the protein to
the organelles (e.g. endoplasmic reticulum or chloroplast
of plant cells) [160, 161] and co-expression of heterologous
recombinant proteins with specific protease inhibitors
without hindering normal plant growth [162]. These strat-
egies may be applicable to the Chlorella platform, although
relevant mechanisms remain to be fully elucidated.
Another challenge is the stability of exogenous
transgenes in Chlorella. The expression level of exoge-
nous genes in Chlorella can be very low and difficult to
predict (instable), despite the fact that all the elements
required for gene expression (promoters and enhancers or
enhancer-like elements) are involved in gene constructs.
This puzzling issue may be contributed by the multiple
gene integration events and subsequent homology-
dependent gene silencing (HDGS), similar to the mecha-
nisms in plants [163]. Transgenes with multiple integra-
tion loci within the genome, arranged particularly as
tandem-linked inverted repeats (IRs), are likely to induce
HDGS and thus suppress transgene expression [163, 164].
Hence, screening for single-copy integrated transfor-
mants in transformed Chlorella population, although dif-
ficult, may be practicable for stable and high transgene
expression [165]. Given that Agrobacterium-mediated
transformation generally possesses the characteristic of
low copy integration of transgenes, it may be used to
render Chlorella transformants for the stable expression of
heterologous proteins in several cases although it is not a
preferred method.
The low efficacy of transformation methods is also a
serious issue that limits the use of Chlorella as an expres-
sion platform. Although several transformation methods
have been developed for Chlorella transformation, these
methods usually lead to low transformation efficiency
compared with other well-developed expression plat-
forms. This low transformation efficiency entails high
failure probabilities and considerable time and effort in
manual handling, thus making the current Chlorella
expression platform less satisfactory for heterologous pro-
teins. Although enhanced transformation efficiency can
be achieved by developing novel transformation methods
or optimizing the transformation parameters, the main
reason for the low efficacy of existing transformation
methods is probably due to the rigidity and robustness of
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the Chlorella cell wall, which makes DNA delivery diffi-
cult. The rigid cell walls of Chlorella species are complex
and still not fully understood. The main chemical compo-
nents of Chlorella cell walls differ dramatically across
intraspecies [166]. The cell walls of Chlorella species can
generally be divided into two types: glucose-mannose-
rigid wall and glucosamine-rigid wall [27, 167]. Accord-
ingly, differential enzymatic digestion may be utilized to
weaken or remove the Chlorella cell wall to facilitate DNA
delivery to cells. For instance, for Chlorella cells with a
glucose-mannose-rigid wall, cellulase and macerase,
which specifically interact with cellulose (consisting of
glucose) and hemicellulose (containing mannose), respec-
tively, can be utilized in enzymatic cocktail for enzymatic
cell wall removal [168]. Chitinase or lysozyme, which spe-
cifically interacts with chitin (consisting of glucosamine),
can be adopted for the glucosamine-rigid wall [166]. Gen-
erally, in Chlamydomonas, the cell wall-deficient mutants
are easy to transform [169, 170]. Increasing the transfor-
mation efficiency for Chlorella by directly developing such
cell wall-deficient Chlorella mutants may also be benefi-
cial. Furthermore, the natural resistance of Chlorella
against many commonly used antibiotics hinders the
development of proper selectable markers for genetic
transformation. Novel dominant selectable markers, such
as the modified PDS gene, are therefore recommended for
further testing and development.
The lack of genetic resource center for Chlorella spe-
cies is also a challenge in the future sustainable use of
Chlorella as a powerful workhorse for the expression of
heterologous proteins. Considering that a genetic resource
center has been already established for model microalgal
species Chlamydomonas (http://chlamycollection.org/),
such a useful center should also be developed for Chlo-
rella. The establishment of this center will be helpful in
maintaining and distributing stocks of wild type and use-
ful mutants of Chlorella species to the algal community. It
will also provide valuable assistance to researchers toward
the attainment of a comprehensive understanding of the
genetic relationship among key factors that determine
the level of heterologous protein expressed in Chlorella.
8    Perspectives
Although many challenges remain to be addressed, pro-
gress in the utilization of Chlorella for heterologous pro-
tein expression is well underway. Many genetic tools for
the expression of transgenes and gene knockdown have
been developed for Chlorella. Moreover, complete genome
sequence information is available for several species of
Chlorella. This scenario will considerably benefit the
future development of a genetic toolbox for improved
protein expression. Compared with the nuclear expres-
sion system, the chloroplast expression system has sig-
nificant advantages, including controlled site-directed
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recombination of constructs, stable and high expression
level without subjecting to gene silencing, and transgene
containment resulting from maternal inheritance [171].
These desirable features make chloroplast expression
ideal for the expression of heterologous proteins, espe-
cially therapeutic ones. Various functional heterologous
proteins have been successfully expressed in the chloro-
plast of plant cells and even microalgae [1, 172–174], but
not in Chlorella plastid. The establishment of a chloroplast
genetic system for Chlorella may provide an effective
alternative approach for improved expression of heterolo-
gous proteins.
Chlorella shows a promising potential for the expres-
sion of heterologous proteins. Given that most Chlorella
species can produce significant amounts of value-added
fine bioactives, integrating the production of these high-
value co-products with heterologous proteins to reduce
the overall production costs is desirable and would help
the Chlorella platform compete with the other expression
platforms. For example, among the Chlorella species
available with genetic engineering (Table 1), C. vulgaris,
C. zofingiensis, and C. minutissima can produce natural
high-value compounds lutein, astaxanthin, and eicosap-
entaenoic acid (EPA), respectively [175–177]. Combined
production of these value-added biocompounds with
heterologous proteins will offset the production cost to a
certain extent and maximize the value of Chlorella for
cost-effective production. Figure 2 shows C. zofingiensis
as an example and proposes such an integrated produc-
tion process in engineered C. zofingiensis. In this inte-
grated system, C. zofingiensis may be cultured either
heterotrophically or mixotrophically for increased bio-
mass production; the heterologous protein and valuable
co-products (astaxanthin and lipids) can be respectively
collected from the aqueous phase and organic phase of
cell extracts. Exploring other Chlorella species, such as
A. protothecoides (C. protothecoides) and C. pyrenoidosa,
which have been rarely transformed but display attractive
traits, for the possible expression of heterologous proteins
is also promising. A. protothecoides (C. protothecoides)
has long been considered a promising candidate for bio-
diesel production. It has excellent capability to accumu-
late a high content of lipid in cells, particularly long-chain
lipid suitable for biodiesel. It can also use sugars as carbon
sources for heterotrophic cell growth [31, 32, 178]. Based
on these traits, further studies can therefore be conducted
by introducing sugar transporters and sugar-degrading
enzymes to its genome to utilize low-cost carbon sources
for cost-effective biodiesel production. Meanwhile,
C. pyrenoidosa is widely used as an important nutrientin-
gredient (mainly as protein sources) for the food industry
and aquaculture [179, 180].
Culture processes can also significantly affect the
expression level of heterologous proteins. These processes
include cultivation methods (e.g. photoautotrophic or het-
erotrophic), media compositions (e.g. carbon and nitrogen
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sources), cultural conditions (e.g. temperature, pH, and air 9    Conclusions

flow rate), and harvesting and refining methods.
Chlorella as an expression platform has achieved certain
progress since the first attempt to express heterologous
proteins in Chlorella. Although the yield of heterologous
proteins remains relatively low, Chlorella offers such sig-
nificant advantages as rapid growth, ease of culture for
high cell density, and ability to perform post-translational
modifications. Further improvement of its production
economics remains the major challenge in the commer-
cialized production of heterologous proteins in Chlorella,
which depends on significant advances in the develop-
ment of a novel genetic toolbox for stable and improved
protein expression, culture systems, and downstream
biorefinery-based integrated production. Innovations and
breakthroughs that occur in these areas will greatly
expand the production capacity with reduced production
costs, thus enabling Chlorella to become a competitive
host for the expression of heterologous proteins. Given the
increased interest in using Chlorella biomass as feedstock
for oils, significant achievements have been obtained in
heterotrophic culture systems and production models for
the algae of this genus, thereby allowing for ultrahigh cell
density that is comparable to that of yeast. To this end,
both the genomes and transcriptomes of several typical
Chlorella strains have been made available, and more
strains are currently underway. This action will benefit
the future rational manipulation of Chlorella for economi-
cally feasible industrial production.

The authors acknowledge the final support provided by

the 985 Project of Peking University, the 863 Plan of
Ministry of Science and Technology of China (No.
­
2012AA023107) and the National Natural Science Foun-
dation of China (No. 31471717and No. 31571807).

The authors declare no financial or commercial conflict of

interest.

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14 © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotecvisions.com
Dr. Jin Liu is a National Youth Thou-
sand Talents Program fellow of China
and an assistant professor at College of
Engineering, Peking University, China.
He obtained his PhD degree in Biotech-
nology from the University of Hong
Kong. Before joining Peking University,
he worked in Arizona State University
and University of Maryland Center for
Environmental Science as a postdoc and a research scientist, respec-
tively. His research interests include algae physiology and biotechnolo-
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