Algal Research 25 (2017) 76–89

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Review article

Microalgae and cyanobacteria as enzyme biofactories MARK

a a
Bruno dos Santos Alves Figueiredo Brasil , Félix Gonçalves de Siqueira ,
Thaís Fabiana Chan Saluma, Cristina Maria Zanetteb,c, Michele Rigon Spierb,⁎
a
Embrapa Agroenergy, Brasília, Federal District, Brazil
b
Federal University of Paraná, Technology Sector, Chemical Engineering Department, Post-Graduation Program in Food Engineering, Curitiba, Parana, Brazil
c
Midwestern State University (UNICENTRO), Food Engineering Department, Guarapuava, Parana, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Microalgae consist of a diverse group that includes prokaryotic cyanobacteria and eukaryotic photosynthetic
Photosynthetic microorganisms microorganisms that inhabit freshwater and marine habitats. Microalgae can be used in different industries,
Enzymes including as food for human consumption, as animal feed, in aquaculture, in cosmetics and as biofuels. Because
Recombinant proteins they are photoautotrophs, with minimal nutritional requirements, microalgae have advantages compared to
Bioactive compounds
other microbial cells. An overview of the great potential of these cells to synthesize enzymes for several
Biomanufacturing
industrial applications is provided. Photosynthetic microorganism-derived enzymes include cellulases, galacto-
sidases, proteases, lipases, phytases, laccases, amylases, antioxidant enzymes and enzyme associated with
carbohydrate accumulation and the carbon concentration. Furthermore, recent reports on microalgae genomics
are revealing a variety of novel genes that should be investigated for biotechnological applications. Exploring
algal genetic diversity will also enable the efficient use of photosynthetic microorganisms as recombinant
enzyme biofactories that will be useful to industry.

1. Introduction year respectively), each with approximate global production values of

Microalgae are microscopic organisms that contain chlorophyll and
are found in freshwater and marine habitats [1]. The term “microalgae”
refers to prokaryotic cyanobacteria and eukaryotic photosynthetic
microorganisms primarily found within the taxa Chlorophyta, Rhodo-
phyta, Glaucocystophyta, Euglenophyta, Chlorarachniophyta, Heterokonta,
Haptophyta, Cryptophyta and Alveolata [2].
Therefore, microalgae consist of a very diverse group with a
significant unexplored genetic potential; the number of known species
represents only 1% of what is believed to exist [3–5]. Moreover, recent
reports on microalgae genomics are finding a variety of novel genes and
a large functional genetic diversity that may be investigated for
biotechnological use [6].
Microalgae have been studied for more than 100 years, and they are
currently being cultivated in large scale, mainly in open pond systems.
The annual worldwide production is approximately 7,000 tons of dry
algal biomass, and the global algae biomass market is worth between
USD 3.8 billion and USD 5.4 billion. The health food sector accounts for
USD 1.5 billion, and aquaculture applications account for USD 0.51
billion [7]. Dietary supplements containing Chlorella and Arthospira
species, which are used as dried whole microalgae, represent the largest
production volumes worldwide (2,000 and 5,000 tons of dry matter/
USD 40 million/year [8].
The potential uses of microalgae extend beyond the products studied to
date. Recent studies emphasize the potential of algal biomass as an
alternative to fossil fuels as raw material for the production of biofuels
such as biodiesel, bioethanol, biohydrogen, and biomethane [9–11]. Several
reports have established that microalgae can be sources of biomass for the
production of fine chemicals production including chlorophylls, β-carotene,
astaxanthin, phycocyanin [4] and omega -3 fatty acids [12,13]. There are
also numerous potential commercial applications of microalgae, including
in the paper industry as fiber/polymer composites and as a filler material
[14,15], in cosmetics formulations [16], for soil restoration, as supplements
in animal feed [17], for phycoremediation [4,18], and in therapeutic
supplements for their polysaccharides (β-glucan) [1], essential amino acids
[19], lipids (tri- and diglycerides, phospholipids, glycolipids), alkenes,
alginates (from brown algae), agar, and carragenates (from red algae)
[20]. Additionally, microalgae are reported to efficiently adsorb and recover
heavy metals [21] and can be used in wastewater treatment in integrated
processes [22–24].
Although some reviews focus on the utilization of microalgae for
value products and biotechnological applications [1,9,21,25,26], none
address the potential of microalgae to be used as producers of
industrially important enzymes.

⁎
Corresponding author at: Chemical Engineering Department, Post-Graduation Program in Food Engineering, P.O. Box 19011, Zip Code: 81531-980, Curitiba, Paraná, Brazil.
E-mail address: spier@ufpr.br (M.R. Spier).

http://dx.doi.org/10.1016/j.algal.2017.04.035
Received 15 August 2016; Received in revised form 6 February 2017; Accepted 28 April 2017
2211-9264/ © 2017 Elsevier B.V. All rights reserved.
B.d.S.A.F. Brasil et al.
Microalgae have several advantages compared to other microbial
cells in industrial enzyme synthesis due to their minimal nutritional
requirements (natural or artificial light, CO2, water, nitrogen source,
and some salts), which reduce cost. Nevertheless, microalgae can have
disadvantages for enzyme production, such as longer cultivation time
compared to bacteria, yeast or fungi and additional stages downstream
to recover intracellular enzymes. Furthermore, the majority of biomo-
lecules are produced intracellularly in small quantities, which makes
analysis and industrial scale-up difficult. However, recent advances in
cell lysis techniques have provided a high extraction yield of several
metabolites from microalgae [27,28].
This review focuses on the great capacity of microalgae cells to
synthesize enzymes for different industrial applications. The enzymes
reported included cellulases, amylases, galactosidases, proteases, li-
pases, phytases, laccases, antioxidant enzymes, and enzymes involved
in carbohydrate accumulation and carbon concentration. Bioprocesses
for enzyme production are addressed. Separation processes are also
discussed, microalgae cell walls are rigid and require specific unit
operations to ensure lysis (cell breaking) while preserving the integrity
of the released cytosolic or membrane-associated enzymes. Finally,
recent advances in recombinant enzyme expression in microalgae and
cyanobacteria are discussed, including the model species and genetic
tools available.
2. Parameters for microalgae cultivation
The primary factors of importance in microalgae cultivation process
are light, nutrients, aeration, temperature and pH. For maximal biomass
yield, the microalgae culture must be supplied with macronutrients and
micronutrients in adequate amounts. Macronutrient requirements
include carbon, nitrogen, phosphorus, sulfur, potassium (also silicon
for diatoms), and trace elements as minerals (e.g., Co, Mo, Mn-enzymes
cofactors) and vitamins (cyanocobalamin, thiamin and biotin) [29,30].
As the carbon source in photoautotrophic microalgae metabolism,
most prefer to utilize free CO2. In an alkaline culture medium, some
microalgae such as Chlorella, Dunaliella and Arthospira can assimilate
bicarbonate [30]. In heterotrophic growth, microalgae grow in the
dark, and cells obtain energy exclusively from organic carbon in the
media. In mixotrophic growth, microalgae can obtain energy from both
light and different organic carbonic sources including glucose, glycerol,
acetate and fructose [31,32].
Few studies have estimated the influence of medium composition on
enzyme production by microalgae. It was observed in Dunaliella salina
cultures that altering the nitrogen source in the medium can increase
the activity of specific enzymes [32]. Guzmán-Murillo et al. [33]
evaluated different agricultural fertilizers as nitrogen sources to scale
up the photoautotrophic production of superoxide dismutase (SOD) by
Phaeodactylum tricornutum. The maximum concentration of intracellular
SOD produced was 79 and 72 U mg− 1 protein using media containing
nitrogen from ammonium sulfate and urea, respectively. These results
indicate that an alternative nitrogen source can be used for the
synthesis of SOD at reduced costs. Table 1 summarizes parameters
related to the production of microalgae enzymes described in the
literature.
3. Algae genetic diversity and potential for enzyme production
Photosynthetic organelles (plastids or chloroplasts) were primarily
derived from the endosymbiotic uptake of a cyanobacterium.
Subsequently, these primary plastids (such as those found in chlor-
ophytes and rodophytes) were spread to other eukaryotic clades
through subsequent rounds of secondary and even tertiary endosym-
biosis [46,47]. These evolutionary events led to the presence of the
“algal phenotype” characterized as photosynthetic unicellular or plur-
icellular (without tissue organization) organisms found in humid
environments within 12 of the 45 major eukaryotic clades and, in the
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Algal Research 25 (2017) 76–89
prokaryotic cyanobacteria clade [46,48,49]. Using conservative ap-
proaches, the number of eukaryotic algal species was estimated to be
72,500 [50] and the number of cyanobacteria species was estimated at
approximately 6,000 [51].
Therefore, simple algal morphologic features hide a huge polyphy-
letic genetic reservoir, orders of magnitude larger than that found
within animals or plant taxa [52–54]. For instance, while Homo sapiens,
Mus musculus and Danio rerio present 52.8% of their gene sets as
orthologs, only 16.3% of orthologous genes are found among Chlamy-
domonas reinhardtii, Thalassiosira pseudonana and Cyanidioschyzon mer-
olae genomes [6].
Uncovering algal genetic diversity will enable access to novel genes,
including industrially valuable enzyme-encoding genes, for future
biotechnological exploration. There are approximately 14,500 anno-
tated sequences from enzyme-coding genes derived from algae species
available in GenBank at present (Figure 1). Furthermore, considerable
advances in algal genomics and in silico predictive capacity are being
made [55]. There are over 60 eukaryotic microalgae and more than 40
cyanobacteria genome sequencing projects either published or under-
way (www.ncbi.nlm.nih.gov/genomes; http://genome.jgi.doe.gov)
[6,55,56]. Bioinformatic tools, such as Kyoto Encyclopedia of Genes
and Genomes (www.genome.jp/kegg), BioModels databases (www.ebi.
ac.uk/biomodels/), CpBase (Chloroplast Genome Database), MitoBase
(Mitochondria Genome Database), and MMETSP (Marine Microbial
Eukaryote Transcriptome Sequencing Project) are enabling the predic-
tion and characterization of gene regulatory pathways and the annota-
tion of de novo genomes of algal species. In addition, an increasing
number of transcriptomics, proteomics, and other systems biology
studies are being reported [25,57]. There are also several in silico
analysis platforms, such as The Greenhouse (https://greenhouse.lanl.
gov/greenhouse/), pico-PLAZA (http://bioinformatics.psb.ugent.be/
pico-plaza/), Phytozome (https://phytozome.jgi.doe.gov/pz/portal.
html), IMG - Integrated Microbial Genomes (https://img.jgi.doe.gov/
), CyVerse (http://www.cyverse.org/), KBase (https://kbase.us/),
EDGE bioinformatics (https://bioedge.lanl.gov/edge_ui/), Galaxy
(https://galaxyproject.org/) and Ergatis (http://ergatis.sourceforge.
net/), that can allow the identification of novel algal encoded variants
(i.e., gene orthologs) from known bacterial or fungal encoded enzymes.
These approaches could also help to identify key biosynthetic genes
for the production of high-value products in microalgae, which could in
turn be altered using molecular tools [55,56,58,59]. An example is the
genetic transformation of the carotenoid-producing microalgae Haema-
tococcus pluvialis with an additional version of the phytoene desaturase
gene, which led to a 26% increase of its astanxanthin content [60].
Another example is the expression of the phytoene synthase genes
derived from Dunaliela salina and Chlorella zoofingensis in transgenic
Chlamydomonas reinhardtii, which increased lutein content more than 2-
fold [61,62].
In addition, the advent of flexible and simple new gene-editing
tools, such as CRISPR/CAS9 technology, offers promising opportunities
in eukaryotic microalgae metabolic engineering. The CRISPR/CAS9
system consists of a specific guide RNA (gRNA) and a non-specific
CRISPR-associated endonuclease protein (Cas9). The Cas9 endonu-
clease promotes a double strand break (DSB) in the DNA loci targeted
by the gRNA sequence. After the DSB, the cell’s DNA repair mechanism
either promotes a non-homologous end joining, which usually leads to
an InDel mutation, or promotes a homology-directed repair that
replaces the targeted sequence with a co-transformed exogenous
sequence [63]. Recent studies have reported the successful use of
CRISPR/CAS9 systems in Phaeodactylum tricornutum, Chlamydomonas
reinhardtii, Nannochloropsis spp. and Thalassiosira pseudonana, establish-
ing novel paths for genomic sequence editing in microalgae [64–68].
4. Microalgal enzymes
Enzymes from microorganisms could be used as catalysts in diverse
 B.d.S.A.F. Brasil et al.

Table 1
Culture conditions and disruption methods for recovering intracellular enzymes from microalgae

Microalgae/cyanobacteria Enzyme Culture conditions Disruption method References

Synechococcus lividus SKP50, S. lividus DSK74, S. Phytase Cultivation at 50 °C under illumination of 2,500 lux. Approximately Not described Klanbut and
bigranulatus Skuja and Chroococcidiopsis phosphate-free medium containing 0.1% (w/v) calcium phytate as the Peerapornpisarn [34]
thermalis geitler sole phosphorus source supplemented with 0.5% (w/v) glucose and 0.02
% (w/v) casamino acids
Poterioochromonas malhamensis peterfi α-galactosidase Nutrient medium containing glucose (10 mg/mL), aerated at 27 °C, and French press and sonicator Dey and Kauss [35]
illuminated with one daylight fluorescent tube at a distance of
approximately 35 cm (2 klux) for approximately 1 h
Anabaena variabilis ATCC 29413 Protease Photoautotrophic with N2 as the nitrogen source Thawed in a buffer and passed through a French press Lockau et al. [36]
at 140 MPa
Anabaena variabilis Prolylendopeptidase Photoautotrophic with N2 or NH4NO3 as nitrogen source in continuously Sonication and centrifugation at 48, 000g for 30 min Strohmeier et al. [37]
illuminated batch culture
Anabaena variabilis Aminopeptidase Grown at ambient temperature under constant illumination at 1,300 lux, Thawed in a buffer and passed through a French press Niven [38]
and cultures were bubbled with air. at 80 MPa
Arthrospira platensis Protease Stirring at 4 °C overnight in buffer pH = 8, Nanni et al. [39]

78
Grown at 30 °C in Erlenmeyer flasks, continuously shaken and aerated
with CO2-enriched (5%) air, irradiance of 70 mmol photon m–2 s–1 centrifugation at 30,000g for 1 h
A. platensis Protease Not described Citrate buffer 50 mM, pH 5 at room temperature for Yada et al. [40]
1 h followed by centrifugation at 14, 000g for 30 min
Porphyridium cruentum SOD Photoautotrophic in salt medium under aeration Thawed in a buffer and passed through a French press Misra and Fridovich [41]
at 20,000 psi
Anabaena cylindrica SOD Grown aerobically in nitrogen-free BG 11 medium at 25 °C and at a Cells were resuspended in potassium phosphate buffer Canini et al. [42]
photon fluence rate of 25 μmol m− 2 s− 1 (pH 7.8) containing EDTA and sonicated in an ice-
bath.
A. platensis SOD Grown at 25 °C under the light intensity of 28 μmol photons m− 2 s− 1 in Sonication operated with Gunes et al. [43]
Pseudanabeana sp. glass bottles for 10 days 80 % power output for 10 min over 8 pulsation cycles
Synechococcus nidulans
Botryococcus sudeticus Lipase Grown in various oils (canola, corn, palm and olive) in modified Bold’s None (lipase is extracellular) Yong et al. [44]
basal medium at ambient temperature with 24 h of synthetic white light.
Nannochloropsis oceanica Lipase Grown in seawater enriched with F/2 medium nutrients at 120 rpm, 20 °C Sonication at 0 °C in 5 1-min cycles, with a cooling Savvidou et al. [45]
and pH 8.0. Continuous illumination at 100 μmol photon m− 2 s− 1 on the time of 1 min between cycles. Centrifugation at 4 °C
culture surface. and 7,000g for 10 min
Algal Research 25 (2017) 76–89
B.d.S.A.F. Brasil et al.
Figure 1. Number of enzyme-coding genes from algal species. Graphics represent the numbers of predicted enzyme-coding genes sequences from eukaryotic algae and cyanobacteria
deposited in GenBank (http://www.ncbi.nlm.nih.gov/gene/) by November, 2016. The six major enzyme classes (hydrolases, oxidoreductases, transferases, ligases, isomerases and lyases)
are represented by colored bars.
industrial processes. The search for new sources of microbial enzymes is
ongoing and requires sustainable solutions [69]. In 2014, the global
market for industrial enzymes was estimated at USD 4.2 billion and is
expected to reach nearly USD 7.1 billion by 2018 [70].
Although there is no industrial production of enzymes from micro-
algae, several reports show the great capacity of microalgae cells to
synthesize enzymes. Different classes of enzymes were reported,
including hydrolases, oxidoreductases and lyases.
4.1. Cellulases
Cellulases, hemicellulases and pectinases can be collectively re-
ferred to as holocellulases; these enzymes are capable of degrading the
cell wall carbohydrate polymers of plants or algae [71]. This enzyme
group is usually found in microbial decomposers of plant and algal
organic matter, e.g., filamentous fungi, on soil and in fresh or salt
water. Cellulose degradation by fungi, for example, occurs via the
synergistic activity of three types of cellulases: endoglucanases (EC
3.2.1.4; endo-β-1,4-glucanases), cellobiohydrolases (EC 3.2.1.91) and
β-glucosidases (EC 3.2.1.21). The main potential applications of
cellulases are in the food (to break down non-digestible carbohydrates),
animal feed (enhancing fiber degradation), textile (biostoning for jeans
and biopolishing for cotton), biofuel (cellulosic ethanol), and chemical
industries [71,72].
Algae and microalgae are the major contributors to the production
of primary energy in the aquatic environment. Some organisms (e.g.,
Euglena) produce polysaccharides for carbon storage such as paramylon
and require β-1,3-glucan hydrolase to metabolize the accumulated
carbohydrates [73].
Aquatic microorganisms are the primary decomposers of polysac-
charides-storage by algae. The degradation of these sugars are per-
formed by enzymatic-path microorganisms (CAZymes), thereby pro-
moting a major carbon cycling processes that is established in the
Earth’s ecological cycles [74]. Cellulases comprise a group of enzymes
known to exhibit features of synthesis (chitosan) or the degradation of
poly-oligo-dimers saccharides [75,76]. However, few manuscripts re-
port the production of cellulases, hemicellulases, and pectinases by
microalgae to degrade these polysaccharides in aquatic environments,
even though microalgae and algae possess genes encoding these
enzymes [77–79].
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Algal Research 25 (2017) 76–89
Anabaena strains were reported as potential organisms that produce
hydrolytic enzymes with possible fungicidal activity [77,80,81]. One
research group identified chitosanase homologs in A. iyengarii and A.
laxa. The cyanobacterial strain A. laxa showed putative genes (end 1
and end 2) encoding β-1,4-endoglucanase, which is in the peptidase
family, and glycoside hydrolase [77]. The algal polysaccharidases
(marine carbohydrase in CAZymes classification) identified to date
(agarases, carrageenases, and alginate lyases) display unique structures
and biochemical features and are distantly related to the currently
known glycoside hydrolases [82]. These findings highlight the potential
for new and original biotechnological uses of these enzymes.
4.2. Amylolytic enzymes
Amylases are a group of enzymes that promote the hydrolysis of
starch, oligosaccharides, and polysaccharides. The best-known amy-
lases are α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), glucoamylase
(E.C 3.2.1.3), isoamylase (EC 3.2.1.68) and glucosidases (EC 3.2.1.20)
[83].
Levis and Gibbs [84] reported α-amylase activity in C. reinhardtii
grown photoautotrophically in 12-h/12-h light/dark cycles. Amylase
reached maximum activity after 4 h of darkness but was active through
the entire cell cycle.
Rismani- Yazdi et al. [85] and Shang et al. [86] studied genes that
encode enzymes involved in the catabolism of starch in the Dunaliella
genus. Two different pathways were identified, a phosphorolytic and a
hydrolytic pathway. In D. tertiolecta, the enzymes associated with
hydrolytic metabolism are α-amylase and oligo-1,6-glucosidase. α-
Amylase and β-amylase were identified in D. parva. These three
enzymes are responsible for starch metabolism to produce D-glucose.
For the phosphorolytic degradation of starch, glycogen phosphorylase
was found in D. parva, and starch phosphorylase was found in D.
tertiolecta.
4.3. Galactosidases
α-D-galactoside galactohydrolase (EC 3.2.1.22), also known as α-
galactosidase, hydrolyzes α-1,6-linked galactose residues found in
oligosaccharides and galactomannan polysaccharides and releases α-
D-galactose [87]. α-Galactosidases are useful in the paper and pulp,
B.d.S.A.F. Brasil et al.
sugar, and food and animal feed industries [88].
Intracellular α-galactosidase (α-gal) activity has been detected in
Poterioochromonas malhamensis, unicellular golden-brown algae that
exhibit a mechanism of osmotic regulation. P. malhamensis α-gal
presents optimal activity at pH 7.0 in cell-free extracts. The intracellular
enzyme activity increases under conditions of high external osmotic
pressure. This condition causes cell shrinkage within a few minutes. As
a mechanism of recovery, the cell biosynthesizes intracellular isoflor-
idoside (α-galactosylglycerol). Higher external osmotic pressure in-
duces the production of α-galactosidase, which then reduces the
intracellular concentration of isofloridoside (O-α-D-galactopyranosyl-
(1 → 1) glycerol), thus releasing glycerol and galactose. Some of the
carbon released is incorporated into β-glucans reserves in the algal cell
or as wall components [35]. Isofloridoside apparently acts as a
substrate to induce α-gal catalysis when the osmotic pressure outside
the cell causes a rapid decrease in its internal levels. The formed
products are used by the cell as osmoregulators or possibly as carbon
sources. Galactose and glycerol accumulate within the cell as an
osmoregulatory mechanism to maintain vital cellular activities.
β-Galactosidase (β-D-galactohydrolase, EC 3.2.1.23), also called
lactase, hydrolyzes D-galactosyl residues from oligosaccharides, poly-
mers and secondary metabolites [89]. The main industrial application
has been the hydrolysis of lactose in milk and dairy products. More
recently, β-galactosidases with transgalactosylation activities have been
extensively exploited for the synthesis of galactooligosaccharides,
which are used as probiotic food ingredients [90,91].
Davies et al. [92] tested macro and microalgae species for β-
galactosidase activity. Eight of nine species from the Chlorophyceae
and Cyanophyceae families presented some β-D-galactosidase activity.
High enzyme activity was shown in Cosmarium sp. and Acutodesmus
obliquus. Girard et al. [93] tested cheese whey permeate as a partial
substitute for culture medium in the mixotrophic cultivation of A.
obliquus. Lactose was metabolized by microalgae, thus promoting a
reduction in lactose concentration and an accumulation of glucose and
galactose in the culture medium and, indicating the extracellular
production of β-galactosidase by microalgae.
Several studies reported α-galactosidase [94,95] and β-galactosidase
production from yeast, molds and bacteria using whey and other agro-
industrial wastes as substrates. These agro-industrial byproducts could
be potential inducers of galactosidase synthesis by microalgae. α-
Galactosidase inducers included D-galactose, melibiose, stachyose and
raffinose. The β-galactosidase inducer is lactose. Future work is
required to increase knowledge about the importance of α- and β-
galactosidases in the metabolism of microalgae.
4.4. Proteases
Proteases constitute a wide group of enzymes that catalyze peptide-
bond cleavage in proteins and peptides [96]. Proteases have a wide
range of metabolic functions and industrial applications, primarily in
the detergent, pharmaceutical and food industries [97].
In microalgae metabolism, protease activity increases during envir-
onmental stress such as light or nutrient limitation and cell apoptosis.
Proteolysis also plays a role in the control of organelle senescence and
heterocyst formation in cyanobacteria [98].
Lockau et al. [36] described a calcium-dependent serine protease
from the cyanobacteria Anabaena variabilis. The enzyme is a trypsin-like
protease that, requires calcium for full activity. Using A. variabilis
mutant IM 141, which lacks this calcium-dependent protease, Stroh-
meier et al. [37] showed that A. variabilis contains a second soluble
trypsin-like protease, a prolylendopeptidase.
The first report of the purification of aminopeptidases (EC
3.4.11.15) from cyanobacteria was performed by Niven using A.
variabilis (1995). Two enzymes (188 kDa and 59k Da) were separated
by gel filtration chromatography and showed different substrate
specificity profiles. The larger enzyme showed specificity for di- and
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Algal Research 25 (2017) 76–89
tri-peptides, whereas the smaller enzyme was capable of hydrolyzing
longer oligopeptides.
Protease production from Arthospira platensis was reported by Nanni
et al. [39] and Yada et al. [40]. In the first study, a novel serine protease
associated with the selective proteolysis of phycobiliproteins was
isolated, purified and partially characterized. Yada et al. [40] purified
a new arginine-specific protease (Sp-protease) that hydrolyzed gelatin
and fibrin, however, phycocyanin was not hydrolyzed. Both enzymes
from A. platensis had a molecular weight of 80 kDa and act indepen-
dently of Ca+ 2.
Because studies have reported the ability of microalgae to produce
proteases and because production is associated with the availability and
nature of the nitrogen source [99], future studies should evaluate
changes in the nitrogen source in the medium, to induce protease
activity in microalgae.
4.5. Lipases
Lipases (triacylglycerol acyl hydrolases, E.C. 3.1.1.3) are enzymes
that naturally hydrolyze triglyceride into fatty acids and glycerol.
However, in aqueous-restricted environments, they can catalyze ester-
ification, transesterification, inter-esterification, and aminolysis
[100–102].
Lipases are among the most important enzymes for biotechnological
applications. They are useful in the detergent, food, flavor, pharma-
ceutical, chemical, agrochemical, and cosmetics industries [103].
Few microalgal lipases and genes encoding lipases have been
investigated, compared to bacterial, fungal, animal and plant lipases.
A lipase from the photosynthetic cyanobacterium A. platensis was
isolated and characterized for the first time by Demir and Tukel
[104]. The lipase presented a pNPP hydrolyzing activity of 45 U
mg− 1 protein after being purified 375-fold, and the protein is mono-
meric and specific for the 3-position in the ester bond. Godet et al.
[105] isolated a new gene from the microalgae Isochrysis galbana that
encode a 49-kDa lipase of 457 amino acids. The deduced protein shares
similarities with known lipases. Phylogenetic analyses showed simila-
rities to fungal lipase sequences.
In the past ten years, the main microalgae species that has been used
as a model to understand lipid metabolism in green microalgae is
Chlamydomonas reinhardtii. Many putative lipases are encoded in the
Chlamydomonas genome. As with many other algae, the photosynthetic
microalgae C. reinhardtii accumulates triacylglycerols (TAGs) when
deprived of nitrogen, a condition that causes genome-wide transcrip-
tional changes. Among the genes regulated by nitrogen deprivation are
numerous genes encoding lipases [106]. Li et al. [107] described the
identification of a mutant from C. reinhardtii with a lesion in a
galactoglycerolipid lipase-encoding gene. This gene is one of the
lipase-encoding genes that is upregulated when microalgae are de-
prived of nitrogen. Disrupting a galactoglycerolipid lipase-encoding
gene caused a reduction of TAG content and galactoglycerolipid
hydrolysis. Mansfeldt et al. [108] also confirmed that a galactoglycer-
olipid lipase gene from a Chlorella sp. strain is upregulated in these
conditions. In C. reinhardtii, one of the downregulated genes was
recently shown to encode a lipase implicated in TAG hydrolysis
[109]. They identified the lipase-encoding gene CrLIP1, and they
suggested that the genes repressed during TAG accumulation might
encode lipases that hydrolyze TAG, DAG (diacylglycerol) or MAG
(monoacylglycerol). When the quantity of the CrLIP1 transcript was
reduced, there was a delay in TAG hydrolysis upon nitrogen resupply,
confirming that CrLIP1 is involved in Chlamydomonas TAG lipolysis.
The data obtained suggest that the lipase encoded by CrLIP1 hydrolyses
the DAG formed from TAG lipolysis, facilitating TAG turnover.
Recently, Savvidou et al. [45] produced and characterized a lipase
from Nannochloropsis oceanica. The microalga was grown in seawater
enriched with nutrients. The cultures were incubated at 20 °C and pH 8
with 24 h of synthetic light. Lipase was extracted by sonication and
B.d.S.A.F. Brasil et al.
characterized. This lipase showed hydrolysis activity against pNP esters
of fatty acids with different chain sizes, and the optimum pH was 7.
Yong et al. [44] also reported the production of lipase from a microalga
strain. They produced, purified, and characterized an extracellular
lipase from Botryococcus sudeticus. They studied the effect of different
oils (palm, canola, corn and olive) in the culture medium and the effect
of agitation on lipase production. Continuous illumination was main-
tained with synthetic white light and cultures were kept at room
temperature. Olive oil in the medium and agitation provided the best
conditions to produce lipases. The lipase was purified and presented a
molecular mass of 120 kDa and maximal activity at 50 °C and pH 10.
The lipase also has good thermal stability, showing its potential to be
applied in different industries.
4.6. Phytases
Phytases (myo-inositol hexakisphosphate phosphohydrolase) initi-
ate the removal of phosphate residues from phytate, the principal
molecule for organic phosphorus (P) storage in grains and plant seeds
[110]. Phytases can be added to animal feed to increase the bioavail-
ability of P, and recent studies have shown the potential use of phytases
in food processing [111,112]. Phytases are classified into the following
three types depending on the position of phytate dephosphorylation
initiation of the phytate: 3-phytases (E.C 3.1.3.8), 6-phytases (E.C
3.1.3.26) and 5-phytases (E.C 3.1.3.72) [113].
Klanbut and Peerapornpisarn [34] found phytase activity in the
intracellular fraction of the following four species of thermotolerant
blue-green algae isolated in Thailand: Synechococcus lividus SKP50, S.
lividus DSK74, S. bigranulatus Skuja, and Chroococcidiopsis thermalis
Geitler. The maximum enzyme activity was 1.83 mU mL− 1, which was
achieved by S. lividus SKP50. Phytase activity was not found in the
extracellular culture broth.
Further studies could be performed to assess whether microalgae
are capable of producing phytase and whether the enzyme is extra-
cellular or cell disruption is necessary for recovery. In addition, medium
composition can be assessed in future studies to induce the synthesis of
enzymes using specific substrates (such as the organic phosphorus
source). It has been reported that phytic acid stimulates microbial
phytase synthesis [114].
4.7. Laccases
Typical laccases (EC 1.10.3.2) are extracellular monomeric glyco-
proteins that belong to the blue multicopper oxidase family that
includes ascorbate oxidase, bilirubin oxidase, ceruloplasmin, and others
[115]. Laccases can promote the oxidation of complex polymeric
structures such as lignin into phenolic compounds [116]. These
enzymes are considered ideal “green catalysts” since they can oxidize
a wide variety of compounds using ambient O2 from the air and
releasing H2O as the only byproduct [115].
The natural functions of laccases include the degradation and
biosynthesis of lignin [117]. They have great relevance as models for
structure/function relationships and as sustainable tools for industrial
processes, demonstrating many applications in the food (brewing, color
enhancement in tea, cork modification), textile (denim bleaching and
finishing), and pulp and paper industries (paper pulp delignification
[118]. Laccases are involved in morphogenesis processes, detoxification
(soil), immunity and plant pathogenicity and bioremediation processes
[119].
Laccases and laccase-like enzymes are found in insects, plants,
microorganism (white-rot fungi), prokaryotes and lichens [120]. Ad-
ditionally, laccase activity has been described in the soil algae
Tethacystis aeria [121] and Shewanella xiamenensis, which show laccase
activity on the substrate 2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic
acid (ABTS) [122]. The Shewanella genus is capable of dissimilatory
manganese and iron oxide reduction ([123–125], apud [122]). The
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Algal Research 25 (2017) 76–89
coccoid green algae T. aeria produces an extracellular laccase-like
enzyme that can oxidize phenolic substances and the anthraquinonic
dye Abu62 [121]. Confirmation of this enzyme as a laccase indicates
that algal laccases might contribute to the bio-transformation of several
natural and xenobiotic aromatic compounds [126]. Species of the
“Moewusinia” clade, including Chlamydomonas moewusii and T. aeria,
secrete putative laccases that might help algae survive in harsh
environments [127].
The laccases of green algae have been reported as biomolecules
capable of breaking down phenolic pollutants in contaminated surface
waters, biodegrading various xenobiotics and textile dyes from water
[128–131]. In addition, algae have been shown to effectively treat
wastewaters from the paper industries without requiring co-substrates
[132].
4.8. Antioxidant enzymes
Reactive oxygen species (ROS) are generated in aerobic organism as
result of respiration and substrate oxidation. Environmental stresses
such as intense light, heavy metals, herbicides, UV radiation, high salt
concentrations, and extreme temperatures stimulate ROS production.
Consequently, microalgae possess antioxidant defense mechanisms that
combat ROS cell damage. Enzymatic antioxidant defenses include SOD,
glutathione reductase, catalase and peroxidase [133].
4.8.1. Superoxide dismutase
Superoxide dismutase (EC 1.15.1.1) are metalloenzymes that con-
vert superoxide radicals (O2•) into oxygen (O2) and hydrogen peroxide
(H2O2). The applications of SOD include therapeutic and prophylactic
applications in humans, in the preservation of biological materials
(organs for transplantation and sperm), in the preservation of perish-
able materials such as foodstuffs and vaccination agents and as an
antigenic agent for the serodiagnosis of pathogens [134].
SODs are present in prokaryotic and eukaryotic cells, but relatively
little information is available regarding SOD activity in microalgae
[135]. Misra and Fridovich [41] purified a Mn-SOD from the micro-
algae Porphyridium cruentum. The enzyme had a molecular weight of 40
kDa and was stable during freezing and thawing.
Canini et al. [42] report the purification of Fe-SOD from the
nitrogen-fixing cyanobacteria Anabaena cylindrica. Gunes et al. [43]
investigated and compared SOD activities in S. nidulans, A. platensis and
Pseudanabeana sp and found the specific activities of 50.4 U mg− 1,
30.0 U mg− 1, and 18.4 U mg− 1, respectively. Guzmán-Murillo et al.
[33] tested different nitrogen medium composition in the scaled up
production of SOD by Phaeodactylum tricornutum and concluded that
SOD production by microalgae is comparable to that by the yeast
Debaryomyces hansenii.
Because SOD is a promising and potent antioxidant enzyme, future
studies should evaluate SOD synthesis in microalgae.
4.9. Enzymes involved in carbohydrate accumulation
Microalgae show the ability to metabolize fatty acids into carbohy-
drates, which is a useful metabolic advantage. González-Fernández and
Ballesteros [136] reported the main enzymes for carbohydrate storage
in microalgae. The enzymes responsible for this conversion are
isocitrate lyase (EC 4.1.3.1), which is also known as isocitrate glyox-
ylate-lyase (succinate-forming) or isocitrase. This enzyme participates
in the decarboxylation step of the tricarboxylic acid cycle and is used by
bacteria, fungi, plants and microalgae. This enzyme acts by cleaving
isocitrate into succinate and glyoxylate. Another enzyme involved in
carbohydrate accumulation is malate synthase, also known as acetyl-
CoA: glyoxylate C-acetyltransferase. It catalyzes the reaction of glyox-
ylate and acetyl-CoA to form malate. After malate enters the cytosol,
subsequent reactions occur to produce glucose [136].
B.d.S.A.F. Brasil et al.
4.10. Enzymes involved in carbon concentration
Carbonic anhydrase (EC 4.2.1.1) is a zinc-dependent metalloenzyme
that catalyzes the formation of carbonic acid from CO2 and H2O [137].
The importance of carbonic anhydrase in carbon fixation metabolism is
being intensely investigated in photosynthetic organisms to increase
biofuel production [138]. A study reported microalgal isolates from the
genera Desmodesmus, Kirchneriella and Acutodesmus, which exhibit
constant high carbonic anhydrase activity during cultivation in closed
photobioreactors at ambient temperature with 0.03% CO2. In higher
levels of CO2, the activity decreases significantly [139]. The authors
observed that CO2 levels modulate the carbonic anhydrase activity. The
increase in enzyme activity correlates with the decrease in free CO2.
Moreover, the increase in carbonic anhydrase activity also correlates
with a decrease in bicarbonate, suggesting its conversion to CO2 by this
enzyme. The study also suggests that a CO2-rich environment increases
the availability of dissolved inorganic carbon (free CO2 and HCO3-) and
modulates the activity of carbonic anhydrase [139].
5. Disruption methods for enzymes recovery
The steps involved in the production of intracellular metabolites
from microalgae start with cell cultivation and biomass recovery
(flocculation, centrifugation, and/or filtration) and are followed by
downstream processing, which includes cell lysis, extraction and
product purification [140]. The downstream recovery of microalgal
products is responsible for 57% of the cost of the final product [141].
Different disruption methods have been used for microalgae, such as
mechanical and chemical methods and enzymatic hydrolysis.
Mechanical methods are preferred since they prevent chemical con-
tamination, are easy to scale up and preserves the functionality of
bioproducts [142]. However, these methods usually have higher energy
requirements than chemical or enzymatic methods. Because heat is
released during mechanical disruption, a cooling system is needed for
thermolabile products [143]. High-pressure homogenization, ultrasoni-
cation, high-speed centrifugation and bead milling are the most
extensively used methods for enzyme recovery on a laboratory scale,
as shown in Table 1. Serive et al. [28] tested nine mechanical disruption
techniques on microalgae cells, and a mixer mill showed the most
efficient cell disruption and recovery of metabolites.
6. Algae and cyanobacteria as recombinant enzyme expression
systems
The characterization of new algal genomes coupled with genetic
tool development has opened paths for engineering these organisms
into platforms for recombinant protein production, including enzymes.
The use of genetically modified organisms to produce industrial
enzymes was the main factor driving the expansion of this market from
USD 1.6 billion in 1998 to USD 5.1 billion in 2009 [195]. Currently,
~ 50% of industrial enzymes are produced using recombinant DNA
technology [144]. Although conventional microbial systems (e.g., E.
coli, S. cerevisiae and P. pastoris) and animal cells have well established
molecular tools and have been used for several years to produce
recombinant proteins, they exhibit significant drawbacks mainly re-
lated to protein processing, solubility and production costs (Table 2)
[144,146,149].
For example, although bacterial host systems are cheap and easily
operated, they lack eukaryotic post-translational modification machin-
ery and may require laborious and costly processing steps for protein
purification from inclusion bodies [150]. Although yeast and filamen-
tous fungi based systems have basic eukaryotic machinery and the
capacity for protein secretion, recombinant protein inactivity due to
issues with fungi-specific post-translational patterns, high concentra-
tions of intracellular proteases, and insufficient secreted protein yields
have been reported [146,151,152]. In addition, mammalian cell and
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Algal Research 25 (2017) 76–89
insect expression systems usually exhibit scaling-up technical difficul-
ties and prohibitive costs for industrial enzymes production [153].
Plant-based recombinant protein expression systems are attractive
because of their scalability and low cost. However, their long-life cycles
and issues related to product recovery from plant tissues/organs make this
method non-ideal for heterologous enzymes expression [148,154,155].
However, photosynthetic microorganisms combine features of both micro-
bial- and plant-based systems that are desirable for recombinant protein
expression, including high growth rate, ease of cultivation, rapid scalability
at the pilot level, protein secretion, and the absence of human pathogens.
Furthermore, eukaryotic algae species are able to perform post-translational
modifications [58,146,149,154–159].
To date, diverse proteins of industrial, nutritional and medical use
have been successfully expressed in photosynthetic microorganisms
(Table 3). Highlights include the use of algae to produce therapeutic
proteins. Human antibodies, for example, are complex polypeptides
that require disulfide bond formation and proper folding, which cannot
be achieved in prokaryotic systems. In addition, issues related to
hyperglycosylation have hampered advances in antibody production
in yeast [176]. Therefore, algae appear to be attractive alternatives to
the costly mammalian cell cultures. Accordingly, the successful expres-
sion of functional human antibodies against Herpes Simplex Virus and
Anthrax in C. reinhardtii chloroplasts has been shown [172,173].
Immunotoxins have also been expressed in C. reinhardtii chloro-
plasts [174,175]. These therapeutic proteins are formed by recombi-
nant antibodies attached to small toxin molecules/proteins, which
target and kill cancer cells. Usually, these toxins cannot be produced
in mammalian expression platforms since they inhibit cell metabolism.
Therefore, algae chloroplasts are an adequate environment for the
expression of these antibody–drug conjugates since they possess
prokaryotic-like cell machinery and yet can properly fold human
antibodies. The C. reinhardtii chloroplast is also uniquely suitable for
the expression of surface proteins of the malarial parasite, Plasmodium
spp., which are not glycosylated but have several disulfide bonds
[169–171].
Furthermore, there is a growing body of studies reporting the
expression of recombinant enzymes for industrial use in microalgae
[160–162,177]. For example, α-galactosidases have been expressed
from genes derived from Lactobacillus acidophilus and Trichoderma reesei
inserted into the chloroplastic genomes of Dunaliella tertiolecta and
Chlamydomonas reinhardtii [161]. Additionally, two phytase-coding
genes, from Escherichia coli and Caulobacter crescentus have been
expressed in the chloroplasts of D. tertiolecta and C. reinhardtii
[161,162].
Yoon et al. [162] demonstrated that the use of whole cells of
transgenic C. reinhardtii expressing the E. coli phytase gene as direct
feed additives for young broiler chicks resulted in higher phosphorous
adsorption. Therefore, even though recombinant proteins expressed
from plastid genomes remain confined within this organelle, the costs
of enzyme recovery and purification can be reduced in some cases. The
gene encoding β-1,4-endoxylanase fromTrichoderma reesei has been
expressed in the chloroplasts of D. tertiolecta and C.reinhardtii [161]
and in the nuclear genome of C. reinhardtii [160]. Rasala and
collaborators (2012) have developed a plasmid construct that allowed
high expression from the nuclear genome by fusing the xylanase gene to
the selection marker gene with the foot-and-mouth-disease-virus 2A
self-cleavage peptide coding sequence between these. In this same
study, the secretion signal sequence from the Arylsulfatase I gene
(ARS1) of C. reinhardtii was fused to the xylanase gene leading to the
release of the bioactive enzyme in the surrounding media. In addition,
Synechocystis sp. 6803 has been used for the expression of a D-lactate
dehydrogenase-coding gene derived from E. coli [178].
7. Eukaryotic microalgae as recombinant protein biofactories
Eukaryotic microalgae hosts offer three distinct contexts for recom-
 B.d.S.A.F. Brasil et al.

Table 2
Comparison of eukaryotic microalgae and cyanobacteria with the most commonly used systems for recombinant protein production

Taxa Cell growth rate Production cost Expression level Advantages Disadvantages

a,b
Eukaryotic algae High (6 h) Very low Nucleus: Scalability; Simple media requirements; GRAS. Low availability of molecular tools.
Low Nucleus: Eukaryotic gene expression machinery; Post-translational modifications (i.e., Nucleus: Low expression efficiency
Chloroplast: disulfide bond formation, phosphorylation, glycosylation); Protein secretion. Chloroplast: Absence of glycosylation; Protein remains in the
Low-high Chloroplast: Post-translational modifications (i.e.; disulfide bond formations, chloroplast.
phosphorylation).
Cyanobacteriaa,b High (4 h) Very low Low-moderate Scalability; Simple media requirements; GRAS; Post-translation modifications (i.e.; Low availability of molecular tools; Absence of glycosylation and some
disulfide bond formations, phosphorylation). types of post-translational modifications; Issues with protein solubility.
Bacteria Very high Moderate Low-high Easy operation; Simple media requirements; Disulfide bond formation (periplasm); Absence of post-translational modifications; Issues with protein
(i.e., E. coli)3,4,5 (30 min) Well-developed molecular tools; solubility; Culture contamination.
Yeastc,d Very high Moderate Low-high Easy operation; Simple media requirements; GRAS; Eukaryotic gene expression Culture contamination; Distinct glycosylation patterns/hyper-
(90 min) machinery; Post-translational modifications; Protein secretion; Well-developed glycosylation.
molecular tools.
Filamentous fungic,d Very high Moderate Low-high Easy operation; Simple media requirements; Eukaryotic gene expression machinery; High intracellular concentration of proteases; Culture contamination;

83
(90 min) Post-translational modifications; Protein secretion. Distinct glycosylation patterns.
c,e
Insect cells Moderate (18 h) High Low-high Eukaryotic gene expression machinery; Post-translational modifications. Demanding culture conditions; Simple glycosylation pattern (no sialic
acid);
Mammalian cellsc,d Low (> 24 h) High Low-moderate Eukaryotic protein processing; Post-translational modifications; Well-developed Demanding culture conditions; Culture contamination; Cell line
molecular tools. instability.
Plantsc,f Low (> 24 h) Low Nucleus: Scalability; Tissue/organ-specific expression. Long life-cycles; Issues with recombinant protein recovery from plant
Low Nucleus: Eukaryotic gene expression machinery; Post-translational modifications organs/tissues
Chloroplast: (disulfide bond formation, phosphorylation, glycosylation); Protein secretion Chloroplast: Absence of glycosylation; Protein remains in the
Low-high Chloroplast: Post-translational modifications (disulfide bond formations, chloroplast.
phosphorylation)

Partly from:
a
Rasala and Mayfield [144];
b
Wang et al., [56];
c
Yin et al., [145];
d
Corchero et al., [146];
e
Assenberg et al. [147];
f
Fischer et al., [148].
Algal Research 25 (2017) 76–89
B.d.S.A.F. Brasil et al. Algal Research 25 (2017) 76–89

Table 3
Examples of recombinant proteins expressed in algae.

Field Application Recombinant protein Host species References

Industrial Enzyme for food and paper
manufacturing
Enzyme for animal feed (increases
phosphorous uptake)
Enzyme for animal feed (increases
oligosaccharide hydrolysis)
Xylanase Dunaliella tertiolecta;
Chlamydomonas reinhardtii
Phytase D. tertiolecta; C. reinhardtii
α-galactosidase D. tertiolecta; C. reinhardtii
Rasala et al. [160]; Georgiana et al.
[161]
Yoon et al. [162]; Georgiana et al.
[161]
Georgiana et al. [161]

Nutritional Supplementation of organic selenium Selenoprotein C. reinhardtii Hou et al. [163]

Fish dietary supplement and growth Flounder Growth hormone Chlorella elipsoidea Kim et al. [164]
promoter

Medical Anti-bacterial and anti-fungal peptide NP-1 peptide C. elipsoidea Chen et al. [165]
Stimulates angiogenesis Vascular endothelial growth factor (VEGF) C. reinhardtii Rasala et al. [166]
Increases resistance to UV-induced stress Metallothionein C. reinhardtii Zhang et al. [167]
Anemia treatment Erythropoietin C. reinhardtii Eichler-Stahlberg et al. [168]
Wound healing High Mobility group Protein 1B (HMGB1) C. reinhardtii Rasala et al. [166]
Anti-malarial vaccines Plasmodium surface proteins (AMS-1, MSP1, C. reinhardtii Dauvillée et al. [169]; Gregory et al.
Pfs25, Pfs28, Pfs48/45, Pfs25) [170]; Jones et al. [171]
Antibody against anthrax Anti-PA 83 anthrax IgG1 C. reinhardtii Tran et al. [172]
Antibody against Herpes Simplex Virus Anti-HSV glycoprotein D lsc C. reinhardtii Mayfield et al. [173]
Imunotoxins against B-cell lymphoma Anti-CD22-gelonin sc C. reinhardtii Tran et al. [174]; Tran et al. [175];
Anti-CD22-ETA sc

The list is not exhaustive. Partly from: Barrera and Mayfield [154]; Rasala and Mayfield [144]; Specht et al. [176].

binant protein expression: the nuclear, the plastid and the mitochon-
drial genomes [55,144,154]. All three algal genomes have been
successfully engineered for recombinant gene expression using trans-
formation methods such as biolistic (nuclear, mitochondrial, and
chloroplastic genomes), glass bead agitation (nuclear and chloroplastic
genomes), electroporation, and Agrobacterium-mediated gene transfer
and silicon carbide whiskers (nuclear genome) (reviewed by [55]). The
gene products expressed from the nucleus can be directed to subcellular
locations or secreted and are subjected to proper folding and post-
translational modifications (i.e.; disulfide bond formations, phosphor-
ylation, glycosylation) by the eukaryotic cell machinery of microalgal
hosts (Table 2).
The possibility of secreting the recombinant products expressed
from the nuclear genome into the surrounding media drastically
reduces the costs of downstream processing, making this genome an
attractive option for the expression of enzyme-coding genes. However,
recombinant nuclear genes frequently shows silencing behavior leading
to low protein expression levels [179].
In contrast, expression levels within the chloroplastic compartment
are usually higher, accounting for up to 10.5% of the total soluble
protein content [159]. The plastid genome is amenable to homologous
recombination, and post-translational modifications such as disulfide
bond formation and phosphorylation can take place in the chloroplast.
However, proteins expressed in this compartment cannot be glycosy-
lated or targeted to secretion or other organelles (Table 2).
The mitochondrial genome also offers an alternative option for
heterologous gene expression, but engineering of this genome has been
poorly explored to date. There are few reports regarding recombinant
protein expression within this organelle in the green microalgae species
Chlamydomonas reinhardtii [180–182].
Species from the Chlamydomonas genus have been studied as a
model for photosynthetic organisms for decades [183]. In addition, the
ease of C. reinhardtii transgene insertion [184], the availability of fully
sequenced nuclear, chloroplast, and mitochondrial genomes [155], and
advanced molecular tools such as well-characterized chloroplast and
nuclear promoter elements [158] make this species the model of choice
for genetic studies and recombinant protein expression in microalgae
(Table 4). Furthermore, the availability of cell wall-deficient strains
coupled with the haploid nature and a well-known sexual life cycle of C.
reinhardtii species make strain engineering relatively straightforward
[144]. Expression at economically viable levels of recombinant vaccines
[187], functional antibodies [172,188] and other proteins of biotech-
nological interest, including enzymes, has been achieved in C. reinhard-
tii ([189–191]).
However, C. reinhardtii presents limited potential for protein
production in large-scale cultivation systems due primarily to limita-
tions in the maximal cell density achieved in stationary-phase cultures
[185,196]. Alternative species, such as Dunalliela salina, Haematococcus
pluvialis, Phaeodactylum tricornutum, Nannochloropsis gaditana and Chlor-
ella sorokiniana are being tested for recombinant protein expression.
Dunalliela salina and Haematococcus pluvialis have been cultivated for
decades at large scale to produce high-value carotenoids (β-carotene
and astaxanthin, respectively) using hypersaline media, a condition
that aids contamination management in open pond systems. Both the
nuclear and the chloroplastic genomes have been transformed in these
two species (Table 4); however, the absence of complete genomic
sequences and an established molecular toolkit hampers their further
development as recombinant protein hosts.
Furthermore, the diatom species Phaeodactylum tricornutum has a
fully sequenced genome and a significant set of molecular tools
available, including genetic transformation methods, the availability
of RNAi vectors, and the possibility of gene editing through transcrip-
tion activator-like effector nucleases (TALENs). In addition, proteins of
therapeutic interest such as monoclonal antibodies and the Hepatitis B
virus surface antigen immunogen (HBsAg) have been successfully
expressed in P. tricornutum [192].
The species Chlorella sorokiniana and Nannochloropsis gaditana are
ranked among the most promising for biomass and biofuel production
in freshwater and saltwater based media, respectively ([193]; NAABB
Final Report, 2014; [185]). Both present high growth rates, high oil
content, and robustness and can achieve high densities in large-scale
cultures. Recent advances in genomic sequencing, the characterization
of promoters, and genetic transformation methods have been reported
for both species [185,193,194]. The increasing interest in genetically
engineering these organisms for biofuels production has led to an
expansion in the knowledge about these species genetic features and
their molecular toolkits.
It is important to highlight, that competing with bacterial/fungal
systems for the production of industrial enzymes is not possible given
the current available knowledge. Advances regarding the optimization
of large-scale phototrophic growth systems, and the development of
powerful genetic tools to drive high levels of enzyme expression pose

84
 B.d.S.A.F. Brasil et al.

Table 4
Selected eukaryotic microalgae and cyanobacteria species with potential for recombinant protein production

Taxon Species Genome sequence Transformation methods available Engineered Key features
available genomes

Chlorophyta Chlamydomonas
reinhardtiia,b,c,h,i
Chlorophyta Chlorella sorokinianac,d,e
Chlorophyta Dunalliela salinaa,c,e
Chlorophyta Haematococcus pluvialisc,e,f
Eustigmatophyceae Nannochloropsis gaditanac
Bacillariophyceae Phaeodactylum
tricornutumc,e,j
Cyanobacteria Synechococcus elongatesg
121 Mb (N, Chl, M) Agrobacterium; Biolistics; Electroporation; Glass N, C, M Well-developed molecular and bioinformatics tools; Cell wall-deficient mutants
bead agitation; Silicon carbide whiskers. available; Metabolic pathways available. Gene targeting by CRISPR/Cas9, TALENs, and
ZFNs available.
60 Mb (N1, Chl, M) Biolistics. N Amenable to large-scale culturing; Fast growth rate.
– Biolistics. N, C Resistant to high salinity; Amenable to large-scale culturing; Lack of rigid cell wall.
– Biolistics; Agrobacterium. N, C Resistant to high salinity; Amenable to large-scale culturing.
34 Mb (N, Chl, M) Electroporation. N DNA integration in the nuclear genome by homologous recombination; Amenable to
large-scale culturing.
27 Mb (N, Chl, M) Biolistics. N Well-developed molecular tools. Gene targeting by CRISPR/Cas9 and TALENs available.
2,7 Mb (Chr, P) Electroporation, conjugation. Chr, P DNA integration in the genome by homologous recombination; Well-developed

85
molecular tools.
g
Cyanobacteria Synechocystis sp. PCC6803 3,6 Mb (Chr, P) Electroporation, ultrasonic transformation and Chr, P DNA integration in the genome by homologous recombination; Well-developed
conjugation. molecular tools.

N: nucleus; Chl, chloroplast; M: mithocondria; chr: Chromosome; P: plasmid; TALENs: transcription activator-like effector nucleases: ZFN: zinc-finger nucleases.
Partly from:
a
Rasala and Mayfield [144];
b
Rosales-Mendoza et al. [158];
c
Stephens et al. [55];
d
Barry et al. [185];
e
Gong et al. [58];
f
Saei et al. [186];
g
Wang et al. [56];
h
Shin et al. [67];
i
Baek et al. [64];
j
Hopes et al. [65].
Algal Research 25 (2017) 76–89
B.d.S.A.F. Brasil et al.
major hurdles that must be overcome. Even the expression of algal
enzyme-coding genes will most likely happen in other systems until, at
some point in the future, algae genetic engineering becomes cost
effective. However, the ability of microalgae to mimic mammalian
post-translational modifications (e.g., glycosylation) promises to accel-
erate the development of algal systems for the production of recombi-
nant proteins for biomedical applications. There is a growing body of
data regarding the expression of antibody-derived drugs, hormones,
growth factors, anti-microbial peptides, and vaccines (Table 3). There-
fore, the market demand for these high-value products might foster the
development of efficient recombinant protein production in photosyn-
thetic microorganisms. Another promising approach for reducing the
costs of algal enzyme production is the concomitant use of the residual
algal biomass and lipids in an integrated biorefinery approach [10]. The
exploitation of the whole algal biomass for the production of high-
added-value products, such as carotenoids and enzymes, together with
low added value products, such as animal feed and/or energy, would
certainly improve the economical sustainability of the process.
8. Future perspectives
The variety of enzymes found among microalgal species demon-
strates the importance of these versatile microorganisms as cellular
factories. Although a large part of microalgae genetic resources remain
unexplored, a growing number of enzymes with potential applications
in the food, pharmaceutical, and chemical industry have been reported.
The development of new strategies and methods to induce and optimize
microalgae enzyme production, including biomanufacturing, is an
important approach for the scale-up and industrialization of potential
enzymes. Some of the most important challenges will be increasing the
concentration of enzymes and to reducing the cost of downstream
operations. Genetic engineering and synthetic biology advances,
coupled with the use of the whole algal biomass to produce several
bioproducts in a biorefinery strategy, offer promising possibilities to
overcome these limitations and achieve the cost-effective production of
industrial enzymes in microalgal cells in the future.
Declaration of interest
The authors report no declarations of interest.
Acknowledgements
The authors are grateful to the Brazilian Agricultural Research
Corporation (EMBRAPA) (grant number 03.12.11.006.00.00) and
National Council for Scientific and Technological Development
(CNPq) (grant numbers 310424/2015-1 and 477891/2013-6) for
supporting this work.
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