The Clinical Promise of Microalgae in Rheumatoid Arthritis: From Natural Compounds to Recombinant Therapeutics

Abstract

Rheumatoid arthritis (RA) is an invalidating chronic autoimmune disorder characterized by joint inflammation and progressive bone damage. Dietary intervention is an important component in the treatment of RA to mitigate oxidative stress, a major pathogenic driver of the disease. Alongside traditional sources of antioxidants, microalgae—a diverse group of photosynthetic prokaryotes and eukaryotes—are emerging as anti-inflammatory and immunomodulatory food supplements. Several species accumulate therapeutic metabolites—mainly lipids and pigments—which interfere in the pro-inflammatory pathways involved in RA and other chronic inflammatory conditions. The advancement of the clinical uses of microalgae requires the continuous exploration of phytoplankton biodiversity and chemodiversity, followed by the domestication of wild strains into reliable producers of said metabolites. In addition, the tractability of microalgal genomes offers unprecedented possibilities to establish photosynthetic microbes as light-driven biofactories of heterologous immunotherapeutics. Here, we review the evidence-based anti-inflammatory mechanisms of microalgal metabolites and provide a detailed coverage of the genetic engineering strategies to enhance the yields of endogenous compounds and to develop innovative bioproducts.

Full text

Citation: Cutolo, E.A.; Caferri, R.;
Campitiello, R.; Cutolo, M. The
Clinical Promise of Microalgae in
Rheumatoid Arthritis: From Natural
Compounds to Recombinant
Therapeutics. Mar. Drugs 2023,21,
630. https://doi.org/10.3390/
md21120630
Academic Editor: Alberto Amato
Received: 17 October 2023
Revised: 4 December 2023
Accepted: 5 December 2023
Published: 7 December 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
marine drugs
Review
The Clinical Promise of Microalgae in Rheumatoid Arthritis:
From Natural Compounds to Recombinant Therapeutics
Edoardo Andrea Cutolo 1, *, Roberto Caferri 1, Rosanna Campitiello 2and Maurizio Cutolo 2
1Laboratory of Photosynthesis and Bioenergy, Department of Biotechnology, University of Verona,
Strada le Grazie 15, 37134 Verona, Italy; roberto.caferri@univr.it
2Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine,
IRCCS San Martino Polyclinic Hospital, University of Genoa, Viale Benedetto XV, 6, 16132 Genoa, Italy;
rosannacampitiello@hotmail.it (R.C.)
*Correspondence: edoardoandrea.cutolo@univr.it
Abstract:
Rheumatoid arthritis (RA) is an invalidating chronic autoimmune disorder characterized
by joint inflammation and progressive bone damage. Dietary intervention is an important component
in the treatment of RA to mitigate oxidative stress, a major pathogenic driver of the disease. Along-
side traditional sources of antioxidants, microalgae—a diverse group of photosynthetic prokaryotes
and eukaryotes—are emerging as anti-inflammatory and immunomodulatory food supplements.
Several species accumulate therapeutic metabolites—mainly lipids and pigments—which interfere
in the pro-inflammatory pathways involved in RA and other chronic inflammatory conditions. The
advancement of the clinical uses of microalgae requires the continuous exploration of phytoplankton
biodiversity and chemodiversity, followed by the domestication of wild strains into reliable produc-
ers of said metabolites. In addition, the tractability of microalgal genomes offers unprecedented
possibilities to establish photosynthetic microbes as light-driven biofactories of heterologous im-
munotherapeutics. Here, we review the evidence-based anti-inflammatory mechanisms of microalgal
metabolites and provide a detailed coverage of the genetic engineering strategies to enhance the
yields of endogenous compounds and to develop innovative bioproducts.
Keywords:
photosynthesis; polyunsaturated fatty acids; carotenoids; oxylipins; xanthophylls;
antioxidants; functional foods; synthetic biology; genetic engineering; inflammation; rheumatoid
arthritis; autoimmunity; docosahexaenoic acid (DHA); eicosapenteanoic acid (EPA); astaxanthin;
rheumatology; interleukins; chloroplast; molecular pharming; novel foods; bioeconomy
1. Introduction
Chronic inflammation is a defining feature of autoimmune diseases, a group of condi-
tions in which immunological self-tolerance is disturbed due to the recognition of autoanti-
gens by immune cells. Rheumatoid arthritis (RA), the most common chronic inflammatory
arthropathy [
1
,
2
], is a systemic autoimmune disorder affecting the synovial joints, with a
higher incidence in women [
3
]. RA displays a complex pathophysiology involving the up-
regulation of pro-inflammatory mediators (interleukins, ILs) and enhanced production of
reactive oxygen species (ROS) [
4
,
5
]. Both genetic and modifiable lifestyle factors contribute
to the risk of RA predisposition [
6
,
7
], with diet highly influencing disease activity [
8
,
9
]. In
particular, a high antioxidant intake is known to reduce onset risk and to ameliorate the
clinical course of the disease [
10
], therefore, the identification of new sources of antioxidant
and anti-inflammatory molecules is of high clinical relevance.
Microalgae are photosynthetic prokaryotes and eukaryotes adapted to diverse envi-
ronments, including extreme habitats [
11
,
12
], which are consumed in human nutrition as
sources of proteins and other bioactive compounds [
13
–
17
]. Several species are non-toxic
producers of essential vitamins, lipids, and pigments of therapeutic value [
18
–
22
], which
could be employed as complementary agents in the management of chronic inflammatory
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Mar. Drugs 2023,21, 630 2 of 27
diseases. Moreover, the fast life cycle and light-powered autotrophic metabolism of mi-
croalgae allows for large-scale cultivation with lower inputs compared with heterotrophic
microorganisms [23].
In this review, we summarize the evidence-based putative interference of microalgal
compounds in pro-inflammatory pathways involved in the pathogenesis of RA and discuss
how bioprospecting for novel pharmacologically relevant strains, and their domestication,
can advance the clinical use of photosynthetic microorganisms. Lastly, we provide an
update on the available genetic engineering strategies to enhance the production of endoge-
nous microalgal metabolites and introduce emerging approaches to achieve light-driven
conversion of CO2into high-value recombinant biopharmaceuticals.
Pathogenesis and Mediators of Rheumatoid Arthritis
Although the exact etiology of RA remains unknown, the balance between immune
cells and the production of inflammatory ILs in the connective tissue that lines the joint
capsule (synovium) is altered in the disease onset and progression [
4
,
5
]. The healthy syn-
ovium consists of a thin lining layer of fibroblasts covering a connective tissue surrounded
by blood vessels and enriched in fibroblasts, and innate and adaptive immune cells: the
sub-lining layer [
24
] (Figure 1A). In RA, the lining layer is hyperplastic while the sub-lining
layer is infiltrated with B-cells, monocyte-derived macrophages, autoantibody-secreting
plasma cells, and differentiated cytotoxic CD4
+
T-cells involved in the breakdown of tissue
tolerance [
25
–
27
]. The release of pro-inflammatory ILs by monocyte-derived M1 macrophages,
and osteoclast activation cause progressive bone resorption [
28
], and autoantibodies pro-
duced by differentiated plasma cells further contribute to joint damage [29].
Mar. Drugs 2023, 21, x 3 of 28
Figure 1. Cellular composition of the synovial membrane, its interaction with bone tissue and
immune cell types, and related mediators involved in inflamed RA joints. (A) In healthy conditions,
the synovium consist of a thin lining layer of lining fibroblasts in association with lining M1 directly
exposed to the bone tissue. The underlying sub-lining layer is a connective tissue enriched in blood
vessels, adipocytes, fibroblasts, and both innate and adaptive immune cells. The inflamed RA
synovium is characterized by a hyperplastic lining layer surrounded by proinflammatory sub-lining
fibroblasts and a massive infiltration of B-cells, monocyte-derived macrophages, autoantibody-
secreting plasma cells, and differentiated cytotoxic effector memory CD4+ T-cells in the sub-lining
layer. The secretion of pro-inflammatory interleukins (IL-1: interleukin 1; IL-6: interleukin 6; TNF-
α: tumor necrosis factor-alpha) by activated immune cells stimulates the production of the soluble
cytokine Mediator Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL), which binds to
its receptor RANK on monocytes and macrophages causing their differentiation into bone-resorbing
osteoclasts. Red arrows indicate pro-inflammatory processes. (B) Interleukin (IL) isoforms produced
by different types of synovial innate immune cells and macrophages (IL-6: interleukin 6; IL-1:
interleukin 1; TNF-α: tumor necrosis factor-alpha; TNF-β: tumor necrosis factor-beta; VEGF:
vascular endothelial growth factor). (C) Differentiation and interconversion of adaptive immune
cells (Treg: regulatory T cells; TH1: T helper 1 cells; TH 17: T helper 17 cells; TH2: T helper 2 cells; B
cells; APCs: antigen-presenting cells). (D) Effects of secreted ILs on bone-remodeling processes
depending on the osteoprotegerin (OPG)–RANKL axis, which regulates the differentiation of
osteoclasts in bone-resorbing osteoclasts. Figures created with BioRender.com, accessed on 15
November 2023.
Figure 1.
Cellular composition of the synovial membrane, its interaction with bone tissue and
immune cell types, and related mediators involved in inflamed RA joints. (
A
) In healthy conditions,

Mar. Drugs 2023,21, 630 3 of 27
the synovium consist of a thin lining layer of lining fibroblasts in association with lining M1 directly
exposed to the bone tissue. The underlying sub-lining layer is a connective tissue enriched in blood
vessels, adipocytes, fibroblasts, and both innate and adaptive immune cells. The inflamed RA syn-
ovium is characterized by a hyperplastic lining layer surrounded by proinflammatory sub-lining
fibroblasts and a massive infiltration of B-cells, monocyte-derived macrophages, autoantibody-
secreting plasma cells, and differentiated cytotoxic effector memory CD4
+
T-cells in the sub-lining
layer. The secretion of pro-inflammatory interleukins (IL-1: interleukin 1; IL-6: interleukin 6; TNF-
α
: tumor necrosis factor-alpha) by activated immune cells stimulates the production of the soluble
cytokine Mediator Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL), which binds to
its receptor RANK on monocytes and macrophages causing their differentiation into bone-resorbing
osteoclasts. Red arrows indicate pro-inflammatory processes. (
B
) Interleukin (IL) isoforms pro-
duced by different types of synovial innate immune cells and macrophages (IL-6: interleukin 6;
IL-1: interleukin 1; TNF-
α
: tumor necrosis factor-alpha; TNF-
β
: tumor necrosis factor-beta; VEGF:
vascular endothelial growth factor). (
C
) Differentiation and interconversion of adaptive immune cells
(Treg: regulatory T cells; TH1: T helper 1 cells; TH 17: T helper 17 cells; TH2: T helper 2 cells; B cells;
APCs: antigen-presenting cells). (
D
) Effects of secreted ILs on bone-remodeling processes depending
on the osteoprotegerin (OPG)–RANKL axis, which regulates the differentiation of osteoclasts in
bone-resorbing osteoclasts. Figures created with BioRender.com, accessed on 15 November 2023.
At the intracellular level, three main pro-inflammatory signaling kinase cascades
responding to soluble mediators are involved in RA, all being influenced by microalgal
metabolites: the Nuclear Factor Kappa-B (NF-kB)-mediated pathway [
30
], the Janus kinase
2/Signal Transducers and Activators of Transcription 3 (JAK2/STAT3) pathway [
31
], and
the Jun N-terminal kinase (JNK)/p38 Mitogen-Activated Protein Kinase (p38 MAPK)
pathway [32], the latter being predominant in the lining layer and endothelial cells.
Pro-inflammatory ILs (Figure 1D) [
33
] stimulate the production of the soluble cy-
tokine Mediator Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) by im-
mune cells. RANKL binds its receptor RANK on monocytes and macrophages causing
their differentiation into bone-resorbing osteoclasts [
34
]. Other ILs produced by anti-
inflammatory M2 macrophages positively influence the osteoprotegerin (OPG)–RANKL
ratio, promoting bone homeostasis [
35
] (Figure 1D), while the IL-10 family (Figure 1D)
appears to play dual roles. Some members stimulate osteogenesis and suppress the syn-
thesis of pro-inflammatory mediators like IL-6, Tumor Necrosis Factor-
α
(TNF-
α
), and
Vascular endothelial growth factor (VEGF) [
36
], while others activate the NF-kB and MAPK
pathways, stimulating TNF-
α
, IL-1
β
, and RANKL production by synovial fibroblasts,
promoting osteoclastogenesis.
In conclusion, given the diversified nature of factors regulating the balance between
pro- and anti-inflammatory mediators in RA, there is a strong interest in discovering novel
molecules to be used as complementary tools alongside conventional therapies.
2. Anti-Inflammatory and Immunomodulatory Metabolites from Microalgae
2.1. Carotenes and Xanthophylls
Like bacteria, fungi, and plants, microalgae synthetize C40 lipophilic pigments con-
sisting of a polyene chain of conjugated double bonds (Figure 2) with terminally linked
ionone rings known as carotenoids [
37
,
38
].
β
-carotene (Figure 2B)—a structural element
of photosystems [
39
]—and the antioxidant lycopene (Figure 2A) are anti-inflammatory
carotenes [
40
,
41
] usually introduced in the diet with carrots (Daucus carota) and tomatoes
(Solanum lycopersicum), respectively, although present also in microalgae [42].
Xanthophylls are oxygenated carotenoids containing hydroxyl and ketone groups
in the ionone rings, which serve different functions in phototrophs. The non-ketolated
xanthophyll lutein (Figure 2C) participates in light-harvesting and photoprotection, while
the ketocarotenoid astaxanthin (ASTX, Figure 2D) scavenges harmful ROS generated by
photosynthetic electron transport under excess light [
43
]. Lutein is an anti-inflammatory

Mar. Drugs 2023,21, 630 4 of 27
carotenoid [
44
] abundantly found in green leafy vegetables and egg yolk, while ASTX is a
potent antioxidant uniquely synthetized by a few microalgal species.
Abiotic stresses induce a hypercarotenogenic response in several chlorophytes, in-
cluding the halophile Dunaliella salina (Chlorophyceae), which overaccumulates
β
-carotene
in lipid bodies (plastoglobules) inside the chloroplast [
45
], and in the freshwater species
Haematococcus pluvialis (Haematococcaceae), which forms haematocysts filled with ASTX-
rich cytoplasmic lipid droplets [
46
]. Other microalgal xanthophylls with anti-inflammatory
and immunomodulatory properties are fucoxanthin and diatoxanthin produced by sev-
eral diatoms (stramenopiles) and by the haptophyte Tisochrysis lutea (Coccolithophyceae)
(Figure 2E,F) [
47
,
48
]. As discussed in the following paragraphs, carotenoids appear to
interfere in all major pro-inflammatory pathways implicated in the onset and progression
of RA.
Astaxanthin: The Red Gold of Algae
With recognized safety for human consumption [
49
], approved Novel Food sta-
tus [
50
], and an established role in promoting bone homeostasis in degenerative skeletal
diseases [51], ASTX is the microalgal pigment of highest biopharmaceutical value.
Clinical studies have shown that ASTX intake reduces the levels of systemic inflam-
matory biomarkers [
52
,
53
] and potentiates the pain-relieving effect of anti-inflammatory
therapies [
54
]. The pharmacological effects of ASTX derive from its strong antioxidant-
activity mediated via ROS quenching [
55
] (Figure 2G, top panel) and direct free radical
scavenging [
56
,
57
] (Figure 2G, bottom panel). This amphipathic molecule is symmetrically
arranged within the lipid bilayer [
58
], thus exerting antioxidant activity on both intra- and
extracellular environments. Notably, the higher number of hydroxyl groups compared with
other carotenoids confers to ASTX superior ROS-detoxifying capacity [
59
]. Early studies
showed that ASTX suppressed ROS production [
60
–
63
] and secretion of pro-inflammatory
ILs by cultured human-activated monocytes [
64
]. Moreover ASTX stimulated the expres-
sion of ROS-scavenging enzymes in chondrocytes challenged with IL-1
β
[
65
], and inhibited
pro-inflammatory and osteoclastogenic gene expression in macrophages challenged with
RANKL [
66
]. Lastly, the administration of ASTX promoted cartilage health in animal
models of arthritis and osteoasthritis [67–69].
Notably, the esterified biological form of ASTX displays higher bioavailability com-
pared with synthetic ester-free derivatives [
70
–
73
], suggesting the need to improve its
production from natural sources. However, a major limitation to the clinical use of ASTX
lies in its low solubility in gastrointestinal fluids [
74
], requiring encapsulation in polysac-
charide, lipid, and protein nanoparticles to enhance its delivery and release [75–80].
2.2. Anti-Inflammatory Mechanisms of Action of Astaxanthin and Other Carotenoids
2.2.1. NF-κB Pathway
ASTX and
β
-carotene interfere with the NF-
κ
B pathway blocking the transloca-
tion of the NF-
κ
B transcription factor to the nucleus, thereby suppressing ROS and pro-
inflammatory gene expression. This effect is likely mediated through targeting the Inhibitor
of the NF-
κ
B
γ
subunit (IKK-
γ
) of the IkB kinase complex [
81
–
84
]. This prevents the phos-
phorylation and subsequent proteasome-mediated degradation of the IkB
α
binding factor,
which abolishes the release of NF-
κ
B [
30
]. A similar inhibitory effect has been proposed for
fucoxanthin and diatoxanthin [85,86].
The Mitogen- and Stress-activated protein Kinase-1 (MSK1) is a nucleus-localized
factor, which activates the NF-
κ
B pathway [
87
] and the transcriptional regulator cAMP-
responsive Element-Binding Protein (CREB) [
88
]. Phosphorylated CREB binds CREB-
Responsive Elements (CRE) promoting pro-inflammatory gene expression [
89
]. These
events are suppressed by ASTX, which inhibits MSK1 autophosphorylation [
90
]. Lastly, in
silico simulations suggested that ASTX and
β
-carotene extracellularly interact with IL-6
and TNF-
α
, preventing their binding to membrane receptors [
91
]. ASTX may also interact

Mar. Drugs 2023,21, 630 5 of 27
with the NF-
κ
B-Inducing Kinase (NIK) and block the phosphorylation of the IKK-
α
subunit
of the IkB αkinase complex, suppressing the NF-κB pathway [92].
Mar. Drugs 2023, 21, x 5 of 28
Clinical studies have shown that ASTX intake reduces the levels of systemic
inflammatory biomarkers [52,53] and potentiates the pain-relieving effect of anti-
inflammatory therapies [54]. The pharmacological effects of ASTX derive from its strong
antioxidant-activity mediated via ROS quenching [55] (Figure 2G, top panel) and direct
free radical scavenging [56,57] (Figure 2G, boom panel). This amphipathic molecule is
symmetrically arranged within the lipid bilayer [58], thus exerting antioxidant activity on
both intra- and extracellular environments. Notably, the higher number of hydroxyl
groups compared with other carotenoids confers to ASTX superior ROS-detoxifying
capacity [59]. Early studies showed that ASTX suppressed ROS production [60–63] and
secretion of pro-inflammatory ILs by cultured human-activated monocytes [64]. Moreover
ASTX stimulated the expression of ROS-scavenging enzymes in chondrocytes challenged
with IL-1β [65], and inhibited pro-inflammatory and osteoclastogenic gene expression in
macrophages challenged with RANKL [66]. Lastly, the administration of ASTX promoted
cartilage health in animal models of arthritis and osteoasthritis [67–69].
Figure 2. Structures of microalgal carotenoids and ROS detoxification mechanisms of astaxanthin.
Lycopene (A), beta-carotene (B), lutein (C), astaxanthin (ASTX, (D)), fucoxanthin (E), and
diatoxanthin (F). Pink and purple bars indicate the hydrophobic and hydrophilic regions of the
molecules, respectively; in red, the oxygen of the keto groups, and in blue, the oxygen of the
carboxylic groups; in green, the R and R’ functional groups of astaxanthin. Panel (G) outlines the
two main routes of ASTX-mediated singlet molecule oxygen (1O2) detoxification. The top pathway
Figure 2.
Structures of microalgal carotenoids and ROS detoxification mechanisms of astaxanthin.
Lycopene (
A
), beta-carotene (
B
), lutein (
C
), astaxanthin (ASTX, (
D
)), fucoxanthin (
E
), and diatoxan-
thin (
F
). Pink and purple bars indicate the hydrophobic and hydrophilic regions of the molecules,
respectively; in red, the oxygen of the keto groups, and in blue, the oxygen of the carboxylic groups;
in green, the R and R’ functional groups of astaxanthin. Panel (
G
) outlines the two main routes
of ASTX-mediated singlet molecule oxygen (
1
O
2
) detoxification. The top pathway is based on an
electron transfer process involving: (i) the formation of a weakly bound ASTX-
1
O
2
complex followed
by direct electron transfer from the highest occupied molecular orbital (HOMO) of ASTX to the lowest
unoccupied molecular orbital (LUMO) of singlet oxygen (
1
O
2
), and the formation of radicals; (ii) a
reverse reaction restoring the electron distribution between the two molecules. The overall process
converts
1
O
2
to its triplet unreactive form (
3
O
2
) upon spin inversion, while ASTX is restored from
3
ASTX via internal conversion [
55
]. The bottom pathway shows the free radical scavenging activity
based on a two-step transfer involving both an electron and proton (H
+
) from ASTX to
1
O
2
. The
formed hydrogen peroxide is readily removed by peroxidase enzymes while ASTX is spontaneously
restored by ascorbate. These mechanisms of action are iterative, meaning that a single ASTX molecule
can perform multiple ROS detoxification cycles. Figures created with BioRender.com, accessed on
15 November 2023.

Mar. Drugs 2023,21, 630 6 of 27
2.2.2. JAK2/STAT3 and JNK/p38 MAPK Pathways
β
-carotene and ASTX further modulate the pro-inflammatory pathways mediated by
the JNK/p38 MAPK [
93
] and JAK2/STAT3 kinases [
84
,
94
], the latter responding to IL-6
in the pathogenesis of RA and osteoarthritis [
31
,
95
]. Phosphorylation of STAT3 dimers
by JAK2 induces nuclear translocation and the differentiation of CD4
+
T cells into the
highly reactive T helper 17 (Th17) cell type [
96
]. TNFs and IL-1 activate the JNK/p38
MAPK pathway starting a phosphorylation cascade ending with JNK/p38 MAPK nuclear
translocation [
97
], and phosphorylation of pro-inflammatory transcription factors (ELK1,
MEF2, ATF2, and STAT1) [
98
] and of the MAPK-activated kinase 2 (MK2), which, in turn,
targets the tristetrapolin (TTP) factor, promoting stabilization of IL mRNAs [
99
]. These
oxidant-sensitive inflammatory pathways are also modulated by lutein [
100
], as reported
using extracts enriched in this xanthophyll from different species of the chlorophyte genus
Tetraselmis (Chlorodendrophyceae) [101].
2.2.3. Other Pro-Inflammatory Pathways Targeted by Microalgal Carotenoids
Mitochondrial disfunction is a key pathogenic driver in RA [
102
], and ASTX was
reported to attenuate organellar ROS production in human chondrocytes treated with
IL-1
β
[
69
]. In addition to suppressing pro-inflammatory pathways, ASTX is also suggested
to promote cartilage homeostasis via the transcriptional regulator nuclear factor-erythroid
2-related factor 2 (Nrf2) [
68
,
93
]. ASTX is suggested to stabilize and promote the nuclear
translocation of Nfr2, which binds so-called antioxidant response elements (AREs), enhanc-
ing the expression of anti-inflammatory and ROS-detoxifying genes [103,104].
2.3. Lipids and Their Derivatives
A hallmark of RA is the altered fatty acid profile of the synovium [
105
,
106
], while the
intake of polyunsaturated fatty acids (PUFAs) correlates with joint health and mitigates
the risk of RA onset [
107
]. Microalgal mass cultivation is a more sustainable way to derive
functional lipids compared with cold water fish [
108
–
112
]. Phytoplankton occupies the
lowest trophic level in oceans and freshwater basins, representing the primary PUFAs pro-
ducer in aquatic food webs [
113
]. Global warning and ocean acidification are predicted to
affect phytoplankton ecology [
114
,
115
], thus reducing PUFAs availability to higher trophic
levels and, eventually, putting at risk the supply for human nutrition [
116
]. Moreover,
upon stress acclimation, microalgae synthetise a wider range of anti-inflammatory and
immunomodulatory lipids compared to animals [117–121].
2.3.1. Long-Chain Polyunsaturated Fatty Acids
Several microalgae accumulate very long-chain PUFAs [
122
,
123
], including the
omega-3
(
ω
-3, n-3) PUFAs [
124
]
α
-linolenic (18:3), docosahexaenoic (DHA, 22:6, n-3, Figure 3A)
,
do-
cosapentaenoic (DPA, n-3, 22:5, Figure 3B), and eicosapentaenoic (EPA, 20:5, n-3,
Figure 3C) acids but also
ω
-6 PUFAs like arachidonic (ARA, 20:4, n-6, Figure 3H),
γ
-linolenic (18:3), linoleic (18:2), and dihomo-
γ
-linolenic (DGLA, 20:3, n-6, Figure 3D) acids.
These molecules are biosynthetic precursors of anti-inflammatory signaling molecules and
interfere with pro-inflammatory pathways [125,126].
The administration of lipids from the DGLA-hyperproducing freshwater chloro-
phyte Lobosphaera incisa (Trebouxiophyceae) suppressed the expression of the NF-
κ
B
pathway-related genes in an animal model of chronic inflammation [
127
]. Similarly, lipid
extracts from the haptophyte Pavlova lutheri (Prymnesiophyceae), an EPA- and DHA-
hyperaccumulating strain, inhibited IL-6 and TNF-
α
production in activated human
macrophages, possibly through suppressing the NF-κB pathway [128].
Several heterotrophic marine microorganisms are strong DHA producers, such as the
dinoflagellate Crypthecodinium cohnii (Dinophyceae) and, above all, the thraustochytrids pro-
tists Thraustochytrium spp., Aurantiochytrium (formerly Schizochytrium) limacinum (Labyrinthu-
lomycetes) [
129
–
133
], and related genera [
134
]. Although these microorganisms can-
not exploit light energy to drive their metabolism, they are capable of fermenting plant

Mar. Drugs 2023,21, 630 7 of 27
biomass hydrolysates, affording cost-effective heterotrophic cultivation using renewable
feedstocks [
135
–
141
]. Arguably, the most valuable heterotrophic sources of DHA are
Schizochytrium spp., which recently obtained Novel Food status [
142
,
143
]. Notably, a recent
human clinical trial investigated the supplementation of an EPA- and DHA-enriched oil
from a Schizochytrium sp. in RA patients, reporting beneficial effects on joint health and the
blood levels of inflammatory markers [144].
Mar. Drugs 2023, 21, x 8 of 28
Figure 3. (A) Docosahexaenoic (DHA, 22:6, n-3); (B) docosapentaenoic (DPA, 22:5, n-3); (C)
eicosapentaenoic (EPA, 20:5, n-3); (D) dihomo-γ-linolenic (DGLA, 20:3, n-6); (E) 1,2-diacylglyceryl-
3-O-4’-(N,N,N-trimethyl)-homoserine (DGTS); (F) 1,2-diacylglyceryl-3-O-carboxy-
(hydroxymethyl)-choline (DGCC); (G) 1,2-diacylglyceryl-3-O-2’-(hydroxymethyl)-(N,N,N-
trimethyl)-β-alanine (DGTA); (H) arachidonic (ARA, 20:4, n-6). Figures created with BioRender.com,
accessed on 15 November 2023.
The administration of lipids from the DGLA-hyperproducing freshwater
chlorophyte Lobosphaera incisa (Trebouxiophyceae) suppressed the expression of the NF-
κB pathway-related genes in an animal model of chronic inflammation [127]. Similarly,
lipid extracts from the haptophyte Pavlova lutheri (Prymnesiophyceae), an EPA- and DHA-
hyperaccumulating strain, inhibited IL-6 and TNF-α production in activated human
macrophages, possibly through suppressing the NF-κB pathway [128].
Figure 3.
(
A
) Docosahexaenoic (DHA, 22:6, n-3); (
B
) docosapentaenoic (DPA, 22:5, n-3); (
C
) eicos-
apentaenoic (EPA, 20:5, n-3); (
D
) dihomo-
γ
-linolenic (DGLA, 20:3, n-6); (
E
) 1,2-diacylglyceryl-3-
O-4’-(N,N,N-trimethyl)-homoserine (DGTS); (
F
) 1,2-diacylglyceryl-3-O-carboxy-(hydroxymethyl)-
choline (DGCC); (
G
) 1,2-diacylglyceryl-3-O-2’-(hydroxymethyl)-(N,N,N-trimethyl)-
β
-alanine (DGTA);
(
H
) arachidonic (ARA, 20:4, n-6). Figures created with BioRender.com, accessed on 15 November 2023.
2.3.2. Betaine Lipids
Betaine lipids are anti-inflammatory and immunomodulatory glycerolipids in which
the phosphate and carbohydrate moieties attached to the glycerol backbone are replaced
with positively charged ether-bond betaine groups. Betaine lipids are widely distributed in
all clades of eukaryotic microalgae, where they act as acyl group donors upon membrane
lipid remodelling and during the accumulation of storage neutral lipids [145,146].

Mar. Drugs 2023,21, 630 8 of 27
Betaine lipids derive from the turnover of membrane phospholipids under abiotic
stresses, mainly temperature and nutrient starvation [
147
–
149
]. The betaine lipid 1,2-
diacylglyceryl-3-O-4’-(N,N,N-trimethyl)-homoserine (DGTS, Figure 3E) is the most abundant
betaine lipid in microalgae, followed by 1,2-diacylglyceryl-3-O-carboxy-(hydroxymethyl)-
choline (DGCC, Figure 3F), and 1,2-diacylglyceryl-3-O-2’-(hydroxymethyl)-(N,N,N-trimethyl)-
β
-alanine (DGTA, Figure 3G), with evidence of DGTS-mediated inhibition of the IKK-
β
kinase causing suppression of the NF-
κ
B pathway and secretion of pro-inflammatory ILs
by Th1 and Th2 cells, as recently reported with extracts from the oleaginous chlorophyte
Chromochloris zofingiensis (Chlorophyceae) [150].
2.3.3. Oxylipins
The eicosanoids, prostaglandins and leukotrienes, and the thromboxanes, are hormone-
like oxygenated metabolites of C20 fatty acids involved in the modulation and resolution
of inflammation in the RA synovium [
106
]. In mammals, oxylipins are produced by
the enzyme phospholipase A2 via release of sn-2 PUFAs from membrane phospholipids.
Typical oxylipin precursors are linoleic,
α
-linolenic, and ARA, which are substrates of
cyclooxygenases, lipoxygenases, and cytochrome P450 enzymes, respectively [151].
Several microalgal species are known to accumulate prostaglandin-like
oxylipins [
152
–
154
]. Their synthesis can occur either enzymatically via animal-like biosyn-
thetic pathways [
155
], as in diatoms Skeletonema marinoi and Thalassiosira rotula (Bacillario-
phyceae) [
156
–
158
], or via spontaneous oxidation of ARA, EPA, and DHA in T. lutea [
159
].
The latter, known as isoprostanoids, are functional lipids which regulate bone health
through preventing osteoclast differentiation [160].
The oxylipins isolated from the chlorophytes Chlamydomonas debaryana (Chlorophyceae)
and Nannochloropsis gaditana inhibited TNF-
α
production in cultured macrophages [
161
],
while the oral administration of biomass of the former suppressed the production of pro-
inflammatory ILs in an animal model of chronic inflammation, possibly through inhibiting
the NF-κB pathway [162,163]
Figure 4summarizes the evidence-based interference of the above-mentioned microal-
gal carotenoids and lipids with the main intracellular pro-inflammatory pathways involved
in the pathogenesis of RA.
Mar. Drugs 2023, 21, x 10 of 28
Figure 4. Summary of evidence-based interference of selected microalgal metabolites with the in-
tracellular pro-inflammatory signaling pathways involved in the pathogenesis of RA. Microalgal
carotenoids and lipids exert inhibitory effects on major intracellular pro-inflammatory signaling
pathways involved in the onset and progression of rheumatoid arthritis. Blunt-ended solid lines
indicate an inhibitory pharmacological effect, while dashed lines suggest proposed interference.
Astaxanthin (ASTX), fucoxanthin, diatoxanthin, and β-carotene target different subunits of the In-
hibitor of κB (IkB) kinase complex, preventing the phosphorylation-dependent release of the pro-
inflammatory transcriptional activator NF-kB and its nuclear translocation. ASTX further acts up-
stream of this pathway through inhibiting the NF-κB-Inducing Kinase (NIK), preventing the auto-
phosphorylation of the Mitogen- and Stress-activated protein Kinase-1 (MSK1), a nucleus-localized
effector which activates NF-κB and the cAMP-responsive Element-Binding Protein (CREB) pro-in-
flammatory transcription factor. ASTX and β-carotene also inhibit the JNK/p38 MAPK signaling
cascade, blocking the nuclear translocation of the JNK/p38 MAPK complex, and thus phosphoryla-
tion of downstream targets: the pro-inflammatory transcription factors ELK1, MEF2, ATF2, and
STAT1, and the MAPK-activated kinase 2 (MK2) responsible for stabilizing IL mRNAs. β-carotene
inhibits the JAK2/STAT3 pathway through blocking phosphorylation of the pro-inflammatory tran-
scriptional activator STAT3 by JAK2 and its nuclear translocation. ASTX positively regulates the
nuclear factor-erythroid 2-Related factor 2 (Nrf2)-mediated pathway involved in antioxidant and
anti-inflammatory gene expression. ASTX and β-carotene are proposed to directly bind interleukin
6 (IL-6) and tumor necrosis factor-alpha (TNF-α), blocking their receptor interaction and activation
of the downstream pathways. Lutein is suggested to inhibit both NF-kB and JNK/p38 MAPK path-
ways. ASTX detoxifies free radicals on both sides of the lipid bilayer and appears to suppress mito-
chondrial ROS production. The betaine lipid DGTS interferes with the activity of the IKKβ subunit
of the IkB kinase complex, while the PUFAs docosahexaenoic (DHA), eicosapentaenoic (EPA), and
dihomo-γ-linolenic (DGLA) acids modulate the NF-kB signaling cascade through targeting un-
known pathway components. Figures created with BioRender.com, accessed on 15 November 2023.
3. Bioprospecting and Domestication of Pharmacologically Relevant Microalgae
Of the over 70,000 estimated existing algal strains [164], only a few species are used
in human nutrition and health. Bioprospecting for novel pharmacologically relevant spe-
cies [165,166] requires scrupulous large-scale screening of phytoplankton biodiversity and
chemodiversity [167], and the establishment of optimal cultivation strategies [168]. Inhos-
pitable environments are excellent ecosystems to discover species with industrial applica-
tions since extremophiles are physiologically adapted to harsh conditions and hyperaccu-
mulate pigments and lipids [11,12,169–171].
Figure 4.
Summary of evidence-based interference of selected microalgal metabolites with the
intracellular pro-inflammatory signaling pathways involved in the pathogenesis of RA. Microalgal

Mar. Drugs 2023,21, 630 9 of 27
carotenoids and lipids exert inhibitory effects on major intracellular pro-inflammatory signaling path-
ways involved in the onset and progression of rheumatoid arthritis. Blunt-ended solid lines indicate
an inhibitory pharmacological effect, while dashed lines suggest proposed interference. Astaxanthin
(ASTX), fucoxanthin, diatoxanthin, and
β
-carotene target different subunits of the Inhibitor of
κ
B
(IkB) kinase complex, preventing the phosphorylation-dependent release of the pro-inflammatory
transcriptional activator NF-kB and its nuclear translocation. ASTX further acts upstream of this path-
way through inhibiting the NF-
κ
B-Inducing Kinase (NIK), preventing the autophosphorylation of the
Mitogen- and Stress-activated protein Kinase-1 (MSK1), a nucleus-localized effector which activates
NF-
κ
B and the cAMP-responsive Element-Binding Protein (CREB) pro-inflammatory transcription
factor. ASTX and
β
-carotene also inhibit the JNK/p38 MAPK signaling cascade, blocking the nuclear
translocation of the JNK/p38 MAPK complex, and thus phosphorylation of downstream targets: the
pro-inflammatory transcription factors ELK1, MEF2, ATF2, and STAT1, and the MAPK-activated
kinase 2 (MK2) responsible for stabilizing IL mRNAs.
β
-carotene inhibits the JAK2/STAT3 pathway
through blocking phosphorylation of the pro-inflammatory transcriptional activator STAT3 by JAK2
and its nuclear translocation. ASTX positively regulates the nuclear factor-erythroid 2-Related factor 2
(Nrf2)-mediated pathway involved in antioxidant and anti-inflammatory gene expression. ASTX
and
β
-carotene are proposed to directly bind interleukin 6 (IL-6) and tumor necrosis factor-alpha
(TNF-
α
), blocking their receptor interaction and activation of the downstream pathways. Lutein is
suggested to inhibit both NF-kB and JNK/p38 MAPK pathways. ASTX detoxifies free radicals on
both sides of the lipid bilayer and appears to suppress mitochondrial ROS production. The betaine
lipid DGTS interferes with the activity of the IKK
β
subunit of the IkB kinase complex, while the
PUFAs docosahexaenoic (DHA), eicosapentaenoic (EPA), and dihomo-
γ
-linolenic (DGLA) acids
modulate the NF-kB signaling cascade through targeting unknown pathway components. Figures
created with BioRender.com, accessed on 15 November 2023.
3. Bioprospecting and Domestication of Pharmacologically Relevant Microalgae
Of the over 70,000 estimated existing algal strains [
164
], only a few species are used
in human nutrition and health. Bioprospecting for novel pharmacologically relevant
species [
165
,
166
] requires scrupulous large-scale screening of phytoplankton biodiversity
and chemodiversity [
167
], and the establishment of optimal cultivation strategies [
168
].
Inhospitable environments are excellent ecosystems to discover species with industrial
applications since extremophiles are physiologically adapted to harsh conditions and
hyperaccumulate pigments and lipids [11,12,169–171].
Among cryophilic species, the extracts of two Antarctic chlorophytes, Chloromonas retic-
ulata (Chlorophyceae) and Micractinium simplicissimus (Trebouxiophyceae), were recently
reported to exert an anti-inflammatory effect on activated macrophages [
172
,
173
]. At
the other extreme, the rhodophyte Cyanidioschyzon merolae (Cyanidiophyceae) thrives at
40
◦
C and low pH, producing heat-stable carotenoids [
174
], while a stress-resilient strain
of the marine chlorophyte species Tetraselmis striata [
175
] accumulates anti-inflammatory
carotenoids and lipids [
176
]. A PUFAs-hyperproducing rhodophyte strain of the Galdieria
genus (Cyanidiophyceae) was identified in acid thermal springs, and its lipid content could
be enhanced via cultivation at temperatures below its optimal range [
177
]. Finally, the
extracts of a chlorophyte Mucidosphaerium sp. (Trebouxiophyceae) isolated from a similar
environment suppressed pro-inflammatory gene expression in human fibroblasts, as well
as mitochondrial ROS production and inflammation in cultured synoviocytes [178,179].
Turning Wild Species into “Unicellular Medicinal Crops”
Although the accumulation of therapeutic compounds in microalgae can be accrued
through abiotic stress challenges [
180
–
185
], wild organisms are usually not suited for
industrial applications. For instance, ASTX production via mass cultivation H. pluvialis is
restrained by its slow growth and elevated risk of pest contamination [186,187].
Adaptive laboratory evolution and random mutagenesis are powerful strategies for strain
improvement based, respectively, on spontaneous and enhanced mutation rates [
188
,
189
]
(Figure 5A). This approach generated lipid- and carotenoid-hyperproducing strains of

Mar. Drugs 2023,21, 630 10 of 27
chlorophyte Chlorella vulgaris (Trebouxiophyceae) [
190
,
191
], enabling the discovery of new
genetic targets to enhance lutein content [
192
]. Similarly, a simultaneous enhancement
of ASTX and EPA was reported in the Nannochloropsis species gaditiana [
193
] and in the
Tetraselmis striata [
194
], while DHA accrual was achieved in Schizochytrium sp. [
195
–
197
],
C. cohnii [
198
], and P. lutheri [
199
]. It should be noted, however, that evolved strains are
susceptible to the risk of retromutation and trait drift [200].
Mar. Drugs 2023, 21, x 12 of 28
Figure 5. Nuclear and chloroplast engineering to produce immunotherapeutics in microalgae. (A)
Foreign DNA sequences can be introduced in the nuclear genome of microalgae resulting in the
stable integration of transgene(s). Trait evolution can be achieved through random mutagenesis us-
ing physical (e.g., UV radiation, violet flash) or chemical agents followed by the identification of
phenotypes of interest. The recombinant protein products synthetized on cytoplasmic ribosomes
can be either accumulated in the cell or secreted in the cultivation medium to facilitate their recov-
ery. Alternatively, recombinant metabolic enzymes can be targeted to the chloroplast to enhance the
biosynthesis of native long-chain polyunsaturated fay acids (PUFAs), or even exotic metabolites
with immunomodulatory/anti-inflammatory properties. (B) Transgenes are introduced in the chlo-
roplast via biolistic delivery and targeted to defined chromosomal loci exploiting homologous re-
combination-enabled homology arm sequences (HA, purple) flanking the gene of interest (GOI, blue
segment). The high copy number of plastid chromosomes ensures a significantly greater synthesis
of different classes of recombinant immunotherapeutics compared with the engineering of the nu-
clear haploid genome. Figures created with BioRender.com, accessed on 15 November 2023.
Figure 5.
Nuclear and chloroplast engineering to produce immunotherapeutics in microalgae.
(
A
) Foreign DNA sequences can be introduced in the nuclear genome of microalgae resulting in the
stable integration of transgene(s). Trait evolution can be achieved through random mutagenesis
using physical (e.g., UV radiation, violet flash) or chemical agents followed by the identification of
phenotypes of interest. The recombinant protein products synthetized on cytoplasmic ribosomes can

Mar. Drugs 2023,21, 630 11 of 27
be either accumulated in the cell or secreted in the cultivation medium to facilitate their recovery.
Alternatively, recombinant metabolic enzymes can be targeted to the chloroplast to enhance the
biosynthesis of native long-chain polyunsaturated fatty acids (PUFAs), or even exotic metabolites with
immunomodulatory/anti-inflammatory properties. (
B
) Transgenes are introduced in the chloroplast
via biolistic delivery and targeted to defined chromosomal loci exploiting homologous recombination-
enabled homology arm sequences (HA, purple) flanking the gene of interest (GOI, blue segment).
The high copy number of plastid chromosomes ensures a significantly greater synthesis of different
classes of recombinant immunotherapeutics compared with the engineering of the nuclear haploid
genome. Figures created with BioRender.com, accessed on 15 November 2023.
4. Biomanufacturing of Immunomodulatory Metabolites
4.1. Engineering Carotenoid Metabolism
Although microalgae can potentially substitute plants in the production of carotenoids like
lutein [
201
,
202
], their yields are still lagging behind heterotrophic microorganisms [
203
–
206
].
Genetic engineering can greatly enhance pigment productivity in microalgae [
207
] but
requires a detailed knowledge of algal genomes and of the transcriptional networks regu-
lating carotenoid biosynthesis to manipulate key metabolic genes [
208
–
211
] (Figure 5A). In
this respect, a model organism for ketocarotenoid biosynthesis is C. zofingiensis [
212
,
213
],
from which the
β
-carotene ketolase (BKT) enzyme was identified as a rate-limiting factor
for ASTX production [
214
], while the chlorophytes D. salina and Desmodesmus spp. (Chloro-
phyceae) are useful resources for
β
-carotene and lutein metabolism, respectively [
215
,
216
].
Carotenoid enhancement and the production of non-native ASTX isomers can be
achieved through different engineering strategies [
217
], such as: (i) heterologous expression
of chaperones stabilizing biosynthetic enzymes [
218
,
219
] and cyanobacterial proteins to im-
prove pigment storage capacity [
220
], (ii) revival of endogenous silent BKT genes [
221
,
222
],
and (iii) multigene overexpression to circumvent several biosynthetic bottlenecks [
223
].
Simultaneous enhancement of fucoxanthin and
β
-carotene was achieved with a similar ap-
proach in the diatom Phaeodactylum tricornutum (Bacillariophyceae) through overexpressing
endogenous biosynthetic genes [
224
,
225
] and a plastoglobule protein to augment pigment
sequestration [
226
]. Ketocarotenoids engineering was recently reported in the industrially
relevant species C. merolae via heterologous expression of two biosynthetic genes [
227
],
and in the thraustochytrid A. limacinum via overexpression of an endogenous
β
-carotene
hydroxylase gene [228].
4.2. Synthetic Long-Chain Carotenoids
Algal chloroplasts provide excellent metabolic chassis to engineer the synthesis of
non-native pigments with superior therapeutic properties [
229
], such as carotenoids with
extended polyene chains and extra hydroxyl groups [
230
,
231
]. In this respect, extremophilic
microorganisms are invaluable genetic resources to implement novel metabolic pathways
in microalgae [
232
–
234
]. Archaea produce long-chain (C50) carotenoids through adding iso-
prene (C5) units to lycopene (C40) [
235
]. This biosynthetic pathway was successfully intro-
duced in bacteria to produce C50 ASTX [
236
,
237
] and non-natural C60 ketocarotenoids [
238
],
and it would be of extreme interest to verify its feasibility in microalgae to accumulate
extremely valuable synthetic metabolites.
4.3. Enhancing PUFAs Accumulation
Microalgae can be engineered to maximize the yields of the endogenous anti-inflammatory
lipids [
239
]. Overexpression of endogenous or heterologous fatty acid biosynthesis genes is
a standard approach to enhance PUFAs productivity [
240
], and the availability of transcrip-
tomes investigating stress adaptation is crucial to identify new factors to modulate lipid
profiles [
241
]. For instance, P. lutheri, different Prasinophyte species of the genus Ostreococcus
(Mamiellophyceae), and the diatom Fragilariopsis cylindrus (Bacillariophyceae) are model
species for studying DHA and EPA biosynthesis [
242
–
244
]. Transcriptomics analysis of
Aurantiochytrium revealed fatty acid synthase isoforms involved in DHA production under

Mar. Drugs 2023,21, 630 12 of 27
nitrogen starvation [
245
], while an acyl-CoA binding protein related to lipid droplet remod-
eling and EPA synthesis in P. tricornutum was recently suggested as a target to enhance the
yields of therapeutic PUFAs [246].
Lipid enhancement was reported in the oleaginous chlorophyte Neochloris oleoabundans
(Chlorophyceae) via heterologous expression of Kennedy pathway genes from the chloro-
phyte Acutodesmus obliquus (Chlorophyceae) [
247
], while DHA was increased in P. tricornu-
tum through overexpressing the
∆
-6 desaturase [
248
], and the heterologous
∆
-5 elongase and
acyl-CoA-dependent ∆6-desaturase from Ostreococcus tauri [249].
DHA enhancement was achieved in Aurantiochytrium sp. via overexpression of glucose-
6-phosphate dehydrogenase to increase NADPH regeneration for fatty acid biosynthesis [
250
],
and in a Schizochytrium sp. via overexpression of ATP-citrate lyase and acetyl-CoA carboxy-
lase [
251
]. Lastly, EPA was increased in D. salina [
252
] with a heterologous
∆
-6 desaturase
gene from T. pseudonana [253].
Prostaglandin biosynthesis was engineered in the oleaginous diatom Fistulifera solaris
(Bacillariophyceae), a natural producer of C20 PUFAs, via expression of a cyclooxygenase
gene from the red macroalga Agarophyton vermiculophyllum (Gracilariaceae), resulting in the
highest reported heterologous prostaglandin production in a photosynthetic host.
5. Production of Heterologous Immunotherapeutics in Microalgae
Microalgae can be genetically engineered [
254
] to produce heterologous biopharma-
ceuticals [255,256], including human immune-related proteins [257–260].
Nuclear transgenesis affords eukaryotic-like post-translational modifications of recom-
binant proteins (mainly N-terminal glycosylation) [
261
,
262
] and extracellular secretion to
facilitate recovery [
258
,
263
] (Figure 5A). Although nuclear transgenesis results in low ex-
pression titers and requires intensive screening efforts to identify high-yielding strains [
264
],
combinatorial transgene assembly and synthetic regulatory elements can greatly improve
translation rates [265,266].
As in plants [
267
], the algal chloroplast genome affords high-level accumulation of
recombinant therapeutics [
268
–
271
] (Figure 5B). The prokaryotic features of the polyploid
chloroplast genome (plastome) enable efficient homologous recombination-based transgene
insertion and multigene expression with synthetic operons [
272
]. A rich genetic toolkit
allows plastome manipulation in non-model species [
273
], including marker-free and
metabolism-dependent selection strategies [
274
–
276
]. Among these, the type D phosphite
dehydrogenase (PtxD) gene, which enables the oxidization of non-assimilable phosphite
ions in phosphate, is a biosafe solution to maintain axenic cultures [
277
,
278
], especially in
mixotrophic conditions where pest contamination risk is high [279].
Fast Tracking Microalgal Immunotherapeutics: The Time Is Now
Among newly proposed therapeutic agents for autoimmune diseases, fragment crys-
tallisable (Fc) multimers against autoantibodies and peptides targeting Fc/Fc
γ
receptors
are highly promising candidates [
280
]. Since microalgae can assemble full-length human
monoclonal antibodies binding Fc
γ
receptors [
281
–
283
], it should be possible to produce
antibodies targeting RA mediators [
284
]. Notably, polycistronic nuclear gene expression
was reported in microalgae [
285
], potentially affording simultaneous production of multiple
immunotherapeutic variants.
Viral nanoparticles are emerging tools with diagnostic and therapeutic applications in
autoimmune diseases [
286
,
287
]. This strategy was pioneered in the plantNicotiana benthamiana
via self-assembled nanoparticles exposing RA autoantigens, which induced immunotol-
erance in animal RA models [
288
]. Although few studies have explored the use of viral
vectors to produce recombinant therapeutics in microalgae so far [
289
,
290
], the ongoing
characterization of microalgae-infecting viruses [
291
–
294
] should facilitate this approach in
photosynthetic microbes [295,296].

Mar. Drugs 2023,21, 630 13 of 27
6. Final Observations, Comments, and Outlook
Microalgae are key players in the transition towards a bioeconomy [
297
], and are
the focus of intense academic and private research [
298
,
299
]. In Europe, the number of
companies engaging in microalgae production is growing [
300
], and so is the list of patents
describing their therapeutic uses in inflammatory diseases [
301
]. Microalgal biotechnology,
however, is still restrained by low product yields and recovery [
302
–
306
], high operating
costs, and barriers to market entry [307,308], especially for engineered strains [309].
6.1. Building a Good Reputation
Arguably, the biggest obstacle to the nutritional uses of photosynthetic microbes
is their recognition as safe [
310
]. Currently, only five eukaryotic microalgae and three
cyanoprokaryotes hold Novel Food status [
311
]. The occasional finding of hazardous
contaminants such as heavy metals, toxins, pathogens, and pesticides in the harvested
algal biomass [
312
,
313
] calls for rigorous adherence to good manufacturing practice in the
microalgal food industry [
314
,
315
], especially in the case of emerging strains [
316
]. An
even greater barrier is the fear of horizontal transfer of antibiotic resistance genes from
engineered strains to the gut microbiome and human pathogens [
317
], thus the adoption of
marker-free selection [
274
] and metabolic markers [
275
] are expected to promote a more
favourable attitude towards engineered microalgae and their derived products.
6.2. A Road Map for Clinical Uses of Microalgae in Chronic Inflammation
To advance the applications of microalgae in autoimmune diseases, the therapeu-
tic potential of novel classes of bioactive compounds should be explored. For example,
some microalgal strains are sustainable sources of the lipid-based prohormone vitamin
D
3
, the biosynthetic precursor of the biologically active 1,25-dihydroxyvitamin D (cal-
citriol) [
318
,
319
] whose deficiency is a major risk factor for RA [
320
,
321
]. Several microalgae
accumulate bioactive ergosterol and
β
-sitosterol [
322
,
323
], precursors of vitamin D
2
(ergo-
calciferol) and D
3
, respectively [
324
,
325
]. Moreover, the chlorophyte Botryococcus braunii
(Trebouxiophyceae) and Schizochytrium mangrovei can be engineered to hyperaccumulate
squalene, an anti-inflammatory triterpene and precursor of ergosterol [326–331].
The photosynthetic protist Euglena gracilis (Euglenoidea), a recently authorised Novel
Food species [
332
], also deserves attention, being a natural producer of anti-inflammatory
carotenoids, DHA [
333
–
335
], and paramylon, an immunomodulatory (1,3)-
β
-glucan [
336
]
with reported inhibitory activity towards Th17 cells [337].
Finally, photosynthetic prokaryotes are still largely unexplored biomanufacturing plat-
forms, despite producing a vast repertoire of unique compounds [
338
], including molecules
with antioxidant and anti-inflammatory properties [
339
,
340
] like the pigment–protein com-
plex phycocyanin, which is a selective inhibitor of pro-inflammatory oxylipin synthe-
sis [
341
,
342
], and various polysaccharides [
343
–
345
] and peptides [
346
–
348
]. Last but not
least, an increasing number of genetic engineering and synthetic biology tools [
349
] can
be employed to produce recombinant therapeutics in cyanoprokaryotes [
350
], as already
reported for immunomodulatory proteins [351].
7. Conclusions
The exploration of microalgal biodiversity and chemodiversity is a promising ap-
proach to discover new complementary therapeutic approaches for the management of
RA and related chronic inflammatory conditions. Indeed, several preclinical studies have
highlighted multiple mechanisms of action of microalgal compounds and their interfer-
ence with pro-inflammatory pathways. Moreover, the availability of advanced genetic
engineering tools holds tremendous potential to develop innovative biopharmaceuticals in
photosynthetic microbes and expand their clinical applications.
Funding: This research received no external funding.
Data Availability Statement: No new data were created or analyzed in this study.

Mar. Drugs 2023,21, 630 14 of 27
Acknowledgments:
E.A.C. acknowledges the support of the post-doctoral research fellowship “Borsa
Valeria e Vincenzo Landi per Ricerche nel Campo della Genetica Agraria” from the Accademia
Nazionale dei Lincei. R.C. acknowledges the financial support of the European Research Council
(ERC) Advanced Grant 101053983-GrInSun (Harvesting Light for Life: Green Proteins at the Interface
between Sun Energy and Biosphere, Scientific Coordinator: Roberto Bassi).
Conflicts of Interest: The authors declare no conflict of interest.
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