CRISPR/Cas9-Mediated Knockout of the Lycopene ε-Cyclase for Efficient Astaxanthin Production in the Green Microalga Chlamydomonas reinhardtii

Abstract

Carotenoids are valuable pigments naturally occurring in all photosynthetic plants and microalgae as well as in selected fungi, bacteria, and archaea. Green microalgae developed a complex carotenoid profile suitable for efficient light harvesting and light protection and harbor great capacity for carotenoid production through the substantial power of the endogenous 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Previous works established successful genome editing and induced significant changes in the cellular carotenoid content in Chlamydomonas reinhardtii. This study employs a tailored carotenoid pathway for engineered bioproduction of the valuable ketocarotenoid astaxanthin. Functional knockout of lycopene ε-cyclase (LCYE) and non-homologous end joining (NHEJ)-based integration of donor DNA at the target site inhibit the accumulation of α-carotene and consequently lutein and loroxanthin, abundant carotenoids in C. reinhardtii without changes in cellular fitness. PCR-based screening indicated that 4 of 96 regenerated candidate lines carried (partial) integrations of donor DNA and increased ß-carotene as well as derived carotenoid contents. Iterative overexpression of CrBKT, PacrtB, and CrCHYB resulted in a 2.3-fold increase in astaxanthin accumulation in mutant ΔLCYE#3 (1.8 mg/L) compared to the parental strain UVM4, which demonstrates the potential of genome editing for the design of a green cell factory for astaxanthin bioproduction.

Full text

Citation: Kneip, J.S.; Kniepkamp, N.;
Jang, J.; Mortaro, M.G.; Jin, E.; Kruse,
O.; Baier, T. CRISPR/Cas9-Mediated
Knockout of the Lycopene ε-Cyclase
for Efficient Astaxanthin Production
in the Green Microalga
Chlamydomonas reinhardtii.Plants
2024,13, 1393. https://doi.org/
10.3390/plants13101393
Academic Editor: Tomoko Shinomura
Received: 5 April 2024
Revised: 9 May 2024
Accepted: 9 May 2024
Published: 17 May 2024
Copyright: © 2024 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/).
plants
Article
CRISPR/Cas9-Mediated Knockout of the Lycopene ε-Cyclase
for Efficient Astaxanthin Production in the Green Microalga
Chlamydomonas reinhardtii
Jacob Sebastian Kneip 1, Niklas Kniepkamp 1, Junhwan Jang 2, Maria Grazia Mortaro 1, EonSeon Jin 2,
Olaf Kruse 1and Thomas Baier 1, *
1Algae Biotechnology and Bioenergy, Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld
University, 33615 Bielefeld, Germany
2Department of Life Science, Research Institute for Natural Sciences, Hanyang University,
Seoul 04763, Republic of Korea
*Correspondence: thomas.baier@uni-bielefeld.de
Abstract:
Carotenoids are valuable pigments naturally occurring in all photosynthetic plants and
microalgae as well as in selected fungi, bacteria, and archaea. Green microalgae developed a complex
carotenoid profile suitable for efficient light harvesting and light protection and harbor great capacity
for carotenoid production through the substantial power of the endogenous 2-C-methyl-D-erythritol
4-phosphate (MEP) pathway. Previous works established successful genome editing and induced sig-
nificant changes in the cellular carotenoid content in Chlamydomonas reinhardtii. This study employs a
tailored carotenoid pathway for engineered bioproduction of the valuable ketocarotenoid astaxanthin.
Functional knockout of lycopene
ε
-cyclase (LCYE) and non-homologous end joining (NHEJ)-based
integration of donor DNA at the target site inhibit the accumulation of
α
-carotene and consequently
lutein and loroxanthin, abundant carotenoids in C. reinhardtii without changes in cellular fitness.
PCR-based screening indicated that 4 of 96 regenerated candidate lines carried (partial) integrations of
donor DNA and increased ß-carotene as well as derived carotenoid contents. Iterative overexpression
of CrBKT, PacrtB, and CrCHYB resulted in a 2.3-fold increase in astaxanthin accumulation in mutant
∆
LCYE#3 (1.8 mg/L) compared to the parental strain UVM4, which demonstrates the potential of
genome editing for the design of a green cell factory for astaxanthin bioproduction.
Keywords:
Chlamydomonas reinhardtii; genome editing; astaxanthin production; microalgal carotenoids;
metabolic engineering; lycopene ß-cyclase; lycopene ε-cyclase
1. Introduction
The green microalga Chlamydomonas reinhardtii is an established model organism for
basic research in photosynthesis, cilia biogenesis, and phototaxis and is a promising next-
generation platform organism for sustainable bioproduction. It combines the ability for
phototrophic cultivation and simple genetic manipulation for the engineered synthesis
of numerous valuable products ranging from platform chemicals [
1
–
3
] to pharmaceutical
proteins [
4
] and high-value secondary metabolites [
5
–
8
]. Cultivation of green microalgae
in simple mineral salt solutions allows the direct conversion of waste carbon (e.g., CO
2
or
acetate) into industrial-relevant products and offers great potential to establish a resource-
efficient, decentralized bioeconomy [8].
C. reinhardtii natively harbors a complex pigment profile composed of chlorophylls
and carotenoids, each with individual value for biotechnological application. They medi-
ate a dynamically regulated capacity for light harvesting, while excess light is effectively
quenched by photoprotection mechanisms. Rapid carotenoid biosynthesis is fueled by
the metabolic power of the methyl-D-erythritol phosphate (MEP) pathway located in
the chloroplast. It provides sufficient amounts of isopentenyl pyrophosphate (IPP) and
Plants 2024,13, 1393. https://doi.org/10.3390/plants13101393 https://www.mdpi.com/journal/plants

Plants 2024,13, 1393 2 of 12
dimethylallyl pyrophosphate (DMAPP) as building blocks for phytoene, the shared precur-
sor of all endogenous carotenoids. The enzymes phytoene desaturase (PDS) and
ζ
-carotene
desaturase (ZDS) catalyze the conversion into bright-red-colored lycopene followed by
pathway branching into
α
- and ß-carotenes with
ε
/
β
- or ß/
β
-ionone-type end groups,
respectively, by the activity of two competing lycopene cyclases (LCYB/E). In C. reinhardtii,
α
-carotene is hydroxylated by the activity of P450
ε
/ß-ring hydroxylases (CYP97A and
CYP97C) [
9
] at both C3 positions for the synthesis of lutein, a common, yellow–red colored
xanthophyll found in all green plants and their progenitors. The native cellular abun-
dance in C. reinhardtii varies depending on the present light intensity [
10
], is reported to
range between 1.77 and 4.13 mg/g [
11
–
13
], and was engineered to 8.9 mg/g [
12
] by the
overexpression of Xanthophyllomyces dendrorhous phytoene-
β
-carotene synthase (crtYB). An
alternative strategy, by the overexpression of C. reinhardtii LCYE, resulted in a 2.6-fold
increased lutein production (to 0.48 mg/L) [
14
]. Lutein can be further hydroxylated at the
C9 position to form loroxanthin by a yet unknown enzyme [
15
], and the lutein–loroxanthin
cycle has a potential function in non-photochemical quenching (NPQ) [10,16–18].
The ß-carotene branch in C. reinhardtii continues by enzymatic hydroxylation via
ß-carotene hydroxylase (CHYB) and results in the formation of zeaxanthin, a frequent
yellow-colored xanthophyll and the start point of the photoprotective xanthophyll cycle.
Zeaxanthin is subject to epoxidation from reactive oxygen species (ROS) in excess light
or via zeaxanthin epoxidase (ZEP) activity to form antheraxanthin and violaxanthin, re-
spectively. A balance of cellular zeaxanthin and violaxanthin levels is an important feature
of NPQ and assists in the dissipation of stimulating light energy [
15
,
19
,
20
]. Additional
photoprotection strategies have evolved in selected organisms to cope with high light
conditions, e.g., by synthesis of astaxanthin, which has a strong antioxidative potential.
The responsible enzyme, the ß-carotene ketolase (BKT), catalyzes the conversion of zeax-
anthin to astaxanthin and ß-carotene to canthaxanthin at high efficiency, and astaxanthin
accumulation is reported to contribute to ROS scavenging and highlight protection [
21
,
22
].
C. reinhardtii does not synthesize astaxanthin in a vegetative state; however, expression
of an evolutionarily silenced ß-carotene ketolase (CrBKT [
23
]) was recently restored by
an innovative gene design [
24
,
25
]. Systematic metabolic engineering identified present
limitations in the activity of involved ß-carotene hydroxylase and phytoene synthase as
rate-limiting steps for increased astaxanthin production in C. reinhardtii [
26
]. By application
of high light intensities (3000
µ
mol photons/m
2
/s) in a high-cell-density cultivation [
2
],
accumulation of up to 23.5 mg/L was achieved after 94 h cultivation [26].
A complex cellular carotenoid profile assists in light-harvesting as well as excitation-
energy quenching [
19
]; however, a tailored carotenoid pathway can support engineered
astaxanthin biosynthesis by redirecting flux from the competing
α
-carotene biosynthesis
and would simplify extraction efforts due to reduced carotenoid byproducts [
27
]. However,
it is essential to maintain light protection activity for robust growth and cellular fitness,
even under elevated light conditions. The application of genome-editing technology is
readily established in C. reinhardtii [
28
–
33
] and was recently applied as a CRISPR-Cas9
ribonucleoprotein (RNP) complex for engineering an increased zeaxanthin accumulation by
a functional double knockout of LCYE and ZEP [
27
]. Zeaxanthin contents were increased
by 60% and resulted in up to 5.2 mg/L (7.3 mg/g) with elevated purity; however, this
mutant showed impaired non-photochemical quenching capacity partly compensated for
by an enhanced cyclic electron flow in phototrophic conditions [
34
]. However, individual
effects from an LCYE knockout have not been tested, yet.
In this study, we demonstrate the potential of a tailored pigment biosynthesis pathway
in C. reinhardtii production strain UVM4. We provide a detailed characterization of the
growth performance of mutants lacking LCYE and the
α
-carotene route of carotenoid
biosynthesis and demonstrate the increased capacity for efficient astaxanthin bioproduction
in this alga.

Plants 2024,13, 1393 3 of 12
2. Results and Discussion
2.1. Cas9-Mediated Knockout of CrLCYE by Integration of Selection Marker aphVII
C. reinhardtii harbors a versatile carotenoid biosynthesis pathway divided into an
α
-
and ß-carotene route resulting from competing
ε
/ß-lycopene cyclase activity (Figure 1A,
LCYB and LCYE). The ß-carotene route provides essential precursors for recently revived
astaxanthin production in C. reinhardtii [
23
], while LCYE activity towards
α
-carotene
displays a substantial competing pathway (Figure 1A, red arrows). In the nuclear genome of
C. reinhardtii, only a single gene locus for LCYE exists (gene ID: Cre06.g267600) and encodes
for a 583 amino acid (aa) large protein, which has a predicted thylakoid luminal transfer
peptide (at aa position 1-41, Pr: 0.4237 targetP 2.0 [
35
]). This gene was already described
by engineered overexpression using genomic DNA amplification [
14
] and was subject
of a double knockout in a
∆
ZEP background for increased zeaxanthin biosynthesis [
27
];
however, effects from an individual knockout have not been described, yet.
Plants 2024, 13, x FOR PEER REVIEW 3 of 13
In this study, we demonstrate the potential of a tailored pigment biosynthesis path-
way in C. reinhardtii production strain UVM4. We provide a detailed characterization of
the growth performance of mutants lacking LCYE and the α-carotene route of carotenoid
biosynthesis and demonstrate the increased capacity for efficient astaxanthin bioproduc-
tion in this alga.
2. Results and Discussion
2.1. Cas9-Mediated Knockout of CrLCYE by Integration of Selection Marker aphVII
C. reinhardtii harbors a versatile carotenoid biosynthesis pathway divided into an α-
and ß-carotene route resulting from competing ε/ß-lycopene cyclase activity (Figure 1A,
LCYB and LCYE). The ß-carotene route provides essential precursors for recently revived
astaxanthin production in C. reinhardtii [23], while LCYE activity towards α-carotene dis-
plays a substantial competing pathway (Figure 1A, red arrows). In the nuclear genome of
C. reinhardtii, only a single gene locus for LCYE exists (gene ID: Cre06.g267600) and en-
codes for a 583 amino acid (aa) large protein, which has a predicted thylakoid luminal
transfer peptide (at aa position 1-41, Pr: 0.4237 targetP 2.0 [35]). This gene was already
described by engineered overexpression using genomic DNA amplification [14] and was
subject of a double knockout in a ΔZEP background for increased zeaxanthin biosynthesis
[27]; however, effects from an individual knockout have not been described, yet.
Figure 1. C. reinhardtii carotenoid pathway and targeted knockout of LCYE. (A) Simplified MEP and
carotenoid biosynthesis pathway in C. reinhardtii. Relevant enzymes are depicted in bold. Red cross
and red color display targeted knockout of LCYE and depleted α-carotene route in carotenoid syn-
thesis. (B) Schematic illustration of RNP-mediated DNA double-strand break and integration of do-
nor DNA containing an expression cassee composed of C. reinhardtii TUB2 promoter, Streptomyces
rimosus aphVII CDS, and C. reinhardtii COP21 terminator. Arrows indicate oligonucleotide binding
sites for amplification. Genetic constructs are illustrated using SBOL3.0 standard and genetic ele-
ments and are not at scale. (C) Exemplary agarose gel after separation of PCR products containing
the LCYE locus from genomic DNA samples of parental cell line UVM4 and an exemplary ΔLCYE
mutant. M—1 kb Plus DNA Ladder (NEB). The table presents the sgRNA binding sequence with
the PAM motif in green and the respective sequences from four selected ΔLCYE mutants (ΔLCYE#1-
4). Integration of aphVII is indicated in blue (functional ORF), inverted (integration in antisense di-
rection), or red (partial integration). Length of integrated aphVII cassee and additional random
DNA fragments at the 5′ and 3′ ends are indicated. G3P—glyceraldehyde 3-phosphate, DXS—1-
Figure 1.
C. reinhardtii carotenoid pathway and targeted knockout of LCYE. (
A
) Simplified MEP
and carotenoid biosynthesis pathway in C. reinhardtii. Relevant enzymes are depicted in bold. Red
cross and red color display targeted knockout of LCYE and depleted
α
-carotene route in carotenoid
synthesis. (
B
) Schematic illustration of RNP-mediated DNA double-strand break and integration
of donor DNA containing an expression cassette composed of C. reinhardtii TUB2 promoter, Strep-
tomyces rimosus aphVII CDS, and C. reinhardtii COP21 terminator. Arrows indicate oligonucleotide
binding sites for amplification. Genetic constructs are illustrated using SBOL3.0 standard and genetic
elements and are not at scale. (
C
) Exemplary agarose gel after separation of PCR products containing
the LCYE locus from genomic DNA samples of parental cell line UVM4 and an exemplary
∆
LCYE
mutant. M—1 kb Plus DNA Ladder (NEB). The table presents the sgRNA binding sequence with the
PAM motif in green and the respective sequences from four selected
∆
LCYE mutants (
∆
LCYE#1-4).
Integration of aphVII is indicated in blue (functional ORF), inverted (integration in antisense direc-
tion), or red (partial integration). Length of integrated aphVII cassette and additional random DNA
fragments at the 50and 30ends are indicated. G3P—glyceraldehyde 3-phosphate, DXS—1-deoxy-D-
xylulose-5-phosphate synthase, IPP—isopentenyl pyrophosphate, DMAPP—dimethylallyl pyrophos-
phate, GGPP—geranylgeranyl pyrophosphate, GGPPS—geranylgeranyl pyrophosphate synthase,
PSY(crtB)—phytoene synthase, LCYB—ß-lycopene cyclase, LCYE—
ε
-lycopene cyclase, CHYB—ß-
carotene hydroxylase, BKT—ß-carotene ketolase, TUB2—ß-2-tubulin promoter, aphVIII—Streptomyces
rimosus aminoglycoside 30-phosphotransferase gene VII, COP21—C. reinhardtii chlamyopsin 2/1.

Plants 2024,13, 1393 4 of 12
A suitable sgRNA binding site was previously predicted in the first exon (Figure 1B
and C, position 185–208 bp past the start codon) using Cas-Designer [
36
]. RNP assembly
was performed using
in vitro
synthesized sgRNAs and a commercial Cas9 protein followed
by electroporation, as previously described [
28
]. Induced double-strand breaks at the
target locus were used for non-homologous end joining (NHEJ)-based integration of a
donor DNA element containing the aphVII resistance gene, including expression elements
for transcription (Figure 1B, C. reinhardtii ß-2-tubulin promoter [
37
] and C. reinhardtii
Chlamyopsin 2/1 terminator [
38
] based on pChlamy3 and used in a previous study [
39
]).
Regenerated transformants exhibit resistance against the antibiotic hygromycin and were
further characterized by PCR amplification (Figure 1B, black arrows).
In total, 96 transformants were isolated and further characterized via PCR amplification
using oligonucleotides spanning the sgRNA target locus. No PCR product was observed
in 28 of 96 transformants (29%), likely due to sequence alteration preventing amplification
(e.g., genomic rearrangements, extended deletions, or insertions) mediated by NHEJ-based
repair, the major mechanism of double-strand break repair events [
40
–
42
]. 64 of 96 transfor-
mants (67%) had similar product sizes compared to parental strain UVM4, indicating an
unchanged LCYE locus (amplification length approx. 716 bp) and aphVII integration at
random positions to confer resistance. However, it is possible that small insertions and
deletions are present in some of these transformants that cannot be identified based on PCR
product size, resulting in an underestimated editing efficiency. Undesired additional DNA
integrations at random positions can cause off-target effects and phenotypical changes but
may be removed by crossing using parental strains if required.
Four of 96 transformants (4%) showed larger PCR product sizes compared to the
parental strain UVM4 (Figure 1C), indicating a potential integration of the aphVII cassette
(length 1.631 bp) into the LCYE target site. The observed editing frequency is in line with
recent findings for NHEJ-based editing [
27
,
43
] but lower compared to recent homology-
directed repair strategies (up to 10–60% [28,30,32]).
Additional oligonucleotides were used to amplify junctions across the inserted DNA
into the genomic LCYE locus (combinations of blue and black arrows, Figure 1B) fol-
lowed by Sanger sequencing for sequence verification (Supplementary Figure S1). Mutants
∆
LCYE#1 and 2 harbor an aphVII cassette with small deletions and few additional nu-
cleotides of random origin at the 5
0
site of the target locus (83 bp and 3 bp for
∆
LCYE#1 and
∆
LCYE#2, respectively). While the aphVII cassette was integrated into an antisense orienta-
tion in mutant
∆
LCYE#2, the integration in
∆
LCYE#4 was found to be only partial (492 bp)
and likely non-functional with a second aphVII integration at a random position. Partial
integration of donor DNA was previously described, using the same approach for LCYE
targeting [
27
], and multiple insertions occur frequently during nuclear transformations of
C. reinhardtii [
44
]. Mutant
∆
LCYE#3 carries a full-length aphVII cassette in sense orientation
followed by two short partial copies, with a short deletion (2 bp) at the 3
0
junction of the
target site. Error-free NHEJ integration without the loss of genomic DNA at the 3
0
junction
of the integration site was frequent in three of the four characterized transformants.
In the analyzed mutants, several junctions of ligated donor DNA ends show short (4 to
6 bp long) microhomologies to each other. In plants and algae, NHEJ is typically favored to
repair DNA damage [
40
–
42
], and DNA ends can either be ligated directly or processed until
terminal microhomologies stabilize DNA ends, a process called microhomology-mediated
end joining (MMEJ) [
45
,
46
]. This could explain why especially fragmental and incomplete
donor DNA integration occurred during genome editing. Despite the successful editing
of LCYE and NHEJ-based integration of donor DNA, recent advantages in homologous
directed repair (HDR) appear more favorable for precise genome editing and typically
result in predictable DNA integrations [
28
,
30
,
32
]. We selected mutant
∆
LCYE#3 for further
characterization of biomass accumulation, endogenous pigment profiles, and engineered
astaxanthin biosynthesis.

Plants 2024,13, 1393 5 of 12
2.2. Growth Characterization and Pigment Profile of Mutant ∆LCYE#3
Cultivation of parental strain UVM4 and mutant
∆
LCYE#3 was performed in mixotrophic
conditions using TAP medium [
47
] and continuous light at different intensities (100 (LL) and
500 (HL)
µ
mol photons/m
2
/s). Cell densities and cell dry weights were recorded regularly
throughout the course of cultivations until the stationary phase (Figure 2) was reached.
Highlight application resulted in rapid growth during the first 48 h past inoculation; how-
ever, final cell densities after 96 h were comparable for both light conditions, which reached
28.3
×
10
6
cells/mL for mutant
∆
LCYE#3 compared to 25.1
×
10
6
cells/mL for parental
strain UVM4 (Figure 2B). The final cell dry weight was comparable for both strains and
reached 0.73 and 0.71 g/L, respectively, indicating no phenotypical difference in growth and
biomass generation for both strains. Exposure to high light intensities directs electron transfer
towards the reduction of molecular oxygen, generating superoxide radicals (O
2−
.) at PSI [
48
]
and, consequently, results in cellular damage. Excess excitation energy can be relieved by
the lutein–loroxanthin cycle, which is absent in mutant
∆
LCYE#3. However, this mutant
was able to cope with selected conditions, likely due to compensatory NPQ mechanisms.
Pulse-amplitude-modulation (PAM) fluorometry indicates comparable photosystem II quan-
tum yields for both strains (UVM4: 0.595
±
0.030;
∆
LCYE#3 0.663
±
0.021) and sufficient
light tolerance.
Plants 2024, 13, x FOR PEER REVIEW 5 of 13
mology-mediated end joining (MMEJ) [45,46]. This could explain why especially fragmen-
tal and incomplete donor DNA integration occurred during genome editing. Despite the
successful editing of LCYE and NHEJ-based integration of donor DNA, recent advantages
in homologous directed repair (HDR) appear more favorable for precise genome editing
and typically result in predictable DNA integrations [28,30,32]. We selected mutant ΔL-
CYE#3 for further characterization of biomass accumulation, endogenous pigment pro-
files, and engineered astaxanthin biosynthesis.
2.2. Growth Characterization and Pigment Profile of Mutant ΔLCYE#3
Cultivation of parental strain UVM4 and mutant ΔLCYE#3 was performed in mixo-
trophic conditions using TAP medium [47] and continuous light at different intensities
(100 (LL) and 500 (HL) µmol photons/m2/s). Cell densities and cell dry weights were rec-
orded regularly throughout the course of cultivations until the stationary phase (Figure 2)
was reached. Highlight application resulted in rapid growth during the first 48 h past
inoculation; however, final cell densities after 96 h were comparable for both light condi-
tions, which reached 28.3 × 106 cells/mL for mutant ΔLCYE#3 compared to 25.1 × 106
cells/mL for parental strain UVM4 (Figure 2B). The final cell dry weight was comparable
for both strains and reached 0.73 and 0.71 g/L, respectively, indicating no phenotypical
difference in growth and biomass generation for both strains. Exposure to high light in-
tensities directs electron transfer towards the reduction of molecular oxygen, generating
superoxide radicals (O2−.) at PSI [48] and, consequently, results in cellular damage. Excess
excitation energy can be relieved by the lutein–loroxanthin cycle, which is absent in mu-
tant ΔLCYE#3. However, this mutant was able to cope with selected conditions, likely due
to compensatory NPQ mechanisms. Pulse-amplitude-modulation (PAM) fluorometry in-
dicates comparable photosystem II quantum yields for both strains (UVM4: 0.595 ± 0.030;
ΔLCYE#3 0.663 ± 0.021) and sufficient light tolerance.
Figure 2.
Growth performance of mutant
∆
LCYE#3. (
A
) Culture of parental strain UVM4 and
∆
LCYE#3 in a 100 mL shake flask 72 h past inoculation. (
B
) Cell-density measurements for UVM4
and
∆
LCYE#3 during a cultivation period of 96 h in TAP medium and constant illumination of
100
µ
mol photons/m
2
/s (LL) and 500
µ
mol photons/m
2
/s (HL). (
C
) Gravimetric cell dry-weight
quantification after 96 h cultivation in HL. (
D
) Total carotenoid content in TAP and HL after 72 h.
(
E
) Pigment quantification via HPLC using acetone extracts from strain UVM4,
∆
LCYE #3, and
commercial standards. (
F
) Thin-layer chromatography of acetone extracts from strain UVM4,
∆
LCYE
#3, and a commercial lutein standard. Signals from
α
/ß-carotene (Car), chlorophyll a/b (Chl),
xanthophylls (Xan), and lutein (Lut) are indicated at the respective positions. The asterisk indicates a
strong change in signal patterns. All quantifications are given as mean values, and error bars display
the standard deviation of three individual measurements from biological replicates.

Plants 2024,13, 1393 6 of 12
Total carotenoid contents were quantified based on absorbance measurements of ace-
tone extracts 72 h past inoculation and results indicate no change in the total amount of
pigments (Figure 2D). HPLC and thin-layer chromatography were performed to character-
ize the endogenous pigment profile. In parental strain UVM4, cellular lutein contents were
2.27 mg/L, while lutein was completely absent in
∆
LCYE#3, confirming the successful
knockout of LCYE.
Our data suggest that knockout of CrLCYE and the subsequent lack of the endogenous
α
-carotene route of carotenoid biosynthesis, including the lutein–loroxanthin cycle, does
not limit cellular fitness compared to parental strain UVM4 under selected conditions. The
carotenoid contents of the xanthophyll cycle were markedly increased, reflected by an
elevated cellular content of zeaxanthin by 1.9 fold (from 0.31 mg/L in UVM4 to 0.59 mg/L
in
∆
LCYE#3), of antheraxanthin by 2.25 fold (from 0.28 to 0.63 mg/L), and violaxanthin by
1.8 fold (from 1.3 to 2.3 mg/L). These findings are in line with the TLC results (Figure 2F),
where the signal intensity was reduced for a band at the running height of a commercial
lutein standard and increased for intermediate carotenoid signals of the xanthophyll cycle
(Figure 2F, asterisk). Likely, increased carbon channeling towards the ß-carotene route and
a compensatory upregulation of xanthophyll biosynthesis to provide light protection are
the main reasons for this increase under vegetative growth conditions.
2.3. Engineering Astaxanthin Biosynthesis in ∆LCYE#3
Engineered overexpression of endogenous ß-carotene ketolase (CrBKT) was enabled
via optimized gene design [
24
,
25
,
49
] and revived evolutionary silenced astaxanthin biosyn-
thesis in C. reinhardtii. Strategic metabolic engineering recently identified phytoene synthase
(PSY/crtB) and ß-carotene hydroxylase (CHYB) as rate-limiting enzymes for increased
astaxanthin bioproduction [
26
]. Nuclear transformations of parental strain UVM4 and
mutant
∆
LCYE#3 were performed to test the capacity for astaxanthin biosynthesis using
previously designed plasmids (Figure 3). Transformation efficiency and selection were
comparable for both strains, indicating no reduced cellular fitness mediated by the knock-
out of CrLCYE. Initial overexpression of CrBKT resulted in 0.61
±
0.14 mg/L astaxanthin
and 3.8
±
0.6 mg/L canthaxanthin for transformants derived from parental strain UVM4
(n = 10), while transformants from
∆
LCYE#3 reached 0.88
±
0.23 mg/L astaxanthin (+44%
compared to UVM4) and 2.9
±
0.6 mg/L canthaxanthin (
−
24%). Increased astaxanthin
levels indicate an increased flux towards zeaxanthin in mutant
∆
LCYE#3 which resulted in
reduced canthaxanthin formation as a byproduct during astaxanthin biosynthesis.
Iterative transformations of parental strains UVM4 and
∆
LCYE#3 were performed
using genetic constructs II and III (Figure 3C). Combined overexpression of CrBKT and
PacrtB (construct II) resulted in an average astaxanthin accumulation of 0.6
±
0.3 mg/L
in selected transformants for both background strains. These values are comparable to
previous findings where subsequent expression of CrBKT and PacrtB increased flux toward
total carotenoids and sufficient activity of CrBKT (reflected by increased canthaxanthin
biosynthesis); however, terminal hydroxylation towards astaxanthin was still limited [26].
Additional co-overexpression of CrCHYB induced a notable reddish coloration of cultures
and increased astaxanthin accumulation to 0.8
±
0.3 mg/L in UVM4 (Figure 3C II + III)
or 1.8
±
0.6 mg/L in mutant
∆
LCYE#3 (II + III), respectively. Astaxanthin biosynthesis
correlated with CrCHYB expression levels based on fluorescence measurements and re-
sulted in up to 2.44 mg/L after 72 h of mixotrophic cultivation. Biomass accumulation
during astaxanthin production was comparable for both background strains (UVM4 and
∆
LCYE#3) and derived transformants based on cell-density measurements and gravimetric
quantifications (Supplementary Figure S2); however, cell densities were slightly increased
for both engineered strains compared to their respective parental cell line.
Our results demonstrate that genome editing technology can successfully be used for
the design of tailored carotenoid biosynthesis in C. reinhardtii, which supports astaxanthin
production and would simplify downstream extraction strategies due to reduced pigment
profile complexity. Astaxanthin accumulation was likely increased by substrate channeling

Plants 2024,13, 1393 7 of 12
towards the ß-carotene route of the endogenous carotenoid pathway and native upreg-
ulation of xanthophyll biosynthesis. Functional knockout of CrLCYE and, consequently,
lack of a lutein–loroxanthin cycle is not affecting the cellular fitness of C. reinhardtii UVM4
and depicts a valuable target for engineering. In a previous study, the introduction of an
additional knockout of ZEP resulted in reduced chlorophyll contents, impaired growth,
and non-photochemical quenching [
27
,
34
]. Further engineering is required to streamline
carotenoid and astaxanthin biosynthesis in C. reinhardtii. Coupled with innovative overex-
pression strategies, these efforts can support the establishment of valuable bioproduction
concepts using C. reinhardtii as a green cell factory.
Plants 2024, 13, x FOR PEER REVIEW 7 of 13
Figure 3. Engineering astaxanthin biosynthesis. (A) Schematic representation of genetic construct I
for overexpression of CrBKT as a fusion with selection marker aadA [26]. Astaxanthin and can-
thaxanthin contents for 10 selected transformants derived from parental strain UVM4 and ΔL-
CYE#3, respectively. The box and whisker plots indicate the distribution of astaxanthin production
data from minimal (lowest line), lower quartile (boom of box), median (central line), mean (cross),
upper quartile (top of box), and maximal (top line) data points. Outliers are depicted as dots. Quan-
tification was performed via HPLC UV/Vis detection (470 nm) from acetone extracts after 72 h mix-
otrophic cultivation in HL. (B) Astaxanthin and canthaxanthin biosynthesis in C. reinhardtii by ex-
pression of CrBKT. (C) Schematic representation of genetic construct II and III for co-overexpression
of P. ananatis crtB and C. reinhardtii CHYB [26]. Astaxanthin contents were quantified for selected
transformants derived from parental strain UVM4 and ΔLCYE#3 in iterative transformations. Sig-
nificance levels from an unpaired, two-sided Student’s t-test assuming non-homogenous variances
are indicated (*** p < 0.01, n.s. p > 0.01). CrBKT—C. reinhardtii ß-carotene ketolase, CrCHYB—C. rein-
hardtii ß-carotene hydroxylase, ZEP—zeaxanthin epoxidase, VDE—violaxanthin de-epoxidase, Pa-
crtB—P. ananatis phytoene synthase, mVenus —yellow fluorescence protein (YFP), mRuby2—red
fluorescence protein (RFP), aadA—spectinomycin adenylyltransferase, PSAD—photosystem I reac-
tion center subunit II, Strep—Strep-tagII epitope, FDX—C. reinhardtii ferredoxin 1 terminator.
Iterative transformations of parental strains UVM4 and ΔLCYE#3 were performed
using genetic constructs II and III (Figure 3C). Combined overexpression of CrBKT and
PacrtB (construct II) resulted in an average astaxanthin accumulation of 0.6 ± 0.3 mg/L in
selected transformants for both background strains. These values are comparable to pre-
vious findings where subsequent expression of CrBKT and PacrtB increased flux toward
total carotenoids and sufficient activity of CrBKT (reflected by increased canthaxanthin
biosynthesis); however, terminal hydroxylation towards astaxanthin was still limited [26].
Additional co-overexpression of CrCHYB induced a notable reddish coloration of cultures
and increased astaxanthin accumulation to 0.8 ± 0.3 mg/L in UVM4 (Figure 3C II + III) or
1.8 ± 0.6 mg/L in mutant ΔLCYE#3 (II + III), respectively. Astaxanthin biosynthesis corre-
lated with CrCHYB expression levels based on fluorescence measurements and resulted
in up to 2.44 mg/L after 72 h of mixotrophic cultivation. Biomass accumulation during
Figure 3.
Engineering astaxanthin biosynthesis. (
A
) Schematic representation of genetic construct I for
overexpression of CrBKT as a fusion with selection marker aadA [
26
]. Astaxanthin and canthaxanthin
contents for 10 selected transformants derived from parental strain UVM4 and
∆
LCYE#3, respectively.
The box and whisker plots indicate the distribution of astaxanthin production data from minimal
(lowest line), lower quartile (bottom of box), median (central line), mean (cross), upper quartile (top of
box), and maximal (top line) data points. Outliers are depicted as dots. Quantification was performed
via HPLC UV/Vis detection (470 nm) from acetone extracts after 72 h mixotrophic cultivation
in HL. (
B
) Astaxanthin and canthaxanthin biosynthesis in C. reinhardtii by expression of CrBKT.
(
C
) Schematic representation of genetic construct II and III for co-overexpression of P. ananatis crtB
and C. reinhardtii CHYB [
26
]. Astaxanthin contents were quantified for selected transformants
derived from parental strain UVM4 and
∆
LCYE#3 in iterative transformations. Significance levels
from an unpaired, two-sided Student’s t-test assuming non-homogenous variances are indicated
(*** p< 0.01, n.s. p> 0.01). CrBKT—C. reinhardtii ß-carotene ketolase, CrCHYB—C. reinhardtii ß-
carotene hydroxylase, ZEP—zeaxanthin epoxidase, VDE—violaxanthin de-epoxidase, PacrtB—P.
ananatis phytoene synthase, mVenus —yellow fluorescence protein (YFP), mRuby2—red fluorescence
protein (RFP), aadA—spectinomycin adenylyltransferase, PSAD—photosystem I reaction center
subunit II, Strep—Strep-tagII epitope, FDX—C. reinhardtii ferredoxin 1 terminator.

Plants 2024,13, 1393 8 of 12
3. Materials and Methods
3.1. Construct Design and Molecular Cloning
Coding sequences of C. reinhardtii BKT (UniProt: Q4VKB4), Pantoea ananatis phytoene
synthase (crtB, UniProt: D4GFK9), and C. reinhardtii ß-carotene 3-hydroxylase (CHYB, UniProt:
Q4VKB5) were previously designed as intron-containing algal transgenes [
24
–
26
] using In-
tronserter [
49
] and chemically synthesized (GenScript Biotech Corporation, Piscataway, NJ,
USA) or PCR amplified via polymerase chain reaction performed using Q5 polymerase
(New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol. All
expression elements were assembled using the pOptimized vector system (Optimus Tech-
nologies, Pittsburgh, PA, USA) [
23
,
25
,
26
,
50
]. Transcription was driven by endogenous
PSAD promoter [
51
] and FDX1 terminator [
52
,
53
]. The endogenous PSAD chloroplast-
targeting peptide was employed to induce post-translational transport [
51
,
53
]. Cloning
was performed using respective restriction enzyme digestion and ligation (T4 Ligase, New
England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol. Assembled
DNA was used for the heat-shock transformation of chemically competent E. coli DH5a
cells followed by selection on an LB medium containing respective antibiotics (300 mg L
−1
ampicillin). Plasmid isolation was performed from overnight cultures using the peqGOLD
Plasmid Miniprep Kit I (VWR International, Radnor, PA, USA) according to the manufac-
turer’s protocol.
3.2. Cultivation and Nuclear Transformation of C. reinhardtii
Chlamydomonas reinhardtii strain UVM4 [
54
] was routinely maintained on solid TRIS
acetate–phosphate (TAP) medium [
47
] and cultivated in liquid TAP medium in microtiter
plates or Erlenmeyer flasks at room temperature and continuous light (200 to 400
µ
mol
photons m
−2
s
−1
). Cell densities were quantified using a Z2 Particle Counter (Beckman
Coulter Life Sciences, Brea, CA, USA) or a Countess II FL Automated Cell Counter (Thermo
Fisher Scientific Inc., Waltham, MA, USA). The cell dry weight (CDW) was gravimetrically
determined from 5–10 mL of culture pellets after centrifugation at 3000
×
gfor 5 min and
drying at 105
◦
C. Optical density was measured via absorbance quantification at 750 nm
using a Genesys UV/Vis spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA,
USA). Nuclear transformation was performed using 10
µ
g linearized plasmid DNA and
glass bead agitation [
55
] followed by regeneration overnight at very low light intensities
(5
µ
mol photons m
−2
s
−1
). The selection was performed on a solid TAP medium containing
appropriate antibiotics (10 mg L
−1
paromomycin, 10 mg L
−1
hygromycin, 200 mg L
−1
spectinomycin, and 2.5 mg L
−1
nourseothricin). For each construct, 288 transformants
were isolated randomly, and the best 20 expressing transformants were identified using
fluorescence measurements in a plant imaging system (NightShade LB 985, Berthold
Technologies GmbH & Co. KG, Bad Wildbad, Germany) with appropriate filter sets
for mVenus (excitation: 504 nm, emission: 530 nm) and mRuby2 (excitation: 560 nm,
emission: 600 nm). Transformants were individually cultivated in microtiter plates and
pooled samples were used for pigment quantification. The selection of strong expressing
transformants for iterative transformations was performed using fluorescence microscopy
(Leica MZ FLIII, Leica Microsystems GmbH, Wetzlar, Germany) with appropriate filter
sets for mVenus (excitation: 510/20 nm, emission: 560/40 nm) and mRuby2 (excitation:
545/30 nm, emission: 620/60 nm). Pulse-amplitude-modulation (PAM) fluorometry was
quantified using a Mini-Pam-II (Heinz Walz GmbH, Effeltrich, Germany).
3.3. CRISPR-Mediated Genome Editing and DNA Integration
Suitable sgRNA binding sites were previously identified [
27
] using the online tool Cas-
Designer (BioTools, Inc., Jupiter, Fl, USA) at standard settings (CRISPR RGENTools, SpCas9,
5
0
-NGG-3
0
,Chlamydomonas reinhardtii v5.0, 20 bp crRNA length [
36
]). The GeneArt
™
Precision sgRNA synthesis kit (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA,
USA) was used according to the manufacturer’s protocol for sgRNA synthesis followed
by purification using RNA Clean and Concentrator (Zymo Research Corporation, Orange,

Plants 2024,13, 1393 9 of 12
CA, USA). Functional RNPs were assembled by mixing 7
µ
g sgRNA with 8
µ
gSpCas9
protein (TrueCut Cas9 Protein v2, Invitrogen, Thermo Fisher Scientific Inc., Waltham,
MA, USA) and incubated at room temperature for 15 min. Donor DNA was amplified
from the aphVII cassette in a previously designed vector (pChlamy3 [
27
,
33
,
56
]) using
the KOD One
TM
PCR Master Mix (Toyobo Co., LTD., Osaka, Japan) and target specific
oligonucleotides (forward 5
0
-GAATGTCTTTCTTGCGCTATGACACTTC-3
0
; reverse 5
0
-
CAAGTACCATCAACTGACGTTACATTCTG-3
0
) followed by extraction from agarose gel
(Dokdo-PrepTM, Gel extraction Kit, Elpis Biotech, Daejeon, Republic of Korea).
A UVM4 culture was routinely diluted and maintained in the early logarithmic phase
(>1
×
10
6
cells mL
−1
) prior to cultivation under nitrogen limitation for 24 h [
28
]. In total,
7
×
10
7
cells were harvested, resuspended in TAP-sucrose (40 mM), and subjected to
heat shock (40
◦
C, 20 min). Prior to electroporation (Gene Pulser Xcel System (Bio-Rad
Laboratories, Inc., Hercules, CA, USA), square-wave protocol, 2 mm electrode-gap cuvettes,
single pulse, 8 ms, and 250 V), the assembled RNPs and 1
µ
g donor DNA were added,
mixed, and transferred to a sterile electroporation cuvette. Cells were regenerated for
10 min at room temperature prior to transfer to fresh TAP-sucrose medium for recovery
at 5
µ
mol photons m
−2
s
−1
. After 24 h, selection was applied by transfer on TAP agar
plates containing appropriate antibiotics (10 mg L
−1
hygromycin) for at least 5 days at
250–350 µmol photons m−2s−1.
Genomic DNA from derived transformants was characterized by colony PCR [
57
] via
amplification of the targeting locus (KOD One
TM
PCR Master Mix (Toyobo Co., LTD., Osaka,
Japan) or Q5 polymerase (New England Biolabs, Ipswich, MA, USA)) and accordingly
from integrated DNA (Figure 1). PCR products were separated in 2% (w/v) agarose gels at
100 V for 30 min, isolated using a peqGold gel-extraction kit (VWR International, Radnor,
PA, USA) and sequence identity was confirmed via sanger sequencing (Sequencing Core
Facility, CeBiTec, Bielefeld University, Bielefeld, Germany).
3.4. Pigment Characterisation via Absorbance and Chromatography
Pigment characterization was performed as previously described [
26
]. Briefly, ab-
sorbance spectra from 350 to 750 nm were recorded for acetone extracts from cell culture
pellets in a NanoDrop One photometer (Thermo Fisher Scientific Inc., Waltham, MA, USA),
and total pigments were determined as previously described [
26
]. Thin-layer chromatogra-
phy was performed with concentrated acetone extracts from cell pellets and separated on
silica gel plates (Nano-ADAMANT 0.2 mm, Macherey and Nagel, Düren, Germany) using
an appropriate running buffer (89.5% (v/v) petroleum, 10% (v/v) isopropanol, and 0.5%
(v/v) water).
High-performance liquid chromatography (HPLC) was performed for quantification
of endogenous pigments (Figure 2E) using a Shimadzu Prominence HPLC model LC-20AD
(Shimadzu, Kyoto, Japan) equipped with a Spherisorb 5.0
µ
m ODS1 4.6
×
250 mm cartridge
column (Waters Corporation, Milford, CT, USA), as previously described [
27
]. Astaxanthin
and Canthaxanthin contents (Figure 3) were quantified using a Dionex UltiMate 3000 HPLC
System (Thermo Fisher Scientific Inc., Waltham, MA, USA) and an Eurospher II 100-2 C18
column (100 mm
×
2 mm, Knauer Wissenschaftliche Geräte GmbH, Berlin, Germany) was
used with a diode array detector measuring at a wavelength of 470 nm. Carotenoids were
separated using a gradient between 9:1 methanol–water (A) and methanol (B) at a flow rate
of 1.0 mL min
−1
: 0 min B: 0%, 10 min B: up to 100%, 65 min B: 100%. Identification and
quantification of chromatography signals were compared to commercially available au-
thentic standards. Statistical evaluation has been performed using an unpaired, two-sided
Student’s t-test assuming non-homogenous variances (significance levels are indicated as
*** for p< 0.01 or n.s. for p> 0.01).
Supplementary Materials:
The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/plants13101393/s1, Figure S1: Sequence information of
LCYE target site and donor DNA integration in four selected transformants. Figure S2: Biomass accu-
mulation for parental strains and engineered production lines for efficient astaxanthin biosynthesis.

Plants 2024,13, 1393 10 of 12
Author Contributions:
Conceptualization, T.B., E.J. and O.K.; methodology, T.B. and E.J.; investiga-
tion, J.S.K., N.K., J.J. and M.G.M.; data curation J.S.K., N.K., J.J. and M.G.M.; writing T.B. and J.S.K.;
visualization, T.B. and J.S.K.; supervision, T.B., E.J. and O.K.; project administration, T.B. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement:
All data generated in this study are available in this published article
and Supplementary Materials.
Acknowledgments:
The authors would like to express their thanks to Ralph Bock for providing
C. reinhardtii strain UVM4. We acknowledge support for the publication costs by the Open Access
Publication Fund of Bielefeld University and the Deutsche Forschungsgemeinschaft (DFG).
Conflicts of Interest:
The authors declare no conflicts of interest. The funders had no role in the
design of the study; in the collection, analyses, or interpretation of data; in the writing of the
manuscript; or in the decision to publish the results.
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