Proteomic characterization of a lutein-hyperaccumulating Chlamydomonas reinhardtii mutant reveals photoprotection-related factors as targets for increasing cellular carotenoid content

Abstract

Background
Microalgae are emerging hosts for the sustainable production of lutein, a high-value carotenoid; however, to be commercially competitive with existing systems, their capacity for lutein sequestration must be augmented. Previous attempts to boost microalgal lutein production have focussed on upregulating carotenoid biosynthetic enzymes, in part due to a lack of metabolic engineering targets for expanding lutein storage.

Results
Here, we isolated a lutein hyper-producing mutant of the model green microalga Chlamydomonas reinhardtii and characterized the metabolic mechanisms driving its enhanced lutein accumulation using label-free quantitative proteomics. Norflurazon- and high light-resistant C. reinhardtii mutants were screened to yield four mutant lines that produced significantly more lutein per cell compared to the CC-125 parental strain. Mutant 5 (Mut-5) exhibited a 5.4-fold increase in lutein content per cell, which to our knowledge is the highest fold increase of lutein in C. reinhardtii resulting from mutagenesis or metabolic engineering so far. Comparative proteomics of Mut-5 against its parental strain CC-125 revealed an increased abundance of light-harvesting complex-like proteins involved in photoprotection, among differences in pigment biosynthesis, central carbon metabolism, and translation. Further characterization of Mut-5 under varying light conditions revealed constitutive overexpression of the photoprotective proteins light-harvesting complex stress-related 1 (LHCSR1) and LHCSR3 and PSII subunit S regardless of light intensity, and increased accrual of total chlorophyll and carotenoids as light intensity increased. Although the photosynthetic efficiency of Mut-5 was comparatively lower than CC-125, the amplitude of non-photochemical quenching responses of Mut-5 was 4.5-fold higher than in CC-125 at low irradiance.

Conclusions
We used C. reinhardtii as a model green alga and identified light-harvesting complex-like proteins (among others) as potential metabolic engineering targets to enhance lutein accumulation in microalgae. These have the added value of imparting resistance to high light, although partially compromising photosynthetic efficiency. Further genetic characterization and engineering of Mut-5 could lead to the discovery of unknown players in photoprotective mechanisms and the development of a potent microalgal lutein production system.

Full text

McQuillanetal.
Biotechnology for Biofuels and Bioproducts (2023) 16:166
https://doi.org/10.1186/s13068-023-02421-0
RESEARCH Open Access
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Biotechnology for Biofuels
and Bioproducts
Proteomic characterization of a lutein-
hyperaccumulating Chlamydomonas reinhardtii
mutant reveals photoprotection-related factors
astargets forincreasing cellular carotenoid
content
Josie L. McQuillan1*, Edoardo Andrea Cutolo2, Caroline Evans1 and Jagroop Pandhal1*
Abstract
Background Microalgae are emerging hosts for the sustainable production of lutein, a high-value carotenoid;
however, to be commercially competitive with existing systems, their capacity for lutein sequestration must be aug-
mented. Previous attempts to boost microalgal lutein production have focussed on upregulating carotenoid biosyn-
thetic enzymes, in part due to a lack of metabolic engineering targets for expanding lutein storage.
Results Here, we isolated a lutein hyper-producing mutant of the model green microalga Chlamydomonas reinhardtii
and characterized the metabolic mechanisms driving its enhanced lutein accumulation using label-free quantita-
tive proteomics. Norflurazon- and high light-resistant C. reinhardtii mutants were screened to yield four mutant lines
that produced significantly more lutein per cell compared to the CC-125 parental strain. Mutant 5 (Mut-5) exhib-
ited a 5.4-fold increase in lutein content per cell, which to our knowledge is the highest fold increase of lutein in C.
reinhardtii resulting from mutagenesis or metabolic engineering so far. Comparative proteomics of Mut-5 against its
parental strain CC-125 revealed an increased abundance of light-harvesting complex-like proteins involved in photo-
protection, among differences in pigment biosynthesis, central carbon metabolism, and translation. Further charac-
terization of Mut-5 under varying light conditions revealed constitutive overexpression of the photoprotective pro-
teins light-harvesting complex stress-related 1 (LHCSR1) and LHCSR3 and PSII subunit S regardless of light intensity,
and increased accrual of total chlorophyll and carotenoids as light intensity increased. Although the photosynthetic
efficiency of Mut-5 was comparatively lower than CC-125, the amplitude of non-photochemical quenching responses
of Mut-5 was 4.5-fold higher than in CC-125 at low irradiance.
Conclusions We used C. reinhardtii as a model green alga and identified light-harvesting complex-like proteins
(among others) as potential metabolic engineering targets to enhance lutein accumulation in microalgae. These have
the added value of imparting resistance to high light, although partially compromising photosynthetic efficiency.
*Correspondence:
Josie L. McQuillan
j.mcquillan@sheffield.ac.uk
Jagroop Pandhal
j.pandhal@sheffield.ac.uk
Full list of author information is available at the end of the article
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Page 2 of 21
McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
Further genetic characterization and engineering of Mut-5 could lead to the discovery of unknown players in photo-
protective mechanisms and the development of a potent microalgal lutein production system.
Keywords Lutein, Norflurazon, Quantitative proteomics, Chlamydomonas reinhardtii, Photoprotection, Pigments,
Light-harvesting complex stress-related proteins, Non-photochemical quenching, Microalgae
Background
Eukaryotic microalgae are photosynthetic microorgan-
isms capable of capturing and transforming light energy
and carbon dioxide (CO2) into high-value products such
as lipids, pigments, and proteins, among other useful
compounds [1–3]. Of these, the yellow-orange carot-
enoid lutein is of particularly high value; it is a power-
ful antioxidant and an essential human dietary nutrient
[4], providing pro-vitamin A and protecting against cer-
tain cancers, cardiovascular diseases, and age-related
macular degeneration [5, 6]. Several microalgal species
naturally accumulate high lutein contents, including two
recently described trebouxiophyceae, Parachlorella sp.
JD-076 and Chlorella sorokiniana FZU60, which pro-
duce up to 11.87 and 11.22mg lutein/g dry cell weight
(DCW), respectively [7, 8], and the chlorodendrophycea
Tetraselmis striata CTP4 strain (3.81mg g−1 DCW) [9].
Lutein is currently produced commercially by extract-
ing oleoresin from the petals of Tagetes erecta (marigold
plants), although microalgae could offer several advan-
tages over marigold farming, including faster growth
rates, reduced land, water and labour requirements,
and less susceptibility to seasonal perturbations [10,
11]. Moreover, microalgal lutein is synthesized in free
form, whereas marigold-derived lutein is esterified and
requires an extra saponification processing step [11].
Although high levels of lutein production have recently
been achieved by optimizing the growth parameters and
extraction methods of some microalgal strains including
those mentioned above [7, 8, 12, 13], applying a com-
bination of strain selection, growth optimization, and
metabolic engineering strategies could further increase
productivity.
In microalgae, lutein is synthesized in plastids, where
it is predominantly bound to light-harvesting complex
(LHC) proteins, among other xanthophyll and chloro-
phyll molecules [14, 15]. Xanthophylls such as lutein par-
ticipate in light-harvesting, are required for the proper
assembly and structural organization of photosystem
II (PSII), and confer high light tolerance by acting as
quenchers of triplet-state chlorophyll, which is responsi-
ble for the production of damaging singlet oxygen radi-
cals [16–18]. Due to its hydrophobic nature, lutein cannot
accumulate freely in the chloroplast stroma and must be
sequestered within membranes or enclosed hydropho-
bic environments; in microalgae, this is mostly limited to
LHCs within the thylakoid membrane [14]. is presents
a threshold to the amount of lutein that can accumulate,
set by the number of LHCs present within the thylakoid
[19]. Discovering a means to overcome this natural stor-
age capacity barrier could therefore improve the com-
mercial viability of lutein production in microalgae.
e green microalga Chlamydomonas reinhardtii nat-
urally produces lutein; although not credited as a high
lutein producer compared to other species [20], this
model alga has the benefit of having fast growth, genetic
tractability, and several decades’ worth of research and
omics data [21]. Furthermore, the nuclear C. reinhardtii
genome is haploid, meaning that all mutations are domi-
nant, and C. reinhardtii can reproduce both asexually and
sexually, enabling genetic crosses between strains exhib-
iting desirable characteristics, i.e. selective breeding.
Improvements in lutein accumulation in C. reinhardtii
may be translatable to other more productive microalgal
species, although recent advances in C. reinhardtii scale-
up suggest that this species may soon be a feasible indus-
trial producer [22, 23].
Several attempts have been made to enhance carot-
enoid production in C. reinhardtii; in most cases, carot-
enoid biosynthesis has been targeted via overexpression
of rate-limiting enzymes in the carotenogenesis pathway,
such as the heterologous expression of phytoene desatu-
rase from Dunaliella salina and Chlorella zofingiensis,
leading to 2.6-fold and 2.2-fold increases in lutein con-
tent, respectively [24, 25]. Otherwise, various forms of
the putative carotenoid biosynthesis regulator ORANGE
were overexpressed in C. reinhardtii, generating 1.7–3.1-
fold increases in lutein content [26–28]. Despite these
increases, the amount of lutein produced by targeting its
biosynthesis alone may, as mentioned above, be limited
by the carotenoid storage capacity of the cells. Overex-
pressing individual enzymes in C. reinhardtii may also
be hampered by notoriously stringent metabolic pathway
regulation at multiple levels, including feedback inhibi-
tion [29–32]. A powerful strategy to rapidly generate
new traits in microalgae, including pigment hyper-pro-
ducing phenotypes, is random mutagenesis followed by
stringent selection [33]. is method, which has already
been applied successfully to enhance the production of
lutein and other carotenoids in microalgae [20, 34, 35],
also provides opportunity to discover novel characteris-
tics within metabolic pathways and their regulation, and
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McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
possibly new targets for metabolic engineering. Here, we
generated a pigment hyperaccumulating C. reinhardtii
mutant (Mut-5) by random chemical mutagenesis, using
a selection process during which cells were simultane-
ously subjected to the carotenoid inhibitor norflurazon
and high light. Mutants were analysed for lutein accu-
mulation by high-performance liquid chromatography
(HPLC), which revealed a very high-lutein phenotype in
Mut-5. To acquire insights into the proteins and pathways
responsible for the increased pigment storage of Mut-5,
we performed comparative label-free quantitative (LFQ)
proteomics, which identified specific LHC proteins
among others as potential genetic engineering targets for
enhancing lutein production in microalgae. We then per-
formed biophysical and biochemical analyses on the new
strain, generating mechanistic insight into positive and
negative metabolic consequences of the high-pigment
and constitutively active-photoprotection phenotype.
Results
Generation andcharacterization ofMut‑5,
ahyper‑pigmented C. reinhardtii mutant
To randomly generate C. reinhardtii carotenoid-
overproducing mutants, CC-125 cells were chemi-
cally mutagenized with ethyl methanesulfonate (EMS)
and spread on to tris-acetate-phosphate (TAP) agar
plates supplemented with the carotenoid biosynthesis
inhibitor norflurazon, and then grown under high light
(1050 ± 150μmol photons m2 s−1). e combined effects
of high light and norflurazon enhance individual nega-
tive effects on C. reinhardtii growth [36], due to the
vital role of carotenoids in protecting cells from strong
irradiation, which damages cells via the generation of
reactive oxygen species [17, 37]. Carotenoid biosyn-
thesis inhibitors such as norflurazon have successfully
been used to isolate carotenoid overproducing micro-
algal mutants, although typically lethal concentrations
of inhibitor were applied, leading to the isolation of
strains carrying mutations in the target carotenoid
enzyme [20, 38, 39]. To avoid restricting mutations to
phytoene desaturase, the enzymatic target of norflura-
zon, sub-lethal concentrations of norflurazon that con-
fer some but not total inhibition of phytoene desaturase
were applied in this study, with the goal of revealing
novel mechanisms that increase carotenoid production.
Of the 648 colonies that survived the combined
selection pressures of norflurazon and high light, nine
mutants exhibited significantly higher relative carot-
enoid contents under ambient conditions compared to
the parental strain CC-125 at the final stage of screen-
ing (Additional File 1). ese mutants were scaled up to
25mL shake-flask cultures and grown under standard
mixotrophic conditions for 96h for pigment analysis
by spectrophotometry and HPLC. ree of the mutants
(Mut-5, Mut-6, and Mut-7) exhibited significantly
higher total chlorophyll (Chl) and total carotenoid con-
tents compared to CC-125, the highest being Mut-5,
which accumulated 3.0-fold more total Chl and 3.6-fold
more total carotenoids per cell than the CC-125 paren-
tal strain (Table1). e lutein content of Mut-5 was
also significantly higher than CC-125, and to a greater
extent than the other mutants, exhibiting 5.4-fold and
2.3-fold higher lutein contents per cell and per g of dry
cell weight, respectively (Table1).
Table 1 Pigment contents of C. reinhardtii CC-125 and nine high light- and norflurazon-resistant mutant strains
Isolated strains were scaled up to 25mL culture volumes and harvested after 4days. Chlorophyll (Chl) a, Chl b, total Chl and total carotenoid (Car) measurements were
estimated using a spectrophotometer [40]. Lutein content was measured by high-performance liquid chromatography against a lutein standard. DCW, dry cell weight.
Data represent mean values (n = 3, except for Mut-6 for which n = 2) ± the standard deviation from the mean. Multiple comparisons were performed using one-way
ANOVA, with a Bonferroni post hoc test, to compare each mutant to the CC-125 parental strain (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)
Strain Chl a (pg cell−1) Chl b (pg cell−1) Total Chl (pg cell−1) Total Car (pg cell−1) Lutein Content
fg cell−1mg g−1 DCW
CC-125 1.13 ± 0.26 0.57 ± 0.12 1.70 ± 0.38 0.25 ± 0.05 151 ± 24 1.77 ± 0.18
Mut-1 1.15 ± 0.20 0.69 ± 0.08 1.84 ± 0.28 0.31 ± 0.07 206 ± 25 1.89 ± 0.08
Mut-2 1.84 ± 0.24 1.02 ± 0.11*2.86 ± 0.35 0.45 ± 0.04 335 ± 7*2.43 ± 0.07
Mut-3 0.76 ± 0.24 0.41 ± 0.15 1.18 ± 0.37 0.20 ± 0.06 133 ± 61 1.90 ± 0.66
Mut-4 1.25 ± 0.24 0.75 ± 0.15 1.99 ± 0.39 0.39 ± 0.06 281 ± 26 2.60 ± 0.12
Mut-5 3.44 ± 0.70**** 1.74 ± 0.25**** 5.18 ± 0.94**** 0.92 ± 0.18**** 806 ± 91**** 4.13 ± 0.4****
Mut-6 2.67 ± 0.19** 1.50 ± 0.09*** 4.16 ± 0.28*** 0.65 ± 0.06** 468 ± 57*** 2.97 ± 0.48*
Mut-7 2.02 ± 0.58 1.10 ± 0.34*3.12 ± 0.92*0.51 ± 0.15*330 ± 123*2.58 ± 0.33
Mut-8 0.86 ± 0.20 0.55 ± 0.18 1.41 ± 0.38 0.29 ± 0.04 190 ± 41 1.98 ± 0.53
Mut-9 0.81 ± 0.07 0.47 ± 0.04 1.28 ± 0.11 0.23 ± 0.03 143 ± 37 2.36 ± 0.32
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McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
Label‑free quantitative (LFQ) proteomics comparing Mut‑5
andparental strain CC‑125
e lutein and total Chl contents of Mut-5 were 5.4-
fold and 3.0-fold higher than the CC-125 control strain,
respectively (Table1). is presents us with several ques-
tions: where is Mut-5 storing its extra pigments, and
what other metabolic processes could be involved in its
increased pigment accumulation? Moreover, how did
Mut-5 simultaneously produce more Chl a, whose exci-
tation produces detrimental singlet oxygen radicals,
while exhibiting better survival under norflurazon and
high light stress during mutant selection? To answer
these questions, LFQ proteomics was performed to com-
pare differentially abundant proteins between Mut-5
and CC-125 strains. Given increasing evidence for the
translational regulation of photosynthesis-related pro-
teins in C. reinhardtii [41–43], a comparative proteomics
approach was implemented to reveal metabolic changes
that may not be ascertained using transcriptomics.
Time‑point selection forcomparative proteomics analysis
e control strain CC-125 was harvested at Day 4,
and Mut-5 harvested at Day 6 for comparing the pro-
teomes. ese time points represent late-log/early-
stationary phase for both strains, with no significant
difference in cells mL−1, as determined during an 8day
growth study (Fig. 1A). e specific growth rate of
Mut-5 (0.027 ± 0.004 h−1) was lower than that of CC-125
(0.039 ± 0.009 h−1); although this was not statistically
significant (p = 0.121; Student’s t-test), the lack of signifi-
cance was due to variance between replicates. e total
carotenoids and Chl per cell for both strains remained
relatively stable across all time points and were consist-
ently higher in Mut-5 (Additional File 2), suggesting that
proteins involved in pigment production and/or storage
would continue to be stably expressed in Mut-5 at the
time points selected.
Proteomics overview
Proteins were extracted from CC-125 and Mut-5 cul-
tures in biological triplicate and analysed by comparative
shotgun LFQ proteomics. Of the 1876 proteins identified
using Maxquant MaxLFQ analysis [44], 1075 proteins
were quantified using LFQ Analyst [45] and compared
between samples. Proteins with Mut-5/CC-125 Log2 fold
changes (Log2FC) > 1 and adjusted p-values < 0.05 were
considered to have significantly differential expression
between strains; 242 (22.5%) of proteins differed signifi-
cantly between Mut-5 and CC-125, of which 124 were
upregulated and 118 were downregulated in Mut-5 com-
pared to CC-125 (Fig.1B). For a list of all detected and
quantified proteins, refer to Additional File 4.
Functional enrichment analysis of the differentially
enriched proteins was performed using ShinyGO [46].
Gene ontology (GO) biological process (BP) terms
including PSII repair and assembly, non-photochemical
quenching (NPQ), and thylakoid membrane organization
were enriched in Mut-5 compared to CC-125, suggestive
of constitutive activation of high light stress responses
in Mut-5 (Fig.1C). GO-BP terms related to acetate and
the tricarboxylic acid (TCA) cycle were significantly
enriched in proteins downregulated in Mut-5 (Fig.1D),
while translation initiation, amino acid metabolism, and
peptide biosynthesis were also downregulated; this com-
bination of decreased translation and carbon metabolism
may explain the extended lag phase and slower growth of
Mut-5 (Fig.1A).
Pigment binding andcarrier proteins are more abundant
inMut‑5
e greatest increase in protein expression in Mut-5 rela-
tive to CC-125 was for light-harvesting complex stress-
related protein (LHCSR) 1 (Fig.2), which had a Log2FC
of 10.75, translating to a steep linear fold change of
1722. Similarly, LHCSR3 exhibited a Log2FC of 3.54. e
higher LHCSR1 and LHCSR3 protein levels were later
confirmed biochemically for three light conditions via
immunoblots as shown in Fig. 3. e LHCSR proteins
are key mediators of the energy-dependent (qE) compo-
nent of NPQ under high light stress [47]. LHCSR1 con-
tains approximately three carotenoid binding sites, two
of which have a high affinity for lutein, and an estimated
Fig. 1 Proteomics data overview. A Growth of CC-125 and Mut-5 cultivated under standard conditions to determine time-point selection
for comparative proteomics. Data points represent the mean of three independent replicates; error bars indicate the standard deviation
from the mean. B Volcano plot showing −Log10-transformed adjusted p-values against the Log2 fold changes for reproducibly quantifiable proteins.
Log2 fold change is presented as Mut-5/CC-125. Data points coloured blue and red represent proteins of increased and decreased abundance
in Mut-5, respectively. Dotted lines represent the cut-offs for significantly differential protein abundance between Mut-5 and CC-125 (adjusted
p-value < 0.05; Log2 fold change < -1 or < 1). C Bar plot showing the top 20 biological process gene ontology (GO) terms enriched in proteins
more abundant in Mut-5 compared to CC-125. The number of significantly upregulated proteins associated with each biological process GO term
is indicated to the right of each bar. −Log10-transformed false discovery rate is indicated by colour. D Bar plot showing the top 20 biological process
gene ontology terms enriched in proteins less abundant in Mut-5 compared to CC-125. The number of significantly downregulated proteins
associated with each biological process GO term is indicated to the right of each bar. −Log10-transformed false discovery rate is indicated by colour
(See figure on next page.)
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McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
Fig. 1 (See legend on previous page.)
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Page 6 of 21
McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
eight Chl binding sites, which preferentially bind Chl a
over Chl b [48, 49]. Likewise, ~ eight Chl a molecules and
three carotenoids, primarily lutein and violaxanthin, are
estimated to occupy each LHCSR3 apoprotein [48, 49].
Two other LHC-like proteins, which contain putative
Chl and carotenoid binding sites and are involved in PSII
assembly and repair, were significantly enriched in Mut-
5: early light-inducible protein (ELIP) 8 and one-helix
protein (OHP) 2 (Fig.2).
Seventeen LHC proteins forming the antenna systems
of PSI and PSII (LHCI and LHCII, respectively) which
bind Chl a and lutein, were detected by proteomics;
although their levels were slightly higher in Mut-5 than in
CC-125, they were not statistically significant (Additional
File 4). is suggests that the increased LHC-like protein
expression may be providing a storage sink for the excess
lutein and Chl a, rather than the LHC antenna proteins.
Two plastid lipid-associated proteins (PLAPs), PLAP9
and Cre16.g674800, displayed increased abundance in
Mut-5 (Fig. 2). PLAPs are associated with plastoglob-
ules and the carotenoid-rich eye-spot in C. reinhardtii
[50]. In plants, PLAPs have roles in photoprotection and
carotenoid storage during chromoplast development and
potentially play a similar role in carotenoid sequestration
in C. reinhardtii [51]. Moreover, two fasciclin-like pro-
teins, FAS3 and FAS2, which are membrane-bound pep-
tides that are also associated with the eye-spot [52], were
significantly upregulated in Mut-5 (Fig.2). e functions
of these proteins in C reinhardtii are as of yet unknown,
but their potential involvement in carotenoid accumula-
tion and storage could be worth further investigation.
Carotenoid biosynthesis enzymes are more abundant
inMut‑5, whilechlorophyll biosynthesis proteins are
lessabundant
Several proteins involved in carotenoid biosynthesis
were more abundant in Mut-5, including a violaxanthin
de-epoxidase and phytoene desaturase (Fig.2). is vio-
laxanthin de-epoxidase is unique to C. reinhardtii and
is bioinformatically predicted to be involved in lutein
and β-carotene biosynthesis, in addition to its role in
NPQ via the xanthophyll cycle [53]. Two putative carot-
enoid biosynthetic enzymes, Cre16.g674950 and Cre13.
g587500, also exhibited significantly higher abundance in
Mut-5 (Fig.2), while the comparative levels of zeta-car-
otene desaturase (Cre07.g314150), prolycopene isomer-
ase (Cre16.g651923), and a flavin amine oxidase (Cre12.
g560900) were higher in Mut-5 but not significantly so,
according to our relatively stringent Log2FC cut-off of 1
(Additional File 4). e increase in carotenoid biosyn-
thetic enzymes begins to explain the higher total carot-
enoid content of Mut-5.
No Chl biosynthesis enzymes were significantly more
abundant in Mut-5, despite its higher Chl content. Con-
versely, Mg-protoporphyrin chelatase subunits CHLD
and CHLI2, which constitute a key enzyme in Chl bio-
synthesis, were significantly lower in Mut-5 (Fig.2). Ten
other predicted Chl biosynthetic enzymes were detected
by proteomics, but their levels were not significantly dif-
ferent to those of CC-125. No Chl catabolic enzymes
were detected by proteomics (Additional File 4). e
incongruence between the observed increase in Chl con-
tent and reduced abundance of Chl biosynthetic enzymes
(See figure on next page.)
Fig. 2 Schematic representation of proteins and pathways differentially enriched in Mut-5 vs CC-125. 73/242 proteins with significantly higher
(blue) or lower (red) abundance in Mut-5 vs CC-125 are shown (Log2 fold change > 1 or < 1; adjusted p-value < 0.05). Pathways coloured blue/red
were significantly more/less enriched in Mut-5 vs CC-125, respectively. Values indicate the mean (n = 3) Mut-5/CC-125 Log2-transformed fold change
for each protein. PSII/PSI, photosystem II/I; Cyt b6f, cytochrome b6f; ATP syn, ATP synthase; (P)Q, (plasta)quinone; PSII aux, PSII auxiliary proteins;
qE NPQ, energy-dependent non-photochemical quenching; CCM, carbon-concentrating mechanism; Chl biosynth, chlorophyll biosynthesis; Car
biosynth, carotenoid biosynthesis; FLVA, Flavodiiron protein-A; psbF/PSBP3/PSB27/PSB28/PSBS2, PSII subunits; CPLD49, Conserved in the Plant
Lineage and Diatoms-49; pafII, PSI assembly factor-II; ELIP8, early light-inducible protein-8; OHP2, ONE-HELIX PROTEIN-2; HCF244/173/136,
HIGH CHLOROPHYLL FLUORESCENCE-244/173/136; TEF5/30/8, thylakoid lumenal protein-5/30/8; HHL1, hypersensitive to high light-1; APE1,
acclimation of photosynthesis to environment 1; CYN38, cyclophilin-38; REP27, repair protein 27; DEG1A/1C, degradation of periplasmic proteins
protease-1A/1C; FTSH1/2, filamentation temperature-sensitive-1/2; SECA1, sorting factor-A1; ALB3.2, ALBINO3-like translocon protein-3.2;
LHCSR1/3, light-harvesting complex stress-related protein-1/3; CAH3/5, carbonic anhydrase-3/5; PDS1, phytoene desaturase-1; AOF8,
flavin-containing amino oxidase-8; VDE, violaxanthin de-epoxidase; CHLD/I2, magnesium-protoporphyrin IX chelatase subunit D/I2; PLAP(s), plastid
lipid-associated protein(s); FAS2/3, fasciclin-like protein-2/3; AMA3, alpha-amylase-3; STA3, soluble starch synthase III; G6P, glucose 6-phosphate;
F6P, fructose 6-phosphate; F1,6BP, Fructose-1,6-bisphosphate; FBP1, Fructose-1,6-bisphosphatase-1; DHAP, Dihydroxyacetone phosphate; SBP,
sedoheptulose-1,7-bisphosphate; Ru5P, ribulose 5-phosphate; RuBP, Ribulose-1,5-bisphosphate; 3PGA, 3-phosphoglycerate; GAPC1, chloroplastic
glyceraldehyde-3-phosphate dehydrogenase-1; FAD4, fatty acid desaturase-4; CGL76; conserved in the green lineage-76; TGL1, triacylglycerol
lipase-1; MSD3, manganese superoxide dismutase-3; TRXx, thioredoxin x; MDAR1, monodehydroascorbate reductase; GRX1/2, glutaredoxin-1/2;
GPX5/3, glutathione peroxidase-1/3; AST1, Aspartate aminotransferase; IDH3, Isocitrate dehydrogenase-3; OGD1, 2-oxoglutarate dehydrogenase
subunit-E1; SCLA1, Succinyl-CoA ligase α-chain; FUM1, Fumarate hydratase; NUOAF4/S1, NADH:ubiquinone oxidoreductase subunit AF4/
S1; TIM17/22, translocase of the inner membrane-17/22; TOM40, 40 kDa translocon at mitochondrial outer envelope membrane; ACS1/2/3,
Acetyl-coenzyme A synthetase-1/2/3; CIS2, citrate synthase-2; ICL1, isocitrate lyase-1; MAS1, malate synthase-1; BCC1, Acetyl-coenzyme A biotin
carboxyl carrier; PCK1, Phosphoenolpyruvate carboxykinase-1
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Fig. 2 (See legend on previous page.)
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Page 8 of 21
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in Mut-5 suggests that the mechanisms driving Chl turn-
over and/or storage may be disrupted in Mut-5.
Photoprotective, reactive oxygen species stress, andredox
homeostasis‑related proteins are more abundant inMut‑5
In addition to the LHCSR proteins, PSII subunit S protein
(PSBS), another protein crucial for qE NPQ, exhibited
high relative abundance in Mut-5 (Fig. 2), as also evi-
denced by immunoblotting using an anti-PSBS serum
specific for the C. reinhardtii protein isoform [54, 55].
e precise function of PSBS is currently unknown in C.
reinhardtii, especially since this gene is usually transcrip-
tionally silent under non-stressing light conditions [55,
56], although its expression is necessary for full LHCSR3
accumulation and acclimation to high light [47, 56, 57].
Various uncharacterized proteins that share homol-
ogy to Arabidopsis NPQ-related proteins were more
abundant in Mut-5, including CPLD42 (Cre01.g004450;
Log2FC = 1.26), which shares 40% sequence identity
(BLAST E-value 8e-43) with NPQ protein FLUCT UAT
ING-LIGHT-ACCLIMATION PROTEIN1 [58], as well
as Cre13.g586050 (Log2FC = 3.32) and CGLD13 (Cre03.
g181250; Log2FC = 1.39), whose amino acid sequences
are similar to Arabidopsis SUPPRESSOR OF QUENCH-
ING1 (At1g56500) and RELAXATION OF QH1
(At4g31530), respectively (Additional File 4).
As shown in Fig.1C, PSII repair and PSII assembly were
the most highly enriched pathways in Mut-5. However,
the core subunits of the PSII reaction centre (RC) did not
differ significantly between Mut-5 and CC-125, with the
exception of cytochrome b559 subunit β and the putative
oxygen-evolving complex protein PSBP3, which were sig-
nificantly enhanced in Mut-5 (Fig.2). Proteins involved
in PSII-RC PsbA (D1) protein turnover were enriched in
Mut-5 (Fig.2), including HIGH CHLOROPHYLL FLUO-
RESCENCE (HCF) 244, HCF173, HCF136, and OHP2,
which cooperate to stabilize psbA mRNA and enhance its
translation [59–61], alongside the high light-induced D1
proteases FTSH1, FTSH2, DEG1A, and DEG1C [62, 63].
Notably, levels of D1 were similar between Mut-5 and
CC-125 (Log2FC = 0.30, adjuste d p-value = 0.052). O ther
PSII assembly and repair proteins enriched in Mut-5
include PSB28, whose orthologue in Synechocystis plays a
role in the biosynthesis of Chl and its incorporation into
the PSII-RC [64], and TEF5, a homologue of Arabidopsis
psb33 that mediates PS-LHCII interactions and energy
transfer under high light [65, 66]. Assembly factors for
cytochrome b6f (Conserved in the Plant Lineage and Dia-
toms 49) and PSI (PSI assembly factor-II) were also more
abundant in Mut-5 (Fig.2).
Proteins involved in reactive oxygen species (ROS)
stress and redox homeostasis displayed particularly high
fold increases in Mut-5 compared to CC-125 (Fig.2). For
example, two known glutaredoxins (glutaredoxins 1 and
2; Fig.2) and one putative glutaredoxin (cre06.g261500;
Log2 = 3.31) were comparatively highly expressed.
Two glutathione peroxidases (GPX3 and GPX5; Fig.2),
which are highly induced by singlet oxygen [67], were
also upregulated. Other enriched redox-related proteins
of note include a chloroplastic manganese superoxide
Fig. 3 Pigment and qE protein accumulation in CC-125 and Mut-5
cultured in three light conditions. Pigment contents and qE protein
accumulation in C. reinhardtii strains CC-125 (blue) and Mut-5 (red)
acclimated to low light (LL; 70 µmol photons m2 s−1), medium
light (ML; 150 µmol photons m2 s−1), and high light (HL; 400 µmol
photons m2 s−1). Total chlorophyll (Chl) content in pg per cell A,
total carotenoid (Car) content in pg per cell B, Chl a/Chl b ratios C,
and Chl/Car ratios D are displayed. Data are represented as the mean
of 3 independent replicates with error bars depicting standard
deviation from the mean. For each parameter, significant differences
between each strain and light condition were calculated by two-way
ANOVA with a multiple comparisons test (compare cell means
regardless of rows and columns) and post hoc Bonferroni correction.
Means marked with the same letter are not significantly different
(p-value > 0.05). E Immunoblots showing LHCSR1, LHCSR3, and PSBS
expression in CC-125 (WT ) and Mut-5 (M5) under LL, ML, and HL.
Antibodies detecting α-ATPase β-subunit were included as a control
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Page 9 of 21
McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
dismutase (Fig.2), whose expression is triggered by Fe/
Mn deficiency or H2O2 stress [68], thioredoxin x, methio-
nine sulfoxide reductase 1B, and monodehydroascorbate
reductase 1 (Fig.2).
Collectively, the induction of photoprotective and
redox homeostasis proteins is indicative of a widespread
stress response in Mut-5 and may have contributed to its
survival during selection in norflurazon and high light.
Central carbon metabolism andrespiration are suppressed
inMut‑5
As indicated by the gene ontology analysis, acetate
metabolism appears to be downregulated in Mut-5 com-
pared to CC-125 (Fig. 1D). Mixotrophic growth using
acetate as a carbon source is reliant upon the glyoxylate
cycle, and crucially the expression of isocitrate lyase 1
[69], which is strongly downregulated in Mut-5 (Fig.2).
Moreover, three acetyl-CoA synthetases (ACS1, ACS2,
and ACS3), which oversee acetate uptake in C. rein-
hardtii, are significantly downregulated in Mut-5, as well
as two glyoxylate cycle enzymes, citrate synthase 2 and
malate synthase 1, pointing towards a trend of downreg-
ulated acetate metabolism in Mut-5 [70](Fig.2). Further
to this, four TCA cycle enzymes were significantly less
abundant in Mut-5 compared to CC-125 (Fig.2), suggest-
ing downregulated central carbon respiration.
Enzymes in the Calvin–Benson–Bentham cycle were
overall slightly downregulated in Mut-5, albeit not sig-
nificantly so (Additional File 4). However, downstream
pathways linked to Calvin–Benson–Bentham cycle prod-
ucts were downregulated in Mut-5. Fructose-1,6-bis-
phosphatase 1, which feeds photosynthetically derived
sugars into the gluconeogenesis and starch pathways,
was reduced, as well as the chloroplastic glyceralde-
hyde 3-phosphate dehydrogenase, which is also linked
to glycolysis and gluconeogenesis (Fig.2). Furthermore,
increased alpha-amylase 3 and decreased soluble starch
synthase III expression in Mut-5 suggests a breakdown
of starch (Fig.2); this, in conjunction with reduced glu-
coneogenesis, is suggestive of carbon limitation, with
Mut-5 inducing glycogen catabolism to compensate for
the sugar deficit. Further to this, levels of key proteins
involved in the carbon-concentrating mechanism were
significantly higher in Mut-5, including two carbonic
anhydrases and two low-CO2-inducible proteins (Fig.2),
which is indicative of carbon limitation, or at least induc-
tion of the carbon limitation response.
Conflicting conclusions can be drawn with regard to
fatty acid metabolism in Mut-5. Fatty acid desaturase 4 is
strongly upregulated in Mut-5, while diacylglycerol lipase
1 was significantly lower, which is suggestive of fatty acid
biosynthesis; this is in contrast with the upregulation
of two enzymes with predicted roles in triacylglycerol
degradation: conserved in green lineage 76 [71] and the
putative triacylglycerol lipase 1 (Fig. 2). While the lack
of characterization of these enzymes makes it difficult
to discern the direction of lipid metabolism in Mut-5,
the combination of starch degradation and general sup-
pression of central carbon metabolism would suggest
that lipid beta-oxidation may be upregulated to generate
acetyl-CoA, given the apparent suppression of acetate
uptake and metabolism.
Two subunits of the mitochondrial oxidative phospho-
rylation complex I were less abundant in Mut-5, along-
side an associated mitochondrial translocase (Fig.2); the
deficit in NADH caused by the downregulation of the
TCA cycle likely had a negative effect on oxidative phos-
phorylation, and thus respiration. e complex I chap-
erone NUOAF4 was, however, upregulated in Mut-5,
which may be related to the general ROS stress response
occurring throughout the cell.
Mut‑5 exhibits dierences intranslation andtranscription
factor expression
e predominant differences in regulatory factors
were related to cytosolic translation (Additional File 4),
which was heavily decreased in Mut-5. Eight subunits
of eukaryotic translation initiation factors 2 and 3 were
significantly lower in Mut-5 (Table2). Cytosolic riboso-
mal subunits and rRNA methylation complex factors
were also lower (Additional File 4). is, in combination
with reduced amino acid biosynthesis, indicates reduced
protein production in the Mut-5 cytosol. Notably, chlo-
roplastic tRNA-aminoacyl synthetase expression and
ribosome biogenesis were increased in Mut-5; this
increase in ribosome biogenesis and amino acid acti-
vation, but not in amino acid biosynthesis, supports
the notion of increased protein turnover in the Mut-5
chloroplast.
Given the importance of translational and post-trans-
lational regulation in C. reinhardtii, it may be fruitful to
consider the as-of-yet uncharacterized RNA processing,
translation factors, and ubiquitin proteasome compo-
nents that are differentially regulated in Mut-5, as regula-
tory factors governing its phenotype may well be among
them. Examples include the programmed cell death fac-
tor with a gene ontology biological process term asso-
ciated with negative regulation of transcription and a
predicted mRNA-interacting domain, a predicted trans-
lation factor FAP244 with a basic leucine zipper domain,
translation elongation factor Tu (EFG) 3 and EFG8,
as well as a 26S proteasome regulatory subunit RPT4,
among others (Table 2; Additional File 4). e expres-
sion of eukaryotic release factor 1 was lower in Mut-5;
interestingly, its higher plant orthologues (e.g. Brassica
oleracea Bo8g065090 and Bo9g007980) are suppressed by
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ORANGE, a carotenoid biosynthesis-inducing regulatory
protein also present in C. reinhardtii [26, 72]. Upregu-
lated translation factors include EFG10 and elongation
factor Ts-like protein (Table2).
At least three potential transcription factors were dif-
ferentially enriched in Mut-5 (Table 2), including an
uncharacterized aldo/keto reductase Cre10.g461900,
which shares homology with the A. thaliana protein
ATB2 (BLAST E-value 3e-26, 30% identity), a light-regu-
lated bZIP transcription factor [73]. Accumulation of the
putative transcription factor nucleolin (Cre06.g275100)
was significantly lower in Mut-5 compared to CC-125.
Its function is currently unknown, but it is upregulated in
low CO2 conditions [74, 75] and in SAK1 mutants, which
lack a ROS response [76]. Levels of a predicted C2H2
transcription factor family protein of unknown function
(Cre03.g152150) were also significantly lower in Mut-5
(Table2). However, no known transcriptional activators/
repressors involved in the control of photoprotective
gene expression were found to be differentially regulated
between Mut-5 and the parental strain [77–79].
LHCSR protein expression andpigment accumulation
are higher inMut‑5 thaninCC‑125 undervarying light
intensities
e strong constitutive expression of NPQ-related pro-
tein expression in Mut-5, particularly LHCSR1 and
LHCSR3, suggests that these proteins may in part explain
the increased pigment content. e proteomics data also
suggest that a high light stress response is triggered in
Mut-5. We performed pigment analyses and immunob-
lots (Fig.3) in CC-125 and Mut-5 under three light inten-
sities: low light (LL; 70μmol photons m2 s−1), medium
light (ML; 150μmol photons m2 s−1), and high light (HL;
400μmol photons m2 s−1). e ML intensity was selected
as this reflects that of the standard conditions used for
the earlier HPLC and proteomics experiments. is ena-
bled us to (i) understand how pigment accumulation
is affected in Mut-5 by altering the light conditions, (ii)
explore the relationship between pigment hyperaccu-
mulation and LHCSR protein expression, and (iii) vali-
date the proteomics data with regards to the constitutive
expression of NPQ-related proteins.
e total Chl and carotenoid contents were signifi-
cantly higher in Mut-5 compared to CC-125 across the
three light conditions tested (Fig. 3A, B). Interestingly,
the total pigments per cell increased in Mut-5 with
increasing light intensity; this contrasts with the CC-125
strain, in which the carotenoids and Chl contents were
highest at ML, decreasing under HL conditions (Fig.3A,
B). e Mut-5 total Chl content under HL was three-
fold higher than that of CC-125 under the same light
intensity, and 1.7-fold higher than that of Mut-5 grown
under ML. e Chl a/Chl b ratio for CC-125 was lowest
Table 2 Predicted translation and transcription factors differentially abundant in Mut-5
Phytozome ID Gene name Log2FC p.adj Description
Predicted translation factors
Cre03.g165000 EFG10 1.64 4.56E−02 Translation elongation factor EFG/EF2, LepA-related
Cre12.g519180 EFT1a 1.04 9.93E−09 Elongation factor Ts-like protein
Cre06.g298100 SUI1A − 1.45 7.05E−03 Translation initiation protein
Cre12.g490000 EIF2A − 1.75 2.47E−04 Eukaryotic translation initiation factor 2 subunit 1
Cre06.g298350 FAP224 − 1.26 2.96E−03 Flagellar-associated protein FAP224
Cre17.g697450 EIF3L − 1.08 3.73E−02 Eukaryotic translation initiation factor 3 subunit L
Cre03.g190100 EIF3B − 1.72 7.88E−03 Eukaryotic translation initiation factor 3, subunit B
Cre04.g217550 EIF3C − 1.59 1.50E−04 Eukaryotic translation initiation factor 3, subunit C
Cre05.g242300 EIF3D − 1.88 6.77E−04 Eukaryotic translation initiation factor 3, subunit D
Cre16.g676314 EIF3H − 1.58 4.40E−03 Eukaryotic translation initiation factor 3, subunit H
Cre06.g259150 EFG8 – 1.09 4.68E−02 Elongation factor Tu
Cre09.g415800 − 1.43 1.97E−02 Programmed cell death protein
Cre06.g284750 EFG3 − 1.22 1.41E−02 Translation elongation factor Tu family protein
Cre13.g587050 ERF1 − 1.41 2.04E−02 Eukaryotic release factor 1
Predicted transcription factors
Cre10.g461900 2.93 6.82E−05 Aldo/ keto reductase; homologous to AtbZIP11/ ATB2
Cre06.g275100 − 3.62 7.57E−04 Nucleolin; Splicing Factor 3B, Subunit 4
Cre03.g152150 − 1.13 2.92E−02 C2H2-type domain-containing protein
Proteasome regulatory subunits
Cre17.g710150 RPT4 − 1.95 1.67E−04 26S proteasome regulatory subunit
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McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
in ML and highest in HL, indicating a slight reduction in
PSII antenna size in response to the higher light inten-
sity (Fig.3C). However, in Mut-5, the Chl a/Chl b ratio
was consistently higher than that of CC-125, increasing
under ML conditions and remaining high under HL con-
ditions. ese differences in Chl a/Chl b ratio suggest
differences in PS structure and antenna size regulation
between CC-125 and Mut-5 in the presence of acetate
(Fig.3C). e Chl/carotenoid ratio was higher in CC-125
compared to Mut-5 under LL, and comparable in both
strains under ML conditions. Under HL, the Chl/carot-
enoid ratio was significantly higher in Mut-5 compared
to CC-125 (Fig.3D).
To investigate the involvement of LHCSR proteins in
pigment composition, immunodecoration experiments of
total algal protein extracts were performed on the same
samples examined for pigment composition to assess
LHCSR1, LHCSR3, and PSBS protein levels (Fig. 3).
Under the three light conditions tested, the immunoblots
indicated that LHCSR1 was consistently highly expressed
in Mut-5, while being barely detectable in the CC-125
control strain (Fig.3E). is is especially apparent under
ML conditions, thus bolstering the validity of the prot-
eomics dataset, which indicated a very high 10.45-fold
increase in LHCSR1 under similar cultivation condi-
tions (Fig.2). e sustained overexpression of LHCSR1 in
Mut-5, even under LL conditions, may indicate disrupted
regulation of light-induced responses, or alternatively
biochemical mimicry of high light stress conditions, e.g.
by increased ROS levels, lower thylakoid lumenal pH,
or an excessively reduced plastoquinone pool. LHCSR3
accumulation increased with increasing light intensity
in both Mut-5 and CC-125, but this effect was more pro-
nounced in Mut-5 (Fig.3E). e expression of LHCSR3 is
also intimately linked to CO2 availability; typically, under
mixotrophic conditions, LHCSR3 accumulation is lim-
ited [80]. e increase in CC-125 LHCSR3 may be due
to the increase in photosynthetic activity with increasing
light intensity, thus reducing the pool of available CO2
and inducing CO2-linked LHCSR3 expression. LHCSR3
expression remained relatively high in Mut-5, even in the
presence of acetate under LL conditions. PSBS was essen-
tially undetectable in CC-125 under any condition, but
bands were visible for all light intensities tested in Mut-5.
PSBS expression appeared to be highest under ML condi-
tions in Mut-5, and lowest under HL.
Mut‑5 exhibits high NPQ butlower photosynthetic
eciency
To investigate how the pigment hyperaccumulation and
increased qE NPQ proteins influenced photosynthetic effi-
ciency and NPQ in Mut-5 under varying light regimes, we
examined NPQ induction and relaxation kinetics. CC-125
and Mut-5 cells were grown under LL, ML, and HL for
8days in TAP media, the latter condition to ensure maxi-
mum expression of the qE NPQ-related proteins in the
CC-125 parental strain. e C. reinhardtii strain npq4/
lhcsr1, a mutant lacking both LHCSR1 and LHCSR3 pro-
teins (hereafter referred to as npq4-1) and which there-
fore completely fails to activate the qE NPQ component,
was included in the experiment to act as a negative con-
trol [81, 82]. After dark adaptation, cells were illuminated
with flashes of saturating white actinic light for 8min to
examine NPQ induction and relaxation (Fig.4). e maxi-
mum PSII quantum yield (Fv/Fm) was significantly lower
in Mut-5 compared to CC-125 and npq4-1 under LL and
ML, indicating reduced photosynthetic efficiency in Mut-5
at lower light intensities (Fig.4A); this was also consistent
with the slower growth rate (Fig.1A) and reduced expres-
sion of respiration and translation-related proteins (Fig.2)
in Mut-5. At HL, Fv/Fm was similar between Mut-5 and
CC-125 (Fig.4A).
e difference in NPQ between Mut-5 and the con-
trol strains acclimated to LL was substantial; by 200s, the
NPQ of Mut-5 was ~ 4.5-fold higher than CC-125 (Fig.4B).
NPQ is typically inactive in low light-acclimated cells and
in the presence of acetate [83]; the constitutive expres-
sion of LHCSR proteins (Fig.3E) and suppression of ace-
tate metabolism (Fig.2) may contribute synchronously to
increase the NPQ activity of Mut-5. e extended NPQ
relaxation of Mut-5 following initiation of the dark recov-
ery period may be indicative of photoinhibition through-
out the 8min actinic light phase (Fig.4B). Under ML and
HL conditions, NPQ was again highest in Mut-5, but to a
lesser extent (Fig.4C, D). Interestingly, NPQ did not reach
a steady-state level in either ML or HL, and instead contin-
ued to rise until dark conditions were induced, which may
suggest a delayed or cumulative NPQ inductive response in
Mut-5.
Discussion
Here, we generated a lutein hyper-producing mutant of the
model green microalga C. reinhardtii with constitutively
active photoprotection via random mutagenesis, which we
characterized using comparative proteomics. Our in-depth
study of the Mut-5 proteome enabled us to interrogate the
metabolic and regulatory pathways that contribute to its
high-pigment and constitutively active photoprotective
phenotype, identifying potential metabolic engineering tar-
gets for amplifying lutein production in the process.
Increased LHC‑like proteins andPSII protein turnover
may contribute tolutein andchlorophyll asequestration
inMut‑5
e increase in Mut-5 carotenoids may be partially
attributed to an increase in carotenoid biosynthetic
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Page 12 of 21
McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
enzymes (Fig.2). However, the same cannot be said for
the increase in Chl a; overall, the abundance of Chl bio-
synthetic enzymes was reduced in Mut-5 (Fig.2; Addi-
tional File 4). is strongly suggests that the high pigment
content of Mut-5 is a result of enhanced pigment storage,
as opposed to upregulated biosynthesis. LHC antenna
proteins, which are the main Chl binding proteins in
Chlorophyta, were slightly enriched in Mut-5, but not
significantly so, nor to an extent that might account for
the Chl content increase. Furthermore, the Chl a/Chl
b ratio of Mut-5 was similar to or higher than that of
CC-125, suggesting that the LHCII antenna size was in
fact slightly smaller in Mut-5 than in CC-125 (Fig.3C).
A possible explanation for the enhanced total pigment
accumulation could be the enrichment of several LHC-
like proteins, namely LHCSR1, LHCSR3, PSBS2, ELIP8,
and OHP2 (Fig.2), which (with the exception of PSBS2)
contain numerous predicted or confirmed carotenoid
and Chl binding sites [48, 49, 61], implying that these
proteins may be acting as pigment storage sinks within
the thylakoid membrane. LHCSR1 exhibited a huge
Log2FC of 10.75, which translates to a linear 1722-fold
increase, in Mut-5 compared to CC-125 (Fig.2). LHCSR1
maintained its comparatively high expression across
three light intensities (70, 150, and 400 µmol photons
m2 s−1), which coincided with high total carotenoid and
Chl accumulation (Fig.3). It would therefore be reason-
able to credit much of the pigment increase to LHCSR1,
which harbours ~ 8 Chl a and 2–4 lutein binding sites [48,
49].
Enrichment of several other LHC-like proteins likely
contributed to the increase in pigment biosynthesis
and storage capacity. LHC-like proteins contain trans-
membrane domains and putative pigment binding
sites and are associated with thylakoid membranes.
In plants, many of these proteins are upregulated
following exposure to stress [84]. With the excep-
tion of LHCSR1 and LHCSR3, the exact functions of
these proteins are currently unclear in C. reinhardtii,
although their importance in photoprotection, stress,
and acclimation processes is becoming increasingly
apparent [84, 85]. ELIP8, an orthologue of the Arabi-
dopsis ELIP2, exhibited significantly higher expression
in Mut-5 compared to CC-125 (Fig. 2). Arabidop-
sis ELIP2 binds Chl a and lutein, and its proposed
functions include scavenging detached Chls from
Fig. 4 Pulse-amplitude modulation (PAM) fluorescence measurements of C. reinhardtii strains CC-125, Mut-5, and npq4-1 cultured at different
light intensities. A Maximum photosystem II quantum yield (Fv/Fm) measurements for CC-125, Mut-5, and npq4-1 acclimated to low light
(LL; 70 µmol photons m2 s−1), medium light (ML; 150 µmol photons m2 s−1), and high light (HL; 400 µmol photons m2 s−1) for 8 days. Data are
represented as the mean of 3 independent replicates with error bars depicting standard deviation. For each parameter, significant differences
between each strain and light condition were calculated by two-way ANOVA with a multiple comparisons test (compare cell means regardless
of rows and columns) with post hoc Bonferroni correction. Means marked with the same letter are not significantly different. B–D Rise and decay
kinetics of non-photochemical quenching (NPQ) in CC-125, Mut-5, and npq4-1 measured following acclimation to B LL, C ML, and D HL conditions.
Dark-adapted cells were subjected to illumination with saturating white actinic light (1630 µmol photons m2 s−1) in intervals for 8 min, followed
by relaxation in the dark. White and black bars above the plots represent illuminated and dark periods, respectively
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damaged LHCII proteins, temporarily carrying pig-
ments during LHCII assembly, and negatively modu-
lating Chl biosynthesis [86–90]. Similar observations
were made in a time-course transcriptomics study of
the lutein-producing alga Desmodesmus sp. JSC3, in
which ELIPs were highly expressed during periods of
increased lutein accumulation [91]. The PSII biogen-
esis protein OHP2, which is required for the stability
of the PSII reaction centre protein D1 [61], was also
highly upregulated in Mut-5 (Fig.2). OHP2 binds Chl
and carotenoid molecules and likely functions as a pig-
ment delivery system for nascent and/or damaged PSII
reaction centres in Arabidopsis [92]. In cyanobacteria,
OHP orthologues retard Chl degradation from PSII
complexes, where they have been proposed to tempo-
rarily hold Chl molecules during damaged PSII protein
replacement [93]. The LHC-like proteins significantly
enriched in Mut-5 may perform similar roles as pig-
ment carriers in C. reinhardtii, and are thus likely to
be key contributors to the hyperpigmentation pheno-
type of Mut-5.
Expanding on the potential roles of ELIP8 and OHP2
in pigment sequestration, increased turnover of PSII
proteins may also have driven the high pigment levels.
High light-induced PSII-associated proteases (FTSH1,
FTSH2, Deg1A, and Deg1C) were significantly upreg-
ulated in Mut-5 (Fig. 2). At the same time, proteins
involved in the synthesis of the PSII-RC D1 protein
(HCF244, HCF173, and HCF136) [61] were signifi-
cantly enriched (Fig.2). is dual upregulation, which
typically occurs during high light stress, may have
increased the turnover of D1 and cytochrome b6f, the
main targets of the FTSH protease [62, 94]. D1 protein
turnover is an important response to high light stress,
as the D1 protein is particularly susceptible to photoda-
mage; the removal and replacement of damaged/aber-
rant D1 is crucial to preventing photoinhibition and
further damage from oxygen radicals [95]. Although D1
levels were comparable between Mut-5 and CC-125, D1
turnover may have been higher in Mut-5; a similar phe-
nomenon was observed in a very high light-resistant C.
reinhardtii strain, which exhibited similar D1 levels to
the wild-type strain but increased D1 degradation and
synthesis [96]. During the presumably increased PSII-
RC degradation and replacement cycle, the pigments
initially bound within the RC may be temporarily held
by the LHC-like proteins ELIP8 and OHP2, of which
there is increased abundance. is may be further pro-
moting Chl and lutein accumulation in the thylakoid.
e assumed increased production and degradation of
D1 may also be connected to the upregulation of plas-
tidial ribosome biogenesis and tRNA aminoacylation
(Additional File 4).
The regulation ofphotoprotection isdisrupted inMut‑5
Many proteins that are comparatively abundant in Mut-5
(Fig. 2, Additional File 4) are upregulated under high
light, ROS stress, or both [97], and many (some overlap-
ping) are regulated by singlet oxygen kinase 1 [76]. High
light stress responses in C. reinhardtii include activa-
tion of NPQ-related gene expression, changes in elec-
tron transport and thylakoid membrane ultrastructure,
altered stoichiometry of PSI:PSII, synthesis and accumu-
lation of xanthophylls and antioxidants such as tocoph-
erol, and turnover of damaged photosynthetic apparatus
components [98]; GO term enrichment (Fig.1C, D) and
individual protein analysis (Fig.2) revealed involvement
of several of these mechanisms in Mut-5. e qE-related
protein levels (Fig.3) and biophysical responses (Fig.4)
were also higher in Mut-5 under three light conditions.
Taken together, we can infer that Mut-5 contains a muta-
tion that affects the regulatory pathways governing high
light and/ or ROS stress responses, leaving the cell in a
perpetually stress-responsive state, even in the absence of
cues. is overzealous stress response was likely respon-
sible for the survival of Mut-5 during the norflurazon
screening step.
e regulation of C. reinhardtii qE-related pro-
teins LHCSR1, LHCSR3, and PSBS, which are notably
increased in Mut-5 (Fig.3E), has recently been a topic
of avid investigation in C. reinhardtii photosynthesis
research [80, 99–102]. Under ambient conditions, the
LHCSR and PSBS proteins are virtually undetectable
[103]. Previous studies show that these proteins are co-
regulated (except in altered CO2 conditions [80, 104]),
and their expression increases under high light, blue
light, and UV irradiation [47, 105, 106]. In a previous
study, a C. reinhardtii mutant overexpressing LHCSR1
and PSBS (similarly to Mut-5) was shown to retain a
missense mutation in a component of a SPA1-COP1 E3
ubiquitin ligase complex, which suppresses qE protein
expression [101]. Similarly, another ubiquitin ligase com-
plex CUL4-DDB1DET1 was found to suppress the induc-
tion of LHCSR and PSBS proteins [99]. is suggests that
part of an E3 ubiquitin ligase complex may have been
disrupted in Mut-5, given its similar phenotype. e
pigment profiles of these mutants, however, were not
reported in these studies [99, 101]. Furthermore, no dif-
ferential expression was detected for any of the proteins
known to be involved in either of the E3 ubiquitin ligase
complexes or their transcription factor targets in Mut-5,
although a mutation that sterically disrupts complex for-
mation but not protein expression may have arisen, or
the regulatory proteins were simply not detected. Many
other proteins implicated in gene expression were differ-
entially expressed in Mut-5, most of which were transla-
tion factors that present decreased relative abundance
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Page 14 of 21
McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
compared to CC-125. It is possible that one or more of
these regulatory factors contributed to the Mut-5 pheno-
type, although due to the lack of a detailed genetic analy-
sis of Mut-5, the potential role of these factors could be
neither confirmed nor ruled out in our mutant at present.
An alternative explanation for the induction of high light
and ROS stress responses that should be considered is
that a mutation may have caused an increase in intracel-
lular ROS levels.
Metabolic engineering targets forenhanced lutein
accumulation
Although the precise causative mutation(s) of Mut-5
phenotype(s) were not identified, some potential leads for
targeted metabolic engineering for enhanced lutein pro-
duction can still be inferred from our study. For exam-
ple, the vastly increased expression of LHCSR proteins in
Mut-5 suggests that their overexpression could increase
lutein storage in C. reinhardtii, which could be achieved
by knocking out components of the CUL4-DDB1DET1
or COP1-SPA1 complexes [99, 101]. Overexpressing
other LHC-like proteins, such as OHP2 and ELIP8, may
also enhance carotenoid storage within the thylakoid.
Another interesting lead for metabolic engineering is the
PAP-fibrillin domain proteins. As discussed above, PAP-
fibrillin domain proteins are localized to plastoglobules in
plants and perform roles in lipid and carotenoid storage
[107, 108]. Interestingly, three PAP-fibrillin proteins were
upregulated in Mut-5 (Fig.2). Recently, a plastoglobule-
associated PAP-fibrillin protein was overexpressed in
the diatom Phaeodactylum tricornutum, leading to a
51% increase in production of the carotenoid fucoxan-
thin [109]. Functionally characterizing PLAPs in C. rein-
hardtii could be worthwhile, as they could potentially act
as a metabolic sink for carotenoids, preventing metabo-
lite-induced feedback inhibition. Moreover, exploring the
functions of the transcription and translation factors that
were significantly differentially abundant in Mut-5 could
reveal unknown regulators of pigment biosynthesis and
more widespread photoprotective responses. ese tar-
gets may additionally be of interest to researchers aiming
to increase the production of other xanthophylls such as
astaxanthin in green algae. Increasing astaxanthin levels
can cause reductions in the number of LHCs and total
carotenoids per cell [110–112]; therefore, our targets may
offer a means to boost the accumulation capacity of valu-
able carotenoids.
Given the pervasive effects of the random EMS chemi-
cal mutagenesis in Mut-5, which creates an array of
single nucleotide polymorphisms scattered across the
genome, it may be difficult to pinpoint the genetic occur-
rences that confer its altered phenotype. Multiple loci are
likely to have been affected by the EMS mutagenesis, as
was found in previous microalgal EMS mutant genera-
tion studies [110, 113, 114], and determining the muta-
tions governing the phenotype(s) of Mut-5 will require
repeated genetic back-crosses, followed by phenotype
segregation analyses and whole genome sequencing of
daughter cells. Hence, these will be the next steps taken
towards identifying the causative genetic lesion(s) con-
tained in the genome of Mut-5.
Increased pigments andphotoprotection may comeatthe
expense ofslower growth andreduced photosynthetic
eciency
Mut-5 displayed a superior ability to accumulate lutein
(5.4-fold) compared to the parental strain (Table1); how-
ever, it also exhibited a reduced growth rate (Fig.1A),
although not statistically significant, and lower maxi-
mum quantum photosynthetic yield (Fig.4A) compared
to CC-125. Several factors may have contributed to the
reduction in Mut-5 growth, including the heightened
Chl accumulation. Lutein is predominantly bound within
Chl-binding LHCs in C. reinhardtii [14], and Chl levels
often correlate with carotenoid content in microalgae
[115–117]; therefore, Chl was used as a selection crite-
rion for isolating potential high-lutein mutants during
the initial round of mutant selection (Additional File
1). However, high Chl contents also reduce the amount
of light that can penetrate algal cultures, reducing light
use efficiency, productivity, and biomass accumulation
[118, 119]. Although the abundance of Chl biosynthetic
enzymes was comparatively lower in Mut-5, total Chl
accumulation was higher, even at high light intensities
(Fig.3A). Additionally, the increased NPQ of Mut-5 may
compete energetically with productive photochemistry,
channelling a disproportionate amount of light energy
towards heat dissipation (Fig.4). ese challenges, how-
ever, may be a small trade-off for large increases in lutein
production, if overall productivity outweighs the growth
effects. Moreover, reducing the photosynthetic antenna
size by targeting processes responsible for pigment–
protein complex accumulation could enhance sunlight-
to-biomass conversion efficiency without completely
removing lutein-binding LHC proteins [120]. is could
be achieved, for example, by suppressing LHC protein
translation or by modulating chlorophyll biosynthesis
using RNA interference-based silencing approaches [121,
122].
Conclusions
Using a random mutagenesis, selection, and quantita-
tive proteomics approach, we discovered new targets for
increasing pigments and photoprotection in the model
green alga C. reinhardtii. is offers a series of candidate
factors that can be exploited to fully realize the potential
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McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
of microalgae to replace both wild-type and engineered
heterotrophic microorganisms for the industrial produc-
tion of high-value carotenoids [123–125]. We have dem-
onstrated that it is physiologically possible to increase the
number of LHC-like proteins in green algae, which likely
increases the number of available carotenoid binding
sites, expanding the cellular carotenoid storage capac-
ity of the photosynthetic membranes and enhancing
photoprotective properties. Further investigation into
the specific causative mutations conferring the photo-
protective and pigment-producing phenotypes of Mut-5
could reveal novel regulatory factors involved in these
processes.
Materials andmethods
C. reinhardtii cultivation andmutagenesis
e C. reinhardtii strain CC-125 was used as the paren-
tal strain for mutagenesis. Under standard conditions,
cultures were grown mixotrophically in tris–acetate-
phosphate (TAP) medium at 25°C with continuous illu-
mination at 150μmol photons m2 s−1 on an orbital shaker
set to 120rpm. Growth was monitored by measuring cell
number with a Neubauer cell-counting chamber (Sigma-
Aldrich, St. Louis, MO, US) or using a Countess II FL
Automated Cell Counter (ermoFisher, Waltham, MA,
US).
To perform chemical mutagenesis, C. reinhardtii
strain CC-125 was cultured to early exponential phase
(1–3 × 106 cells mL−1) and harvested by centrifugation
at 2000 × g, 5min. Cultures were concentrated tenfold in
0.1M phosphate buffer (pH 6.8), and EMS was added to a
final concentration of 0.3M. Cells were incubated for 2h
while shaking, after which the EMS mutagenesis reaction
was stopped by adding 10 mL sterile 5% sodium thio-
sulphate (w/v), followed by vortexing and centrifugation
(same settings). e pellet was washed with 5% sodium
thiosulphate (w/v), followed by washes with 0.1M phos-
phate buffer (pH 6.8) and TAP, and lastly resuspended
in TAP. All supernatants following EMS treatment were
discarded in a beaker containing sodium thiosulphate
crystals . For selection, ~ 1 × 106 cells were spread on to
TAP-agar plates supplemented with 0.5, 1, or 3µM nor-
flurazon and grown inside a high light box with constant
illumination at 1050 ± 150μmol photons m2 s−1. e 648
colonies that survived the combined pressures of norflu-
razon and high light were transferred to liquid culture on
96-well plates supplemented with 1µM norflurazon and
grown for 5days within the high light box. One hundred
and forty-four strains exhibiting high specific growth
rates and/or high Chl a fluorescence, with ‘high’ defined
as one standard deviation above the average growth rate/
Chl fluorescence intensity of each individual 96-well plate
(Additional File 1), were transferred to 24-well plates
and grown under standard conditions, after which their
relative pigment contents were estimated using a fluores-
cence plate reader (Additional File 1).
Pigment extraction andanalysis
To analyse and compare the pigment compositions of
Mut-1–9 and CC-125 (Table1), 4mL of each 25mL cul-
ture was harvested in triplicate after 96h of growth under
standard conditions by centrifugation at 2000 × g, 5min,
4°C; pellets were frozen at −20°C. All of the following
steps were completed in the dark with samples kept on
ice. Pellets were resuspended in 1mL cold 100% acetone,
mixed with 425–600µm diameter glass beads, and incu-
bated on ice for 15min. Samples were vortexed for 2min,
then incubated on ice for 2min, for a total of five times,
followed by centrifugation at 10,000 × g, 5min, 4°C. e
green supernatant was frozen at −80°C prior to analysis.
Total Chl and total carotenoid contents were estimated
using extinction coefficients as described previously [40].
HPLC was performed on a Dionex UltiMate 3000 HPLC
machine with a Hyperselect C18 reverse phase column
(125Å pore size, 5μm particle size, 250 × 4.6 mm), using
a previously described method [126]. e separation
programme, in which solvent A was ethyl acetate and
solvent B was acetonitrile:water 9:1 (v/v), was as follows:
0–16min, gradient from 0–60% solvent A; 16–28min,
60% solvent A. Injection volume was 10 μL, and flow
rate set to 1.0mL min−1. Carotenoids were detected at
450nm absorbance wavelength. Lutein analytical stand-
ard (Sigma) was suspended in 100% acetone. Pigment
concentrations were determined by interpolating to
standard curves generated with known pigment con-
centrations and normalizing to cell number. For the lat-
ter pigment analyses (Fig. 3), pigments were extracted
from algal cells using 85% acetone buffered with Na2CO3
as previously described [127]. Absorption spectra
were recorded at room temperature using an Aminco
DW-2000 spectrophotometer. Quantification of cellular
pigment content, Chl a/b ratio, and Chl/Car ratio were
calculated from the deconvoluted spectra of five biologi-
cal replicates following an established method [128].
Protein extraction forlabel‑free quantitative (LFQ)
proteomics
Twenty mL of late-exponential phase cultures were har-
vested by centrifugation (2000 × g, 18 °C, 5 min) and
pellets frozen at −20°C. Samples were thawed and resus-
pended in 1mL lysis buffer (2% SDS [w/v], 40mM Tris
base, 60mM dithiothreitol) with 10µL Halt™ protease
inhibitor cocktail (ermo), frozen at -80°C for > 24 h,
then thawed at 37°C for 2 min. Each sample was vor-
texed with 425–600µM acid-washed glass beads at high
speed for 30s and cooled on ice for 30s for 10 cycles.
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Page 16 of 21
McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
Lysed samples were centrifuged at 18,000 ×g, 4°C, 5min,
and stored at −20°C. 100µL of each lysate was purified
using a protein 2-D Clean-Up Kit (GE Healthcare, Chi-
cago, IL, US) following the manufacturer’s instructions.
In‑solution protein reduction, alkylation, anddigestion
forLFQ proteomics
Pellets from the 2-D protein clean-up were resuspended
in 50µL urea buffer (8M urea; 100mM Tris–HCl [pH
8.5]; 5mM dithiothreitol) and sonicated into suspension.
Protein concentration was estimated using a NanoDrop™
2000 spectrophotometer (ermoFisher), and ~ 50 µg
protein was transferred to a fresh 1.5mL protein LoBind
Eppendorf tube. Protein samples were reduced by dilut-
ing samples up to 10µL with urea buffer and incubating
at 37°C for 30min. Proteins were S-alkylated by adding
1µL 100mM iodoacetamide and incubating in the dark
at room temperature for 30 min. Two micrograms of
trypsin/LysC enzyme mix (Promega, Madison, WI, US)
was added to the protein solution and incubated at 37°C
for 3h for LysC digestion, after which the solution was
diluted with 75 µL 50 mM Tris–HCl (pH 8.5)/10 mM
CaCl2 and incubated overnight for trypsin digestion. e
digestion was stopped by acidification with 0.05 volumes
of 10% trifluoroacetic acid. To desalt the samples, Pierce®
C18 spin columns (ermoFisher) were used accord-
ing to the manufacturer’s instructions, achieving a pep-
tide yield of ~ 30µg. Samples were dried using a vacuum
evaporator and stored at −80°C. Method was adapted
from a previous study [129].
LC–MS/MS anddata analysis
Peptide sample pellets were thawed and resuspended in
15µL loading buffer (97% acetonitrile, 3% H2O, 0.1% tri-
fluoroacetic acid [v/v]). Following 5min centrifugation
(room temperature, max speed), 500 ng of sample was
then analysed using a nanoflow LC (Dionex UltiMate
3000 RSLCnano system) coupled online to a Q Exactive
HF mass spectrometer (ermoFisher). Two technical
replicates were analysed per biological replicate.
Raw MS data files were processed using MaxQuant
version 1.5.2.8 software [44], using the MaxLFQ function.
Data were searched against the C. reinhardtii proteome
(UniprotKB proteome ID UP000006906, last modified
December 2019; 18,829 proteins). MaxLFQ parameters
were set accordingly: fixed modifications: carbamidome-
thyl; variable modifications: acetyl (Protein N-term), oxi-
dation; decoy mode: revert; peptide spectrum matches,
protein, and site false discovery rates (FDRs): 0.01; spe-
cial amino acids: arginine and lysine; MS/ MS tolerance
(Ion trap MS): 0.5 Daltons (Da); MS/MS tolerance (Fou-
rier transform MS): 20ppm; MS/MS tolerance (time of
flight): 0.5 Da; minimum peptide length: 7; minimum
score for modified peptides: 40; peptides used for protein
quantification: razor; minimum peptides: 1; minimum
razor peptides: 1; minimum unique peptides: 0; mini-
mum ratio count: 2.
e raw MS data files were processed using the label-
free quantification option in MaxQuant software (Max-
LFQ), which matched MS peaks to peptides in the C.
reinhardtii proteome using the Andromeda search
engine, ten calculated LFQ intensities for identified
peptides [130]. As part of the MaxLFQ analysis, com-
mon contaminants and non-unique/ razor peptides
were filtered out. Matched and quantified proteins were
statistically analysed using LFQ Analyst [45] with the
following parameters: adjusted p-value cut-off, 0.05;
Log2 fold change cut-off, 1; Perseus-type imputation;
Benjamini Hochberg-type FDR correction. GO analysis
was performed using ShinyGO [46] with the following
parameters: FDR cut-off = 0.05; # pathways to show = 20;
and removed redundancy selected. Proteins were anno-
tated and classified using Mercator4 [131], then refined
manually using Phytozome 13 [132] and Uniprot [133].
Immunoblotting oftotal algal protein extracts
For immunodecoration experiments, total cellular
extracts were obtained using a previously described
protocol starting from approximately 3 × 107 pelleted
cells [134]. Approximately 5 µg of proteins were sepa-
rated via SDS-PAGE and transferred to a nitrocellulose
membrane, and subsequently probed using the follow-
ing primary antibodies α-LHCSR1 (AS142819, Agrisera,
Vännäs, Sweden) α-LHCSR3 (AS142766), and α-ATPase
β-subunit (AS05085), as well as the previously described
[55] primary α-PSBS antibody, which was kindly pro-
vided by Prof. Stefano Caffarri (Aix-Marseille Universitè,
France). Blots were developed using an anti-rabbit sec-
ondary antibody conjugated to alkaline phosphatase.
Chlorophyll uorescence analysis
Chl fluorescence analyses were performed using a Dual
PAM 101 fluorometer (Walz, Effeltrich, Germany). Cells
were acclimated for seven days under LL, ML, and HL
regimes until stationary phase was reached. Measure-
ments were performed on dark-adapted cells kept under
agitation on a rotary shaker for > 30 min prior to the
analysis. Two millilitres of cells at 1 × 108cells mL−1 was
used for measuring standard photosynthetic parameters
and NPQ induction curves using an established protocol
[127]. Dark-adapted algal cells were exposed to a satu-
rating actinic light pulse (1630µmol photons m2 s−1) to
determine maximum (Fm) and minimum (Fo) fluores-
cence emission. During the light phase of NPQ activation
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Page 17 of 21
McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
and in the dark relaxation phase, each lasting 8 min,
saturating light pulses were applied to monitor NPQ. Far
red light was used throughout the entire light treatment
interval to oxidize the electron transport chain and thus
to maximize the contribution of NPQ activation to PSII
fluorescence quenching. Maximum PSII quantum yield
was calculated using the equation (Fm−Fo)/Fm. NPQ was
calculated as previously described [135].
Statistical analyses
Statistical analyses were performed by one- or two-way
ANOVA with Bonferroni post hoc tests using GraphPad
Prism software. Error bars represent standard devia-
tion. For one-way ANOVA, statistical significance is rep-
resented by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001,
****p < 0.0001. For two-way ANOVA, significance is rep-
resented by letters, whereby each letter represents statis-
tical similarity.
Abbreviations
Chl Chlorophyll
CO2 Carbon dioxide
DCW Dry cell weight
ELIP Early light-inducible protein
EMS Ethyl methanesulfonate
Fv/Fm Maximum quantum yield of photosystem II
HL High light
HPLC High-performance liquid chromatography
LFQ Label-free quantitative
LHC Light-harvesting complex
LHCSR Light-harvesting stress-related protein
LL Low light
Log2FC Log2 fold change Mut-5/CC-125
ML Medium light
NPQ Non-photochemical quenching
OHP One-helix protein
PLAP(s) Plastid lipid-associated protein(s)
PSBS Photosystem II subunit S protein
PSII Photosystem II
qE Energy-dependent non-photochemical quenching
RC Reaction centre
ROS Reactive oxygen species
TAP Tris-acetate-phosphate
TCA Tricarboxylic acid cycle
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13068- 023- 02421-0.
Additional le1: Lutein hyperaccumulating mutant screening and selec-
tion workflow. A Decision tree for initial round of mutant screening. SGR,
specific growth rate; mean, average value for microplate on which mutant
was grown; SD, standard deviation; ChlFlu, chlorophyll fluorescence; Y, yes;
N, no. Strains that fit the criteria for the green boxes were sub-cultured
into 24-well plates for the second round of screening. B Normalized
chlorophyll fluorescence of mutant strains grown for first round of selec-
tion. Chlorophyll fluorescence measurements taken by plate reader after
4 days’ growth with the following parameters: excitation 440 nm, emission
680 nm, gains 50. Chlorophyll fluorescence readings for each mutant
were normalized to 1/average fluorescence reading for its respective
microplate. Each black dot represents an individual mutant strain in an
individual well of a 96-well plate. Red line shows the average chlorophyll
fluorescence normalized to 1; green line shows the average chlorophyll
fluorescence for each plate + 1 standard deviation. B Specific growth rates
of mutants grown for first round of selection. Specific growth rates were
calculated from chlorophyll fluorescence measurements taken by plate
reader for each well between Days 1 and 2. Each black dot represents an
individual mutant strain in an individual well of a 96-well plate. Green line
represents the average mutant growth rate per plate + 1 standard devia-
tion. D Total carotenoid (Cars) content of 144 mutant C. reinhardtii strains
adjusted to OD750. Total carotenoids were calculated following pigment
extraction in pure acetone and subsequent spectrophotometer analysis
[40]. Total carotenoid contents were adjusted to cell density at OD750.
Each circle represents an individual mutant strain. Red line shows average
total carotenoid value/ OD750 for control strain CC-125.
Additional le2: Pigment and growth data for proteomics time-point
selection and cultures. A Total carotenoid (Cars) and C total chlorophyll
(Chl) contents were measured daily for CC-125 and Mut-5 grown under
standard conditions on Days 3–8, and are expressed here in pg per cell.
Pigment concentrations were estimated using previously described
extinction coefficients following acetone extraction and spectrophotom-
eter analysis [40]. B Growth curves of samples harvested for proteomics
analysis. DFinal cell density measurements (in cells per mL) for the CC-125
and Mut-5 cultures harvested for proteomics analysis, between which
there was no significant difference (p = 0.1888; Student’s t-test).
Additional le3: Principle component analysis of the Mut-5 vs CC-125
comparative proteomics data. Principle component analysis (PCA) plot
showing six technical replicates of both CC-125 (blue) and Mut-5 (orange).
The two strains cluster separately along the PC1 axis, indicative of prot-
eomic differences between the two strains.
Additional le4: Differential abundance analysis and functional annota-
tion of Mut-5 vs CC-125 proteomics data. Table S1 List of all proteins
quantified with LFQ Analyst, including GO and MapMan annotations.
Table S2 Proteins with significantly higher abundance in Mut-5 compared
to CC-125 grouped according to functional annotation. Table S2 Proteins
with significantly lower abundance in Mut-5 compared to CC-125
grouped according to functional annotation.
Author contributions
JM conceived, designed and performed the investigation, and wrote
the manuscript. EAC performed immunoblot and pigment analyses, and
reviewed and revised the manuscript. CE ran peptide samples on LC–MS/MS,
and reviewed and revised the manuscript. JP supervised the investigation, and
reviewed and revised the manuscript. All the authors have read and approved
the final manuscript.
Funding
JM and JP would like to acknowledge funding from an EPSRC Doctoral
Training partnership award (EP/N509735/1) and a BBSRC travel award (SA-
AU012001). EAC acknowledges the support of the post-doctoral research fel-
lowship “Borsa Valeria e Vincenzo Landi per Ricerche nel Campo della Genetica
Agraria” from the Accademia Nazionale dei Lincei. The QExactive HF orbitrap
mass spectrometer was funded by BBSRC UK (award no. BB/M012166/1).
Availability of data and materials
The datasets used and analysed during the current study are available from
the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Page 18 of 21
McQuillanetal. Biotechnology for Biofuels and Bioproducts (2023) 16:166
Author details
1 Department of Chemical and Biological Engineering, University of Sheffield,
Mappin Street, Sheffield S1 3JD, UK. 2 Laboratory of Photosynthesis and Bioen-
ergy, Department of Biotechnology, University of Verona, Strada le Grazie 15,
37134 Verona, Italy.
Received: 13 June 2023 Accepted: 28 October 2023
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