Bioresource Technology 341 (2021) 125830

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Ectopic expression of bacterial 1-aminocyclopropane 1-carboxylate

deaminase in Chlamydomonas reinhardtii enhances algal biomass and lipid
content under nitrogen deficit condition
Ramachandran Srinivasan a, Parthiban Subramanian b, Srikanth Tirumani c,
Kodiveri Muthukaliannan Gothandam d, Mohandass Ramya a, *
a
Molecular Genetics Laboratory, Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar,
Kattankulathur 603203, Chengalpattu District, Tamil Nadu, India
b
Department of Biotechnology and Microbiology, National College, Karumandapam, Thiruchirapalli 620001, Tamil Nadu, India
c
Indian Institute of Science Education and Research, Karkambadi Road, Mangalam (P.O), Tirupati 517507, Andhra Pradesh, India
d
Department of Biotechnology, School of Bio-Sciences and Technology, Vellore Institute of Technology, Vellore 632014, Tamil Nadu, India

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Overexpression of acds enhanced the

nitrogen stress tolerance in
Chlamydomonas.
• Ethylene level was significantly reduced
in transgenic lines compared to wild
type.
• ACC deaminase-expressing lines exhibi­
ted higher biomass and lipid content.
• Reduced the ROS by improving antiox­
idant enzymes in acds-expressing lines.

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

Keywords: 1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a well-known bacterial producing enzyme that helps
ACC deaminase plants to overcome stress conditions by modulating ethylene biosynthesis. However, the functional role of ACC
Antioxidant enzymes deaminase and ethylene in microalgae during stress remains to be explored. In this study, to investigate the role
Ethylene
of ACC deaminase (acds) from Pseudomonas putida UW4 in enhancing the biomass and lipid content of Chla­
Lipid
Nitrogen deficit
mydomonas under nitrogen deficit condition. The synthetic codon-optimized acds gene was cloned into vector
pChlamy_4 and introduced into Chlamydomonas. Results indicated that Chlamydomonas-expressing acds lines
showed significant tolerance to nitrogen-deficit by reducing the ethylene content. The biomass, chlorophyll
content and photosynthetic activity of acds-expressing lines were significantly increased during nitrogen deficit
condition. Moreover, the intracellular lipid and fatty acid content were much higher in acds-expressing lines than
the wild-type. In terms of stress alleviation, the transgenic lines displayed increased antioxidant enzymes,
reduced ROS and lipid peroxidation levels.

* Corresponding author at: Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar,
Kattankulathur 603 203, Chengalpattu District, Tamil Nadu, India.
E-mail address: ramyam@srmist.edu.in (M. Ramya).

https://doi.org/10.1016/j.biortech.2021.125830
Received 13 July 2021; Received in revised form 18 August 2021; Accepted 19 August 2021
Available online 24 August 2021
0960-8524/© 2021 Elsevier Ltd. All rights reserved.
R. Srinivasan et al.
1. Introduction
In the present scenario, microalgae are the most promising alterna­
tive feedstock for third and fourth generation biofuels (Abdullah et al.,
2019). They constitute 40–70% of lipids in terms of dry weight when
grown under favourable environmental conditions. Microalgae can
accumulate neutral lipids such as triacylglycerol (TAGs) in the form of
droplets in their cytosol which serve as a carbon and energy source
(Radakovits et al., 2010). Lipid production can be enhanced in micro­
algae by subjecting them to nutrient or abiotic stresses that enhance the
metabolic flux directing to TAG biosynthesis (Park et al., 2019). Among
them, nitrogen depletion is the commonly reported factor for inducing
lipid accumulation and several studies have reported a significant in­
crease in lipid production in microalgal species under nitrogen starva­
tion conditions (Chen et al., 2017). However, this strategy acts as a
double-edged sword as nitrogen starvation which increases lipid pro­
duction, also leads to oxidative stress that suppresses the microalgal
growth which could eventually reduce the lipid accumulation. There­
fore, to overcome this situation, there are certain recent methods such as
supply of growth promoting substances, co-culture with beneficial
bacteria and overexpression or ectopic expression of certain genes in
microalgae (Chu, 2017).
Aquatic environments are co-inhabited by both bacteria and micro­
algae which play an essential role in nutrient cycling as well as energy
flow. It has been established in the past that several bacteria promote the
growth of microalgae under various extreme conditions (Yao et al.,
2019). In general, bacteria exhibit traits such as synthesis of phytohor­
mones, siderophores, vitamins and low molecular weight compounds or
enzymes to enhance growth of microalgae and plants (González-
González and De-Bashan, 2021; Vandana et al., 2020). These growth
promoting factors can stimulate growth and aid accumulation of valu­
able products in plants during abiotic stress. Previous study reported
that certain growth promoting bacteria are able to synthesize the
enzyme ACC deaminase which cleaves the ethylene precursor ACC,
which in turn reduces the ethylene levels in stressed plants (Glick,
2005). The phytohormone ethylene is produced when plants were sub­
jected to biotic and abiotic stressors and is synthesized from L-methio­
nine via a two-step process that intermediates S-adenosyl-L-methionine
(SAM) and ACC (Glick, 2014). Elevated levels of ethylene in plants affect
normal plant growth and development, while plants treated with bac­
teria producing ACC deaminase are reported to display high resistance
to stress conditions (Sapre et al., 2019). Moreover, it also enhances plant
growth, photosynthetic activity and antioxidant enzymes to counteract
the reactive oxygen species (ROS) induced during stress condition
(Gupta and Pandey, 2019). However, till date, the detrimental effects of
ethylene in microalgae present in aquatic environments remains largely
unknown.
Only few experiments in the past have been conducted in microalgae
on exogenous application of ethylene or ACC. In the microalgae Hae­
matococcus pluvialis, production of ethylene was increased upon addition
of L-methionine and SAM, suggesting that microalgae also follow a
similar pathway as observed in plants (Maillard et al., 1993). A report
by, Lee et al. (2016) observed changes in levels of astaxanthin content in
H. pluvialis on addition of ethylene precursor ACC to the growth media.
However, they did not study the effect of exogeneous ACC on the
microalgae at different stages of growth. Later on, Vo et al. (2016)
observed that a specific two-step application of ACC during vegetative
stage enhanced the biomass and astaxanthin accumulation during the
early red stage on dose-dependent manner. However, few studies have
reported that elevated level of ethylene coupled with ROS induce cell
death in Chlamydomonas (Yordanova et al., 2009; Yordanova et al.,
2010). Uji et al. (2020) have shown the detrimental effects of exoge­
nously supplied ACC, on the growth rate and high ethylene synthesis in
marine red algae. In this context, we investigated the effect of ectopi­
cally expressing bacterial ACC deaminase gene from P. putida UW4 into
Chlamydomonas, in terms of growth, biomass and lipid accumulation
2
Bioresource Technology 341 (2021) 125830
under nitrogen deficient condition. Moreover, we also examined the
ROS, lipid peroxidation levels and antioxidant defense mechanisms in
acds-expressing lines compared with wild type.
2. Materials and methods
2.1. Strains, plasmid and environmental conditions
Chlamydomonas CC124 strain (wild type) was acquired from Chla­
mydomonas Resource Center, Minnesota, USA. The obtained strain was
maintained in TAP medium at 25 ◦ C under continuous light of 50 µE m− 2
s− 1. Transgenic strains were also grown under the same conditions with
zeocin (25 µg/mL) as selection antibiotic in the TAP medium. The
expression vector pChlamy_4 used in this study was purchased from
Invitrogen (Thermo Scientific, USA). Escherichia coli DH5α (New
England-Biolabs) cells were maintained in Luria-Bertani medium at
37 ◦ C and used for cloning as well as plasmid amplification.
2.2. Codon-optimized gene synthesis of acds and cloning into pChlamy_4
expression vector
To generate overexpressing construct of acds (pChlamy-acds), the
sequence of acds from P. putida UW4 (AY823987.1) was codon opti­
mized for the microalgae C. reinhardtii, followed by chemical synthesis
at Macrogen Inc. (South Korea). The codon optimized acds gene was
amplified using the primers GATCGAATTCATGAACCT­
GAATCGTTTTGA and GTCGCTCGAGTCAGCCGTTGCGAAACAGGA.
The underlined sequences indicate the EcoRI and XhoI restriction sites.
The amplified PCR product of 1017 bp was purified and ligated onto
pChlamy_4 expression vector between EcoRI and XhoI site using DNA
ligase (New England Biolabs, USA). The subsequent plasmid construct
was transformed into DH5α cells and confirmed using colony PCR.
Further, overexpressing construct pChlamy-acds was confirmed by DNA
sequencing (Eurofins Pvt Ltd., India).
2.3. Electrotransformation of Chlamydomonas and selection
The early log phase culture of Chlamydomonas cells were harvested
and resuspended in GeneArt™ MAX Efficiency™ transformation reagent
(Invitrogen, Thermo Scientific, USA) to final cell concentration of 2.5 ×
108 –3 × 108 cells/mL. A 250 µL aliquot of cell suspension was trans­
ferred into a disposable electroporation cuvette with a 0.4 cm gap (Bio-
Rad laboratories, USA). Two micrograms of SspI-linearized plasmid was
subsequently added the cell suspension and incubated at 4 ◦ C for 5 min.
The cells were electroporated using a Gene Pulser Xcell Electroporation
System (Bio-Rad Laboratories, USA) with the appropriate settings rec­
ommended by the Invitrogen manufacturer’s protocol (500 V, 50 µF,
800 Ω) and an electro pulse duration of 30 ms. The cells were transferred
into a 50 mL conical flask containing 10 mL of TAP-40 mM sucrose
medium and incubated at 25 ◦ C for 24 h. Next day, the cells were har­
vested and spread on selective TAP medium containing zeocin (25 µg/
mL). The plates were incubated at 25 ◦ C with continuous light of 50 µE
m− 2 s− 1 for 5–8 days or until transgenic C. reinhardtii colonies were
clearly visible. The zeocin-resistant colonies were screened for the
presence of transgene by colony PCR using acds primers.
The selected lines were analysed further by nucleic acid extraction,
expression level of acds and transgene copy number in the transgenic
lines. The acds-expressing lines gDNA was isolated by the phenol–­
chloroform extraction method (Jang et al., 2015). Total RNA was ob­
tained from acds-expressing transgenic lines and wild type using TRIzol
Reagent (Invitrogen, Thermo Scientific, USA) and quantified the RNA
concentration using NanoDrop 2000 spectrophotometer (Thermo Sci­
entific, USA). The cDNA was synthesized from 1 µg of total RNA using
with RevertAid cDNA synthesis kit according to the manual (Thermo
Scientific, USA). The semiquantitative RT-PCR was performed to analyse
the expression of acds in transgenic lines using gene-specific primers. As
R. Srinivasan et al.
an internal control, Chlamydomonas G protein ß-subunit-like poly­
peptide (CBLP) transcripts were examined using primers CBLP-F and
CBLP-R, as described previously (Ibuot et al., 2020). For southern blot
analysis, 5 µg of genomic DNA from wild type and acds-expressing lines
digested completely with the enzyme EcoRI. The digested DNA product
was separated on a 0.8% agarose by gel electrophoresis and transferred
to a positively nylon membrane. Probe labelling and hybridization were
carried out using the DIG High Prime DNA Labelling and Detection
Starter Kit I (Roche, USA) as per the instruction’s manual. For nitrogen
deficit tolerance assay, a series of diluted exponentially grown trans­
genic cells were spotted on TAP or nitrogen free (TAP-N) plates and
incubated for 4 days under illumination of 50 µE m− 1 s− 1 at 25 ◦ C. The
C. reinhardtii CC-124 wild type strain with empty pChlamy_4 vector was
used as control.
2.4. ACC deaminase enzyme activity and ethylene content
Ten millimetres of exponential phase grown cells were harvested and
resuspended in TAP and TAP-N medium for 24 h respectively. After 24 h,
cells were centrifuged at 5000 × g and resuspended in 1X Phosphate
buffered-saline (PBS) which contained 0.1% Tween and 1 mM phenyl­
methylsulfonyl fluoride. Cell lysis was carried out through sonication
and cells were spun at 12000 × g at 4 ◦ C for 10 min. The total protein
concentration of the supernatant was measured using a BCA protein
assay kit (Thermo Scientific, USA). One hundred microliters of total
protein was added in a microfuge tube to which 0.5 mL of 0.56 M HCl
was added. The tubes were then vortexed followed by centrifugation at
12000 × g for 5 min. To the final reaction mixture, 0.4 mL of 0.56 M HCl
at and 150 µL of 2,4-dinitrophenylhydrazine reagent was added to 0.5
mL of supernatant, then followed by incubation at 30 ◦ C for 30 min in
dark. At the end of incubation, 1 mL of 2 N NaOH was added and
measured the absorbance at 540 nm (Honma and Shimomura, 1978).
The final ACC deaminase activity was calculated in terms of micromoles
of α-ketobutyrate protein− 1h− 1.
For ethylene content, 5 mL of exponential phase grown wild type and
transgenic cells in TAP and TAP-N medium were transferred to sterile
10 mL serum vials, sealed with a sterile rubber stopper, and incubated at
25 ◦ C with 50 µE m− 1 s− 1 illumination. After 24 h, 100 µL of head space
gas sample was collected from serum vials using 500 µL gas-tight syringe
(Hamilton Company, USA) and injected manually into a gas chromato­
graph packed with a Porapak-Q column (1.8 m length and 2.1 mm inner
diameter, 80/100 mesh; Agilent Technologies, USA) and equipped with
flame ionization detector (Clarus 580 PerkinElmer GC/MS, USA). Ni­
trogen gas was used as the carrier at a flow rate of 20 mL min− 1. The GC
program was adjusted at 40 ◦ C, 120 ◦ C and 200 ◦ C for oven, injection
and detection temperature, respectively (Durall et al., 2020). The
amount of ethylene produced was calculated in terms of nanomoles of
ethylene per litre of culture (nmol/L) by comparing the standard curve
of pure ethylene (Sigma, India).
2.5. Physiological measurements
Growth analyses of wild type and acds-expressing lines were deter­
mined by sampling 1 mL of culture at two day intervals and measuring
the absorbance at 750 nm. The algal biomass was determined by dry
weight measurements. Ten millilitres of algal cells were collected in pre-
weighed vials and washed three times using distilled water. The cell
pellets were freeze-dried using lyophilizer (Lark Freeze Dryer, India)
overnight at − 40 ◦ C. Finally, the dry biomass was measured gravimet­
rically. For determination of the chlorophyll content, both wild type and
transgenic cells were harvested and extracted with 80% acetone at room
temperature (Lichtenthaler and Wellburn, 1983). The chlorophyll con­
tent was determined by measuring OD values at 664 and 647 nm using
UV spectrophotometer (GE Healthcare, India). The maximum photo­
synthetic efficiency of PSII (Fv/Fm) of transgenic lines and wild type
cells were quantified using cuvette adapted model AquaPen AP-100
3
Bioresource Technology 341 (2021) 125830
fluorometer (Photon Systems Instruments, Republic of Czech) as
described in Srinivasan et al. (2017). The experiment was carried out at
25 ◦ C and 2 mL of algal cultures were dark-adapted for at least 15 min
prior to analysis. The maximum excitation light intensity was approxi­
mately 1500 µE m− 2 s− 1 during the measurement and the recording time
was 5 s. The photosynthetic efficiency was expressed as Fv/Fm (Fv,
variable fluorescence; Fm, maximum fluorescence).
2.6. Analysis of lipid accumulation
Microscopic analysis of lipid accumulation in Chlamydomonas cells
stained with BODIPY 505/515 (Thermo Scientific, USA) was observed
under Leica fluorescence microscope (Leica Microsystems, Germany) as
reported by Govender et al. (2012). To detect the lipid droplets, 0.5 mL
of cells (1 × 106) were harvested and resuspended in 0.5 mL of 1X PBS
with 1 µM BODIPY. The cells were incubated at 25 ◦ C for 10 min and
washed thrice with 1X PBS. Then, the cells stained with BODIPY were
examined under a fluorescence microscope and excitation given at 488
nm followed by recording the emission at 550 nm for BODIPY and be­
tween 650 nm and 730 nm for chlorophyll autofluorescence. The in­
tensity of fluorescence in BODIPY-stained cells (100 µL/well) was
quantitatively measured using Synergy HTX Multi-mode Plate Reader
(BioTek, USA) to determine the accumulation of intracellular lipid.
For determination of lipid content, acds-expressing transgenic lines
and wild type cells were grown in TAP or TAP-N and subjected to lipid
extraction was performed according to Bligh and Dyer (1959) with slight
modifications. Twenty milligrams of freeze-dried cells were extracted
with addition of 0.9 mL chloroform:methanol (2:1) and 100 µL of 0.9%
potassium chloride. The sample mixtures were centrifuged at 5000 × g
for 5 min at room temperature. The bottom chloroform phase containing
the lipids was collected and dried by evaporation under nitrogen (N2)
steam for further analysis. For triacylglycerols (TAGs) analysis, dried
lipids were dissolved in chloroform and 20 µL of extracted lipids were
spotted onto a thin-layer chromatograph plate (TLC Silica gel 60, F254,
Merck, India) and developed with 100 mL solvent (hexane/ether/acetic
acid, 70:30:1). The separated TAGs were observed by exposing the TLC
plates to iodine vapour at 37 ◦ C for 5 min (Miao et al., 2019). TAG
corresponding bands were recovered from plate and quantified by using
GC–MS after being converted to fatty acid methyl esters (FAMEs).
Similarly, total fatty acid content of lipids from acds-expressing lines and
wild type cells were also analysed. The FAMEs were dissolved in 200 µL
hexane and analysed by GC–MS using PerkinElmer Clarus 580 Gas
Chromatograph and equipped with a SQ8 mass spectrometry and
TRACETM TR-WAX column (30 m × 0.25 mm × 0.25 µm; ThermoFisher
Scientific, USA) as described earlier (Liu et al., 2017). Helium gas was
used as a carrier and the ionization energy was 70 eV. One microliter
aliquots of each sample was analysed with a 1:5 split injection and
constant flow rate of 1.5 mL min− 1. The oven temperature program used
as follows: initial temperature hold at 45 ◦ C for 1.5 min, increased to
150 ◦ C at 15 min, then by 240 ◦ C at 30 min, and a final temperature hold
at 240 ◦ C for 3 min. The retention time and the peaks were calibrated
using a FAME mixture standard (Sigma, India) and quantified using
heptadecanoic acid (C17:0) as the internal standard. The data was
processed using the TurboMass GC–MS software (v5.4, Perkin Elmer,
USA) and FAMEs were identified with the National Institute of Stan­
dards and Technology (NIST v20) mass spectral library.
2.7. Analysis of ROS, lipid peroxidation and antioxidant enzymes
The accumulation of ROS in Chlamydomonas cells was assessed by
fluorescent staining using oxidant-sensing probe 2′ ,7′ -dichlorodihydro­
fluorescein diacetate (H2DCFDA) as described in Upadhyaya et al.
(2018). Briefly, 0.5 mL of acds-expressing and wild type cells (1 × 106)
were collected from TAP or TAP-N medium, washed once and resus­
pended in 0.5 mL of 1X PBS containing 5 µM H2DCFDA fluorescent
probe. After incubation at 25 ◦ C for 30 min, then the cells were washed
R. Srinivasan et al. Bioresource Technology 341 (2021) 125830

Fig. 1. Molecular characterization of acds-overexpressing lines in Chlamydomonas. Schematic representation of acds construct in pChlamy_4 vector (A). Confirmation
of transgene (acds) in the genome of acds-expressing lines (B). Transcript expression of acds in the acds-expressing lines (C). Southern analysis of transgene copy
number in the transgenic lines (D) and Tolerance of acds-overexpressing lines under nitrogen deficit condition (E).

three times with 1X PBS. The H2DCFDA-stained cells were visualized centrifuged and supernatant was used as enzyme extract for further
under fluorescence microscope for the detection of intracellular DCF analysis. Total protein content of the supernatant was determined by
fluorescence upon excitation at 488 nm and emission at 530 nm. The BCA protein assay kit (Thermo Scientific, USA). The enzyme activities of
DCF fluorescence intensity of cells (100 µL/well) was also collected with Superoxide dismutase (SOD), Catalase (CAT) and Ascorbate peroxidase
Synergy HTX Multi-mode Plate Reader (BioTek, USA). The intracellular (APX) were assessed using commercially available antioxidant assay kits
accumulated H2O2 was determined using the horseradish peroxide- from Nanjing Jiancheng Bioengineering Institute, Nanjing, China and
linked Amplex Red assay kit (Invitrogen, Thermo Scientific, USA) as carried out the experiment as per manufacture’s instruction. All the
described in manufacture’s protocol. One millilitre of algal cells were colorimetric measurements were carried out at 25 ◦ C using Gene Quant
harvested and resuspended in 100 µL of 1X reaction buffer, sonicated 1300 UV spectrophotometer (GE Healthcare, India)
briefly. The resulting solution was centrifuged and the supernatant
taken separately. The supernatant was mixed with horseradish peroxi­
2.8. Statistical method
dase and Amplex reagent at 25 ◦ C for 30 min and measured the absor­
bance to determine the intracellular H2O2 content. H2O2 concentration
All experiments were carried out by using three biological replicates
was measured by a standard curve using 0.2 to 2.5 µM of H2O2.
and the data represents the Mean ± standard deviation (SD). The
Lipid peroxidation in algal cells was observed using fluorescent dye
experimental data were analysed by one-way ANOVA and Bonferroni’s
C11-BODIPY581/591 (Invitrogen, Thermo Scientific, USA) as described in
post hoc correction using GraphPad Prism statistical software v5.0.
Cheloni and Slaveykova (2013). 0.5 mL of algal cells (1 × 106) were
Statistical significant p < 0.05 between the acds-expressing lines and the
harvested and resuspended with 0.5 mL of 1X PBS containing 5 µM C11-
wild type cells are designated with lowercase alphabets.
BODIPY581/591. After incubation at 25 ◦ C for 30 min, cells were washed
thrice with 1X PBS and visualized under fluorescence microscope with
3. Results and discussion
excitation at 488 nm and emission at 535 nm. For quantitative analyses,
the C11-BODIPY stained cells were aliquoted in microtitre plates (100
3.1. Generation and characterization of acds-overexpressing
µL/well) and fluorescence intensity of cells was measured using Synergy
Chlamydomonas lines
HTX Multi-mode Plate Reader (BioTek, USA). Also, the level of lipid
peroxidation products in Chlamydomonas cells was determined by the
For generation of acds overexpression construct, the codon optimized
quantification of thiobarbituric acid reactive substance (TBARS) (Heath
ACC deaminase of P. putida UW4 was cloned into pChlamy_4, fused with
and Packer, 1968). Five millilitres of algal cells were harvested by
hybrid promoter (HSP70 and RbcS2) and ble gene encodes for zeocin as
centrifugation at 50000 × g for 5 min and homogenised with 1.5 mL of
selection marker (Fig. 1A). The overexpression construct was trans­
trichloroacetic acid (0.1%) using ultra-sonication for 10 s (Vibra-Cell
formed into Chlamydomonas by electroporation and randomly selected
ultrasonic processor, USA, 50% duty cycle). The homogenate was
zeocin-resistant colonies. To evaluate the transgene in Chlamydomonas
centrifuged at 10000 × g for 10 min and 1 mL of supernatant was mixed
lines, more than 30 colonies were chosen and screened for the transgene
with 1 mL of TBA solution (0.5% w/v thiobarbituric acid in 20% tri­
(acds) by colony PCR using gene specific primers and ACC deaminase
chloroacetic acid). In a water bath, samples were heated to 95 ◦ C for 30
enzyme activity. From the results, three independent transgenic lines
min and then cooled at room temperature. The samples were centrifuged
(acds_13, acds_26 and acds_28) were selected for further molecular
at 10000 × g for 5 min and TBARs in the supernatant was measured at
characterization. The independent transgenic lines showed the presence
535 nm. The quantity of TBARS was determined by using the extinction
of transgene in the genomic DNA and RNA. In contrast, no transgene was
coefficient of 155 mM− 1 cm− 1 and expressed in pmoles per 106 cells
observed in wild type (Fig. 1B and 1C). For integration of transgene,
For antioxidant enzyme activities, fifty millilitres of wild-type and
southern blot analysis was carried out using DIG labelled acds gene as a
acds-expressing cultures were harvested and homogenised with 5 mL of
probe. This result clearly indicates the stable integration of two copy
extraction buffer (50 mM of potassium phosphate buffer with 1% (w/v)
transgene in their genome (Fig. 1D). To investigate the nitrogen stress
polyvinylpyrrolidone) using sonication at 4 ◦ C. The sample mixture was
tolerance, the wild type and acds-overexpressing cells were spotted on

4
R. Srinivasan et al.
Fig. 2. ACC deaminase activity (A) and ethylene content (B) of acds-overexpressing lines and wild-type cells. The each vertical bar represents the means ± SD (n =
3). Different lowercase alphabets on the bar graph indicate significant differences among the acds-expressing lines versus the wild type (p < 0.05, Bonferroni’s post
hoc test).
solid TAP and TAP-N in series of dilutions. Both the acds-expressing lines
and wild type cells were showed similar growth in TAP medium how­
ever; the cells overexpressed acds gene had exhibited higher rate of
growth compared to wild type in TAP-N medium. These results proven
that acds enhanced tolerance under nitrogen deficit condition in Chla­
mydomonas cells (Fig. 1E).
3.2. Overexpression of acds modulates ethylene production in
Chlamydomonas
The ACC deaminase activity in the transgenic lines was assayed
under TAP and TAP-N deficit conditions by estimating the amount of
α-ketobutyrate. Under nitrogen replete or deficit condition, acds-
expressing lines showed a significant increase in enzyme activity, which
was absent in wild type cells. However, 4.5 fold increase in ACC
deaminase activity was observed in the transgenic cells grown in TAP-N
compared to TAP medium (Fig. 2A). As indicated in Fig. 2B, the ethylene
content was distinctly increased in wild type cells grown in TAP-N when
compared to cells grown in TAP. The acds overexpressed lines had
significantly lower ethylene production (by more than 45%) when
grown in TAP-N. These results suggested that stress induced ethylene
production occurs during nitrogen deficit condition which is overcome
by the acds-overexpressing lines which exhibited tolerance to the
imposed stress by modulating the ethylene levels in Chlamydomonas. In
support to our findings, few studies have been reported involvement of
ethylene during oxidative stress induced regulated cell death in Chla­
mydomonas (Yordanova et al., 2009; Yordanova et al., 2010). Recently,
Uji et al. (2020) reported that upon treatment with exogenous applica­
tion of ACC promotes the ethylene production and resulted in decreased
growth rate in marine red alga Pyropia yezoensis.
3.3. Physiological characteristics of acds-expressing lines
The wild type and acds-expressing lines grown in both TAP and TAP-
N were also studied for their growth, biomass, chlorophyll and photo­
synthetic activity (Fig. 3). An earlier study has established that detri­
mental effect of N starvation on the growth and biomass in microalgae
(Sathe et al., 2019). Results from the present study demonstrate that
acds-expressing lines exhibited higher growth and biomass compared to
wild type under TAP-N. However, under TAP, there was no significant
difference was observed (Fig. 3A), suggesting that overexpression of
acds gene is important to confer stress tolerance in Chlamydomonas.
Regulation of ethylene in plants by application of ACC deaminase
expressing bacteria to improve the growth during stress conditions has
been well established (Gupta and Pandey, 2019). From our results it can
be understood that ethylene produced in microalgae can also be
5
Bioresource Technology 341 (2021) 125830
downregulated using ACC deaminase enzyme. In photosynthetic or­
ganisms, chlorophyll is an essential accessory pigments that absorbs the
light energy and transferring to the photosynthetic reaction centres and
their concentration is directly attributed to photosynthetic efficiency
(Yokoya et al., 2007). In our results, there was drastic decrease in total
chlorophyll of wild type cells in TAP-N, whereas in acds-expressing lines
chlorophyll content was significantly enhanced (Fig. 3B). Further, to
determine if there also an improvement of the photosynthetic activity in
acds-expressing lines, the maximum photosynthesis efficiency yield of
photosystem II under TAP and TAP-N was studied by Fv/Fm measure­
ment using a PSI Aquapen fluorometer. A 2.8-fold increase of Fv/Fm in
acds-expressing lines compared to wild type was observed when the cells
were grown in TAP-N (Fig. 3C). However, no significant difference was
found between wild type and transgenic lines grown in TAP. Therefore,
the reason behind the improvement in biomass under nitrogen deficit
condition was likely due to the increase in the maximum efficiency yield
of photosystem II by ACC deaminase expression. Similarly, in a previous
study, transcriptome analysis revealed that genes associated with pho­
tosystems were upregulated in the Camelina roots-expressing ACC
deaminase during stress condition (Heydarian et al., 2021).
3.4. Lipid accumulation in acds-expressing cells
There are several approaches to augment the lipid content in
microalgae among which nutrient starvation is an established technique
to significantly increase lipid production in microalgae. Nitrogen star­
vation is a highly effective method for increasing lipid content in
microalgae, presumably via inducing the formation of ROS (Zhao et al.,
2019; Zhang et al., 2020). According to Shi et al., 2017, ROS may act as
decisive signalling molecules that regulating several biological processes
and cellular responses to stress. In particular, nitrogen starvation causes
an increase in ROS; these increased ROS levels play a dual function in
microalgae: (1) ROS is a harmful by-product that causes oxidative
damage; (2) ROS is a signalling molecule that mediates the lipid pro­
duction (Zhao et al., 2019; Zhang et al., 2020). Despite lipid production,
nitrogen deprivation has been shown to impair cell growth and biomass
productivity in microalgae. Therefore, it is important to increase lipid
production without impairing cell growth, as well as to reduce oxidative
damage during nitrogen deficiency. Several approaches, namely chem­
ical priming and genetic engineering, have been employed to improve
the lipid production and nitrogen stress tolerance in microalgae (Sun
et al., 2019; Zhao et al., 2019; Kang et al., 2019). Recent research has
shown that heterologous or overexpression of transgene improves lipid
accumulation and stress tolerance in microalgae by increasing antioxi­
dant system enzyme activity and reducing oxidative damage (Wang
et al., 2020; Zhang et al., 2018). Through the current study we
R. Srinivasan et al. Bioresource Technology 341 (2021) 125830

Fig. 3. Physiological characteristics of acds-expressing Chlamydomonas lines and wild-type cells. Growth curve at 750 nm and biomass was determined for every two
days interval (A), Chlorophyll content (B) and (C) PSII activity (Fv/Fm). The experimental data are shown as mean ± SD (n = 3).

investigated the lipid and fatty acid content in C. reinhardtii strain
overexpressing the ACC deaminase gene. In this study, BODIPY505/515
used to specifically stain neutral lipids by exhibiting green fluorescence
under blue light excitation was used. The fluorescence of BODIPY also
showed an increase in the overall number and size of lipid droplets that
positively correlated with total lipid content in Chlamydomonas cells. As
shown in Fig. 4A, all the three acds-expressing lines exhibited higher
fluorescence intensity than wild type cells under TAP and TAP-N con­
ditions. The total lipid percentage indicated that the transgenic lines
exhibited significant increase in lipid content of up to 2.5 fold (35 to

6
R. Srinivasan et al. Bioresource Technology 341 (2021) 125830

Fig. 4. Lipid and total fatty acids content in acds-overexpressing Chlamydomonas lines. Lipid staining of transgenic lines by Bodipy 505/515 and fluorescence in­
tensity of Bodipy 505/515 was measured by Synergy HTX Multi-mode Plate Reader (A), Lipid content (B), TAG content (C) and total fatty acid content (D). The each
vertical bar represents the mean ± SD (n = 3). Different lowercase alphabets on the bar graph indicate significant differences among the acds-expressing lines versus
the wild type (p < 0.05, Bonferroni’s post hoc test). Scale bar = 10 µm.

40%) compared to wild type under N deficit condition. Interestingly, the
lipid content was also significantly increased even under N replete
conditions where the acds overexpression lines showed 10% increase
compared to wild type cells (Fig. 4B). This result correlates with results
obtained from fluorescence microscopy and fluorescence intensity of
BODIPY stain. Similarly, we analysed TAG and total fatty acid content in
acds_13, acds_26, acds_28 and their parental line CC-124 after 48 h
under N deficit and replete conditions using GC–MS. The acds over­
expression lines accumulated increased amounts of TAG and fatty acid
content under both N replete (1.5-fold) and deficit conditions (2.0-fold),
respectively (Fig. 4C and 4D). Overall, these results showed that over­
expression of ACC deaminase from bacteria could significantly increase
the lipid accumulation and fatty acid content in microalgae. This result
is corroborated by findings of Heydarian et al., (2016) who observed
that acds-expressing lines showed a significant increase in seed oil
content of Camelina sativa. Similarly, recent studies also revealed, bac­
teria that produce ACC deaminase, were capable of improving the lipid
content in oil seed crops (Novinscak and Filion, 2018; Novinscak and
Filion, 2019; Jiménez et al., 2020).
3.5. ROS accumulation and lipid peroxidation in acds-expressing lines
Excessive accumulation of ROS causes peroxidation of unsaturated
lipids, it resulting in loss of membrane integrity and cellular damage in
microalgae during stress condition including nitrogen starvation (Zhang
et al., 2020). In this study, we examined the level of ROS along with lipid
peroxidation in the acds-expressing lines and the wild type under N
deficit (Fig. 5). The cellular level accumulation of ROS in

7
R. Srinivasan et al. Bioresource Technology 341 (2021) 125830

Fig. 5. ROS and lipid peroxidation accumulation in wild type and acds-expressing lines. (A) ROS accumulation was detected using the fluorescent dye H2DCFDA and
imaged using fluorescence microscope. (B) H2O2 level was determined by Amplex Red assay kit. (C) Lipid peroxidation accumulation was detected using C11-
BODIPY581/591 and TBARS level was quantified by enzymatic assay (D). The each vertical bar represents the mean ± SD (n = 3). Different lowercase alphabets on the
bar graph indicate significant differences among the acds-expressing lines versus the wild type (p < 0.05, Bonferroni’s post hoc test). Scale bar = 10 µm. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Chlamydomonas cells was analysed using ROS-sensitive fluorescence dye
H2DCFDA. DCF fluorescence signals can be captured with fluorescence
microscope and intensity of signal reveals the intracellular level of ROS
accumulation including H2O2. The results showed that ROS levels were
significantly decreased in acds-expressing lines than the wild type cells
under N deficit conditions, suggesting that acds expression can also
reduce the ROS generation and prevent the cellular damage in the
transgenic lines (Fig. 5A). Previous reports revealed significant increase
in lipid production during oxidative stress induced by N deficit condi­
tions in microalgae. However, the accumulated ROS, at high levels also
leads to cell damage which reduces the lipid production (Shi et al., 2017;
Zhao et al., 2019; Zhang et al., 2020). In this study, acds-expressing lines
demonstrated a significant reduction in ROS levels of more than 20%
when compared to wild type. This creates a low ROS environment
during N deficiency, which may have favoured cell metabolism in acds-
expressing lines, resulting in higher biomass and lipid production.
Similarly, several studies have shown that reducing ROS accumulation
improves lipid production in microalgae (Zhao et al., 2019; Cui et al.,
2021). Our results also corroborates with these findings. Furthermore,
we also determined the H2O2 level in cells using Amplex Red-

8
R. Srinivasan et al.
Fig. 6. The enzyme activities of SOD (A), CAT (B), APX (C) in acds-expressing transgenic lines and wild type under nitrogen deficit condition. The each vertical bar
represents the mean ± SD (n = 3). Different lowercase alphabets on the bar graph indicate significant differences among the acds-expressing lines versus the wild type
(p < 0.05, Bonferroni’s post hoc test).
Horseradish method. No significant change was detected in H2O2 con­
tent between acds-expressing lines and wild type grown in TAP medium.
However, H2O2 content was significantly reduced in acds-expressing
cells than wild type upon N deficit condition (Fig. 5B), which is
consistent with the results obtained during ROS staining (Fig. 5A).
The lipid peroxidation and its by-products are common biomarkers
for in vivo oxidative stress (Niki, 2008). Lipid peroxidation was evalu­
ated in the current study using C11-BODIPY581/591 and TBARS content
in transgenic lines. Upon N deficit, the intensity of C11-BODIPY was
significantly increased in wild type than the acds-overexpressing lines
(Fig. 5C). Similarly, TBARs content was increased significantly in wild
type when compared to transgenic cells under TAP-N (Fig. 5D). Though,
no substantial difference was observed in fluorescence intensity of C11-
BODIPY staining and TBARS content between acds-expressing lines and
wild type under N replete conditions. These results suggest that nitrogen
stress leads to lipid peroxidation but the effect is counteracted in the
acds-expressing transgenic lines that reduce the lipid peroxidation and
protect the cells from ROS damage through the enzyme ACC deaminase.
Our results accordance with prior findings that heterologous expression
of ACC deaminase reduces the ROS accumulation and lipid peroxidation
level in plants by modulating ethylene during stress conditions (Sapre
et al., 2019; Singh et al., 2021).
3.6. Antioxidant enzyme activities in acds-expressing
Under adverse conditions, microalgae usually accumulate ROS,
leading to induction of oxidative stress which in turn damages proteins,
DNA and membrane lipids (Rezayian et al., 2019). To scavenge the ROS,
microalgae employ several antioxidant defence mechanisms during
stressful conditions. The antioxidant enzymes including APX, CAT and
SOD are important factors that help in quenching ROS in microalgae
(Rezayian et al., 2019; Ugya et al., 2020). In the current study, SOD, CAT
and APX activity levels revealed no significant changes were observed in
the acds-expressing lines and the wild type during N replete condition
(Fig. 6A-6C). However, under N stress, acds-expressing transgenic cells
showed 1.5-fold increase of SOD activity, 1.6-fold increase of CAT ac­
tivity and 1.4-fold increase of APX activity compared to wild type cells.
In order to control ROS levels and maintain redox homeostasis, micro­
algae can develop tolerance to abiotic stress by improving the antioxi­
dant enzyme activity. In accordance with the reports emphasizing the
importance of antioxidant enzymes in nitrogen condition, our study
reported that acds expression reduces ROS accumulation and damage
during N stress by improving antioxidant enzyme activities.
4. Conclusion
Control of ethylene levels using ACC deaminase has been proven to
improve growth, reduce stress and improve productivity in plants
9
Bioresource Technology 341 (2021) 125830
(Glick, 2014; Gupta and Pandey, 2019). However, the potential of ACC
deaminase to improve growth and productivity in microalgae has not
been studied. The current study demonstrates the potential role of ACC
deaminase in enhancing biomass and lipid content under nitrogen
deficit conditions in Chlamydomonas. Further, it also improved the
antioxidant defence mechanism by reducing ROS accumulation.
Therefore, expression of ACC deaminase in microalgae can be consid­
ered as a new strategy for enhancing the productivity of microalgae.
CRediT authorship contribution statement
Ramachandran Srinivasan: Methodology, Data curation, Investi­
gation, Funding acquisition, Writing - original draft. Parthiban Sub­
ramanian: Software, Validation, Writing - review & editing. Srikanth
Tirumani: Formal analysis, Visualization. Kodiveri Muthukaliannan
Gothandam: Formal analysis, Writing - review & editing. Mohandass
Ramya: Supervision, Project administration, Funding acquisition,
Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The author acknowledges National Post-doctoral Fellowship Scheme
(NPDF) by Science and Engineering Research Board (SERB), Govern­
ment of India (Grant No. PDF/2018/000789). The authors express their
sincere thanks to SRM Institute of Science and Technology for facilities
to perform the experiments. We also express our deep gratitude to SRM-
DBT platform for Advanced Life Science Technologies for carrying out
fluorescence microscopy analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2021.125830.
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