Introduction
Chlamydomonas reinhardtii is a haploid unicellular ciliated
chlorophyte from the Viridiplantae lineage. As a model organism, it is
used to study of and produce recombinant proteins, metabolites, and
biofuels. One of its greatest advantages over yeast or bacteria is the
presence of a photosynthetic system and its ability to fix
CO2. C. reinhardtii can also be used to clean
wastewater and require low space of land for its growth. Several tools
have been developed to efficiently transform this microorganism
(Jinkerson and Jonikas, 2015; Schroda, 2019). Despite these advances,
studies exposed that nuclear transgene expression and metabolite
production remain a challenge, as the microalgae had the ability to shut
down transgene expression (Cerutti et al., 1997). This led to a very low
yield of protein expressing colonies, a necessity to screen a huge
number of transformants (Wang et al., 2022; Nouemssi et al., 2020), and
a progressive loss of expression, costing time and money.
Epigenetic silencing is believed to be a main contributor of inefficient
expression (Cerutti et al., 1997; Yamasaki and Ohama, 2011) hindering
the exploitation of C. reinhardtii as a model system for
synthetic biology or other applications. Interestingly, epigenetic
modulation has been proposed as a possible defense mechanism to prevent
expression of foreign DNA (Neupert et al., 2020; Shaver et al., 2010).
In eukaryotic cells, this mechanism can be modulated by the level of
chromatin condensation, through histone and DNA methylation and histone
deacetylation leading to gene expression regulation (Casas-Mollano et
al., 2007). These post-translational modifications drive a
repressive-condensed chromatin state that prevents transcription factors
access to promoters (Shaver et al., 2010).
Studies have reported that nearly
all detectable histone 3 (H3) included lysine methylation in C.
reinhardtii , i.e. very little unmethylated H3 were
found (Khan et al., 2018; Potdar
et al., 2018; Waterborg et al., 1995). The majority of lysine residues
in the position 4 were monomethylated (H3K4me1), while only a minority
were trimethylated (H3K4me3). A strong positive correlation was found
between H3K4me3 and a high level of H3 acetylation, while H3K4me1 was
associated with a low level of H3 acetylation (Rommelfanger et al.,
2021). Consistently, H3K4me1 was associated with inactive euchromatin at
transgenic loci, whereas H3K4me3 was enriched in highly transcribed
genes promoters (Strenkert et al., 2013; Strenkert et al., 2022). In
addition to nucleosomal mechanisms, RNA-mediated interference has also
been demonstrated to modulate gene expression in C. reinhardtii(Rohr et al., 2004).
To overcome the gene silencing, several strategies have been developed
like: hybrid promoters optimization (Schroda et al., 2000; Specht et
al., 2015), addition of endogenous introns into coding sequences (Baier
et al., 2018) and transgenes codon optimization. Moreover, mutant
strains with enhanced protein expression have been generated by UV
mutagenesis (Kurniasih et al., 2016; Neupert et al., 2009). These
strains harbor mutations in histone deacetylase (hdac ) andDNA methyltransferase genes. Although these breakthroughs
contributed to an increase in expression, 50% of the transformant
clones still do not express transgenes following transformation (Neupert
et al., 2009). Surprisingly, Neupert et al showed that treated strains
with HDAC inhibitors (sirtinol, trichostatin A and OSS_128167) did not
lead to an upregulation of transgene expression (Neupert et al., 2020).
By contrast, Kaginkar et al. showed that treatment with some metal ions,
light, curcumin, cinnamic acid, quercetin, sodium butyrate and
5-aza-2’-deoxycytidine could impact on stress-induced gene silencing
phenotypes, using antibiotic resistance as a readout for
transgene expression/silencing (Kaginkar et al., 2021).
In this study, we focused on deacetylation-dependent silencing mechanism
in C. reinhardtii transformants. We hypothesized that chemical
treatment with HDAC inhibitors could be used to restore high transgene
expression. For that, we used nuclear transformants containing themVenus reporter gene under the hybrid HSP70/RBCS2 promoter
and showed different levels of mVenus expression over time and in
between clones by flow cytometry, a process that was unrelated to the
number of transgene integration events, as measured by qPCR. A
progressive loss of transgene expression was noted in most clones and in
parallel mVenus expression fluctuated over the growth cycle reaching a
maximum level just before stationary phase in all clones. Moreover, we
showed that the addition of HDAC inhibitors (HDACi) of the hydroxamate
family, such as vorinostat (suberoylanilide hydroxamic acid, SAHA) at
initiation of C. reinhardtii culture, increased the frequency of
transgene-expressing cells and the level of transgene expression per
cell over the whole growth cycle. Thus, this study proposes a new tool
to successfully trigger the expression of heterologous proteins, a
simplified method to overcome nuclear gene silencing in the microalgae