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