Discussion

In this study, we propose a new tool to successfully trigger the expression of silenced heterologous proteins in the green microalgae C. reinhardtii . Integration of exogenous DNA into the microalgae nuclear genome predominantly occurs randomly, via non-homologous-end-joining (NHEJ), leading to a large and heterogeneous population of transformed cells with varied expression levels (Zhang et al., 2014; Nouemssi et al., 2020). Despite antibiotic selection, not all transformed cells express the targeted transgene at a desired level. This loss of expression occurred even though the resistance marker was downstream of the reporter expression cassette in the transformation vector. Hence, screening of mVenus+transformants by plate reader or flow cytometry becomes useful to target the transformants with the highest expressing level of transgene. As usually observed with C. reinhardtii nuclear transformants, many (97.4%) early positive transformants expressing the transgene of interest, which was not essential for survival, underwent transcriptional and/or post-transcriptional gene silencing mechanisms (Tran et Kaldenhoff, 2020). Thus, the number of positive transformants falls overtime after rounds of subcultures despite antibiotic selection. Our report demonstrated for the first time, to our knowledge, the use of HDAC inhibitors of hydroxamate-class like SAHA in C. reinhardtiito trigger protein expression.
Upon SAHA treatment, the expression level of the mVenus reporter protein can be recovered at doses that do not affect the cell growth. Treatment at initiation of cultures lead to a maximal production of mVenus at the end of the exponential phase (around day 6). However, SAHA did not prevent the progressive decline in mVenus production when transformants advanced into stationary phase, although cultures treated with SAHA still expressed more transgene.
The overall growth pattern of microalgae was not impacted upon treatment with 5 µM SAHA, but a significant decline in chlorophyll fluorescence was observed. In addition, cell motility was progressively lost ≥2.5 µM SAHA, while palmelloid formation was induced.
Some studies have shown that abiotic stress could transiently induce palmelloid in C. reinhardtii without impacting viability (Cheloni et Slaveykova, 2021; de Carpentier et al., 2022).
Cheloni et Slaveykova examined palmelloid colony formation upon micropollutants (MPs) exposure. The number of palmelloid and their size were dependent on MP concentration and exposure duration. Cells kept growing and dividing within the palmelloid and reverted to their unicellular lifestyle when colonies were harvested and inoculated in fresh medium, indicating that palmelloid formation is a common (and not specific) plastic response to different micropollutants. In mixed populations cultures, the unicellular population exhibited chlorophyll bleaching, membrane damage and oxidative stress, whereas palmelloids were unaffected. In a different study, Carpentier et al. reported on the characterization of an abiotic stress response that the algae can trigger, forming massive multicellular structures called aggregates, which are different from palmelloids. Aggregates are formed by a few tens, to several thousand cells, held together in an extracellular matrix, whereas palmelloids are composed of 4 to 16 cells surrounded by a cell wall. These aggregates constitute an effective bulwark within which the cells are efficiently protected from the toxic environment. Aggregation is not the result of passive agglutination, but rather of genetic reprogramming and substantial modification of the algae’s secretome. Hence, the induction of aggregates and/or palmelloid following SAHA treatment, could help concentrate proteins (and even metabolites) within fewer cells linked together in these multicellular structures, which would not hinder significantly usual cellular metabolism.
High doses of SAHA (20, 40 and 80 µM) strongly increased the number and promoted the larger size of the palmelloids and/or aggregations formation and inhibited the cell growth. There have been some studies using HDAC inhibitors of hydroxamate-class like SAHA in plant cell cultures (Medicago truncatula and Bambusa multiplex ) to boost protein expression (Santos et al., 2017; Nomura et al., 2021), which reported cell toxicity induced by the tested HDACi. Toxicity was lower when the cell culture was treated at day 3, at the onset of exponential phase. In a different study, Nomura et al. boosted the production of two endogenous specialized metabolic compounds (3-O -p -coumaroylquinic acid and 3-O -feruloylquinic acid in a cell line culture of B. multiplex treated with Suberoyl bis‑hydroxamic acid (SBHA), an analog of SAHA. Production of both compounds was induced by SBHA at concentrations between 2 µM and 100 µM, but production decreased at concentrations ≥ 50 µM, mainly because of the cytotoxicity of SBHA. Interestingly, in their study, smaller initial cell density of 5% SCV (sedimented cell volume) lead to the strongest induction of both metabolites. This suggests that inoculum size might be an important contributing factor to the success of silencing reversal.
Upon SAHA-treatment, the relative mVenus mRNA expression and protein levels, together with fluorescence intensity increased in most clones. These results are consistent with SAHA modus operandi at the DNA levels, inducing gene expression. Transcriptional gene silencing is believed to be the main cause for transgene expression inhibition inC. reinhardtii , which is largely mediated by protein factors that place specific histone modifications onto nucleosomes at the transgene loci to trigger the formation of a repressive chromatin structure, a mechanism that may have evolved to protect the genome from invading DNA (Schroda, 2019). H3K4 and K9 monomethylated are some of the histone marks known to occur on nucleosomes in promoter regions of silent genes in C. reinhardtii , while H3K4 trimethylated and H3K9 acetylated appear in promoter regions of active genes (Yamasaki et al., 2008; Shaver et al., 2010; Strenker et al., 2013; Barahimipour et al., 2015; Schroda, 2019). SAHA triggered an increase in histone acetylation level of H3K9 (determined by western-blot) in all clones, while the level of H3 remained consistent. This further confirms that SAHA treatment restored expression by increasing histone acetylation levels, or by preventing histone deacetylation. In addition, Kaginkar et al.,using antibiotic resistance as readout for transgene expression inC. reinhardtii suggested that the use of some metal ions, light, curcumin, cinnamic acid, quercetin sodium butyrate, decitabine (5-aza-2’-deoxycytidine) could reverse stress-induced silencing, through inhibition of DNA methylation or histone deacetylation (Kaginkar et al., 2021). By contrast Neupert et al. could not induce expression using sirtuin inhibitors and other HDACs inhibitors, despite increasing histone acetylation levels (Neupert et al., 2020). In our hands, inhibition of methylation did not yield to an increase in mVenus expression, while hydroxamate-family HDAC inhibitors were very efficient. Multiple and distinct mechanisms responsible for silencing could occur in different clones. It might depend on the promoter, implying that triggering expression with some inhibitors might work for some clones and not others.
In this study, mVenus gene expression was driven under the hybridHSP70A-RBCS2 fusion promoter. Strenkert et al. (2011, 2013) demonstrated that the transgenic HSP70A promoters harbor lower levels of active chromatin marks than the native HSP70A but more than transgenic RBCS2 promoters (Strenkert et al., 2013; Strenkert et al., 2011). The authors found that, first, heat shock transcription factor 1 (HSF1) binds to the promoter, second histone acetylation occurred, then nucleosomes were remodeled, and transcript accumulated. This suggested that the HSF1 recruits histone acetyltransferase (and other histone-modifying enzyme activities) to target promoters. HSF1 could constitutively form a scaffold at the transgenic HSP70A promoter, presumably containing mediator and TFIID, from which local chromatin remodeling and polymerase II recruitment to downstream promoters is realized. However, the authors also observed HSF1-independent histone H3/4 deacetylation at theRBCS2 promoter after heat shock, suggesting interplay of specific and presumably more generally acting factors to adapt gene expression to the new requirements of a changing environment. Interestingly, in the case of the HSP70A-RBCS2 fusion promoter, the chromatin state at the HSP70A promoter was dominantly transferred to RBCS2 by HSF1, to recruit all the machinery necessary for transcription. Here, we show that HDAC inhibitors of hydroxamate-class like SAHA can further help maintain active chromatin marks at the HSP70A-RBCS2 fusion promoters.
In summary, we uncovered a new tool to successfully trigger the expression of heterologous proteins in C. reinhardtii . This method could also be useful and applicable for recombinant production in other microalgae species and open the field to new studies.