Results

Screening of mVenus+transformants by flow cytometry

Electroporated cells with pOpt_mVenus_Paro linearized vector (Figure S1) were allowed to recover in liquid media for 24 hours and plated on Petri dishes containing TAP agar medium supplemented with paromomycin, as antibiotic selection, for 5-7 days. Cells were transferred twice in fresh media before fluorescence assessment in 6-day old liquid culture. Flow cytometry was used for the initial detection and screening of mVenus+ positive colonies transferred to liquid medium. The initial gating strategy to screen mVenus+clones was as follow: >1% mVenus+ total cells and MFI > 1.5-fold compared to the WT (Figure 1 B). A total of 88 (22.9%) colonies over the 384 antibiotic-resistant were mVenus+ at day 6 of culture (Figures 1A and S2). Among these positive colonies, 23 were selected and tested again to assess fluorescence stability 6 weeks later; 10 colonies (43.5%) were still mVenus+, while the others lost detectable levels of mVenus fluorescence signal (Figure 1C). We hypothesized that the loss in mVenus expression, despite antibiotic resistance, was due to silencing events.

mVenus expression levels is modulated by hydroxamate family of HDAC inhibitors

Five transformants (numbered here as 16, 18, 20, 21, 22) were selected with different levels of fluorescence in order to assess if chemical treatment with silencing inhibitors could increase mVenus expression. C. reinhardtii WT was used as negative control. We used HDACi targeting HDACs classes I to IV. Sirtinol, nicotinamide target HDAC class III/sirtuin, and OSS-128167 is specific to sirtuin 6. SAHA inhibits HDAC class I, II & IV; and sodium butyrate is known as a HDAC class I & IIA inhibitor (Wang & al., 2019). HDACi were added at initiation of culture.
For most of the tested clones, treatment with SAHA (2.5 µM) for 24 hours increased the frequency of cells expressing mVenus and its level of expression per cell (Figures 2A and 2B).
The % of mVenus+ cells increased by 5.1-fold in clone 16 (mean: 13.3 to 68.1%) and there was a 4.6-fold increase in protein fluorescence intensity (MFI), when cells were treated with SAHA (Figure 2A). The rise was less marked in other clones (1.1- to 1.8-fold in frequency) with higher basal fluorescence, consistent with a baseline decreased silencing. OSS-128167 and sodium butyrate had a slight effect on the frequency and the mean fluorescence intensity of mVenus+ cells, while none of the other inhibitors consistently improved expression levels of mVenus.
We further evaluated a 6-day treatment of SAHA and a new generation HDAC inhibitors, i.e. belinostat, dacinostat and panobinostat of hydroxamate-class like SAHA, mocetinostat and entinostat, two benzamide derivatives (HDAC class I inhibitor), and romidepsin, a bacterial-derived cyclic tetrapeptide (HDAC class I inhibitor) (Karagiannis et Rampias, 2021). All treatments were done with 5 µM of inhibitors at initiation of culture (Figures 3A and 3B).
Clone 22 was replaced by clone 17 that exhibited less fluorescence at basal levels, i.e mVenus expression was suspected to be more silenced in clone 17 compared to 22 (Figure 1C). Relative copy number ofmVenus was assessed to verify if the differences between the clones were not due to the number of mVenus copies integrated into the genome. There were no differences in the amount of integrated mVenus in between the tested clones, as measured in copy number or relatively to endogenous gene (Figure S3).
Results showed that the hydroxamate class of HDACs inhibitors was the most potent in boosting both the frequency of mVenus cells in silenced clones (8.6-fold in clone 16 for SAHA (8,5 to 73,1%), 27-fold in clone 17 for SAHA (2,2 to 59,9%)) and its level inside each cell (MFI, 3-4-fold in clone 16 for SAHA, 2-5-fold in clone 17) at day 3 post-treatment (Figures 3A, 3B and S4A). Comparable results were obtained with the other inhibitors from this class, but SAHA was usually less toxic than dacinostat and panobinostat (growth curves and chlorophyl levels, although not to statistically significant levels) and was generally more potent than belinostat (Figure S4B and S4C).
Thus, we continued investigating the efficiency of this molecule (SAHA) to reverse silencing in C. reinhardtii .

mVenus levels are modulated over time and treatment

Expression of mVenus was followed using both a microplate reader and a flow cytometer for 12 days after treatment with SAHA. In untreated, or solvent-treated cells, overall fluorescence peaked around day 6 in all samples (day 5-7), corresponding to the end of the exponential phase and the start of the stationary phase (Figures 4 A-C, S5A and B). Fluorescence levels of mVenus progressively decreased over the following 6 days of culture. When cells were treated with SAHA, the maximum detection of mVenus was also measured at the end of the exponential phase (day 4-7), reaching more than 74% of producing cells in all the clones (Figure 4C), and a maximum of fluorescence intensity overall and per cell (Figure 4A and 4B). Consistently with the low number of cells impacting on the flow cytometer sensitivity in the first 3 days, the analysis of the % of mVenus+ cells varied at early time point (Figure 4C).
Nonetheless, SAHA treatment at initiation of culture steadily increased fluorescence around day 4-5 in all clones, but did not prevent the progressive decline in production when transformants advanced into stationary phase around day 7-8. At day 12, most clones declined to <10% of mVenus+ cells (except 16 and 21) but had still more frequent population of mVenus expressing cells and more mVenus fluorescence signal per cells compared to untreated or DMSO treated cells (Figure 4A, 4B and 4C). SAHA did not seem to impact on overall growth of microalgae (Figure S5A) but a significant decline in chlorophyll fluorescence was noted (Figure S5B, One-way Anova, Dunett’s multiple comparison test), suggesting that some metabolic pathways related to photosynthesis are altered. This prompted more investigation on SAHA’s impact on cell fitness.

SAHA’s effect on viability, cell growth, palmelloid and chlorophyll levels

We treated cells at initiation of culture with increasing concentrations of the inhibitors and incubated them for 6 days. Viability was analyzed through membrane integrity confirmation using propidium iodide (PI) staining at day 6 on a flow cytometer. SAHA’s addition at concentration of 2.5, 5 and 10 µM seemed to slightly increase the % of PI+ cells, but ~90% of the transformant cells remained intact and there was no significant increase compared to background levels in WT cells (Figure 5A). Different stress conditions have been reported to transiently induce palmelloid colonies in C. reinhardtii without impacting viability (de Carpentier et al., 2019). We observed that 6 days of treatment with 5 and 10 µM of SAHA lead to the accumulation of small palmelloid colonies in C. reinhardtii (Figure 5C and D compared to Figure 5B), while motility was also progressively lost above 2.5 µM. We quantified palmelloid cells formation by flow cytometry when transformants were treated with 5 µM of SAHA and showed that in most cases palmelloid colonies increased at day 3 and 6 post-treatment (Figure 5E and S6A). These results suggest that SAHA modifies chlorophyll content and colony morphology but does not alter cell growth kinetic nor viability at concentrations of 10 µM and lower.
SAHA’s addition at higher concentration of 20, 40 and 80 µM was also tested to see at which degree it could affect the growth kinetic and viability. Larger palmelloid colonies (or aggregates) were observed (Figure S6B) with increasing concentrations, while growth kinetic was inhibited partially with 20 and 40 µM SAHA, and totally with 80 µM SAHA (data not shown). Chlorophyll content was also strongly modified, as observed by the progressive loss of the usual dark green color ofC. reinhardtii healthy culture in favor of a more yellow tint (20 and 40 µM SAHA) or even red tint (80 µM SAHA).

SAHA upregulates mRNA transgene levels by inhibiting histone deacetylation.

Then, we measured the impact of SAHA treatment on transgene mRNA levels. SAHA being a HDAC inhibitor, its addition should increase the availability of the chromatin to transcription factors, and hence the mRNA levels. The relative mVenus mRNA expression over the housekeeping gene h3 transcript expression increased 5.7- and 6.6-fold in clone 16 and 17 respectively at day 6 of cultures, when cells were treated with SAHA compared to the DMSO control (Figure 6A); the rise was also observed in other clones, although less marked (1.2- to 2.8-fold).
Finally, we verified by western-blot that histones acetylation was restored upon SAHA treatment (Figure 6B), leading to brighter band using the H3K9Ac antibody in all transformants, while the amount of histone 3 remained consistent. In addition, we could detect an increase in mVenus protein accumulation in clones 16, 17 and 18 at day 6 of cultures, but not in 20 and 21 the two transformants with the highest level of mVenus basal expression. Without treatment, mVenus was barely detectable in clones 16, 17, and 18, consistently with the low fluorescence signal detected by plate reader and FACS.