Abstract
Chlamydomonas reinhardtii has been successfully engineered to produce
compounds of interest following transgene integration and heterologous
protein expression. The advantages of this model include the
availability of validated tools for bioengineering, its photosynthetic
ability and its potential use as biofuel. Despite this, breakthroughs
have been hindered by its ability to silence transgene expression
through epigenetic changes. Histone deacetylases (HDAC) are main players
in gene expression. We hypothesized that transgene silencing can be
reverted with chemical treatments using HDAC inhibitors. To analyze
this, we transformed C. reinhardtii, integrating into its genome the
mVenus reporter gene under the HSP70-rbcs2 promoter. From 384
transformed clones, 88 (22.9 %) displayed mVenus positive (mVenus+)
cells upon flow-cytometry analysis. Five clones with different
fluorescence intensities were selected. The number of integrated copies
was measured by qPCR. Transgene expression levels were followed over the
growth cycle and upon SAHA treatment, using a microplate reader, flow
cytometry, RT-qPCR, and western blot analysis. First, we observed that
expression varies with the cell cycle, reaching a maximum level just
before the stationary phase in all clones. Second, we uncovered that
supplementation with HDAC inhibitors of the hydroxamate family, such as
vorinostat (suberoylanilide-hydroxamic-acid, SAHA) at the initiation of
culture increases the frequency (% of mVenus+ cells) and the level of
transgene expression per cell over the whole growth cycle, through
histone deacetylase inhibition. Thus, we propose a new tool to
successfully trigger the expression of heterologous proteins in the
green algae C. reinhardtii, overcoming its main handicap as an
expression platform.