Summary
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 theHSP70-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.
Keywords: Chlamydomonas , transgene, epigenetic silencing,
hydroxamate-type histone deacetylase inhibitor, vorinostat, mVenus, flow
cytometry