Light-driven recombinant protein (RP) production
in eukaryotic microalgae offers a sustainable
alternative to other established cell-culture systems. RP
production via secretion into the culture medium enables
simple product separation from the cells adding a layer of
process value in addition to the algal biomass, which can be
separately harvested. For the model microalga Chlamydomonas
reinhardtii, a broad range of molecular tools have been
established to enable heterologous gene expression; however,
low RP production levels and unreliable purification from
secretion concepts have been reported. Domesticated C. reinhardtii strains used for genetic engineering are often cell-wall
deficient. These str... More
Light-driven recombinant protein (RP) production
in eukaryotic microalgae offers a sustainable
alternative to other established cell-culture systems. RP
production via secretion into the culture medium enables
simple product separation from the cells adding a layer of
process value in addition to the algal biomass, which can be
separately harvested. For the model microalga Chlamydomonas
reinhardtii, a broad range of molecular tools have been
established to enable heterologous gene expression; however,
low RP production levels and unreliable purification from
secretion concepts have been reported. Domesticated C. reinhardtii strains used for genetic engineering are often cell-wall
deficient. These strains nevertheless secrete cell-wall components such as insoluble (hydroxy)proline-rich glycoproteins into the
culture media, which hinder downstream purification processes. Here, we attempted to overcome limitations in secretion titers
and improve protein purification by combining fusion partners that enhance RP secretion and enable alternative aqueous twophase
(ATPS) RP extraction from the culture medium. Protein fusions were strategically designed to contain a stably folded
peptide, which enhanced secretion capacities and gave insights into (some) regulatory mechanisms responsible for this process
in the algal host. The elevated protein titers mediated by this fusion were then successfully applied in combination with a fungal
hydrophobin tag, which enabled protein purification from the complex microalgal extracellular environment by ATPS, to yield
functional recombinant human epidermal growth factor (hEGF) from the algal host.