Method for the production of recombinant proteins by green microalgae

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The present invention relates to a method for the production of recombinant proteins by algal cells, which are fresh water unicellular green microalgae belonging to the order of Sphaeropleales. The present invention further provides recombinant algal cells, wherein said algal cells are fresh water unicellular green microalgae belonging to the order of Sphaeropleales, and wherein said algal cells are capable of producing a recombinant protein. The invention is also directed to a method for selecting recombinant algal cells. The invention also relates to the use of recombinant algal cells as described herein for producing recombinant proteins.

The invention is in the field of molecular biology, and relates to recombinant engineering and protein expression. In particular, the invention relates to a method for producing recombinant proteins by fresh water unicellular green microalgae. More specifically, the present invention relates to a method for producing a recombinant protein by a recombinant algal cell of the order of the Sphaeropleales. The invention also relates to a recombinant algal cell of the order of the Sphaeropleales capable of producing recombinant proteins. The invention further also relates to the use of recombinant algal cells for producing recombinant proteins.

There is a growing demand for recombinant proteins, and industry is looking for new production systems with the following characteristics: high production capacity at low cost, straight forward to use, able to produce active eukaryotic proteins and GRAS status (absence of animal or human pathogen and of any toxic compound).

Unicellular algae could meet these requirements while providing the market with adequate production capacity. Algae propagate by vegetative replication, lack pollen, and have no potential for gene transfer to food crops. They can easily be grown in containment, again reducing any chance of environmental contamination. Their controlled growth also assures that external contaminants, like pesticides or pollutants, do not contaminate the protein being produced. Algae are eukaryotes, meaning that unlike bacteria, they are efficient at producing complex proteins and have the machinery necessary to fold and assemble multi-component complexes into functional proteins. In addition, green algae are generally regarded as safe (GRAS), posing little risk of viral, prion or bacterial endotoxin contamination. Thus, algae would seem to be an ideal system for biologic production, as long as high levels of protein expression can be achieved, and that the expressed protein can be shown to function in a bioactive manner.

However, the full potential of algae can only be fully developed with an increase in the ability to genetically tailor algae for specific purposes.

Unfortunately, molecular biological manipulation of algal systems has seriously lagged behind other systems. Many of the techniques that have been developed for the introduction of DNA into bacterial, yeast, insect and animal cells cannot be easily adapted to algal systems. There have been some reports in the art of genetic engineering of algal cells. However, there are no universally applicable techniques for transforming, selecting and growing recombinant algae that can be applied in all types of algal systems.

WO 97/39106 for instance discloses the genetic engineering of eukaryotic marine algae, i.e. algae that grow in sea water, i.e. under marine tidal influence. However, the techniques described for engineering marine algae cannot simply be extrapolated or used for fresh water algae, especially in view of the specific growing requirements of marine algae.

The Sphaeropleales are an order of green algae that include non-motile unicellular microalgae that have biflagellate zoospores with flagella that are directly opposed in direction. This type of microalgae can be cultivated and shows benefits, including the absence of animal or human pathogens and of any toxic compound when naturally cultured. However, up to date no adequate techniques have been described for the transformation of this type of algal cells in an economically and industrially applicable way.

In view of the above, it is clear that there remains a need in the art for improved methods and tools for genetically engineering of fresh water eukaryotic algae in order to manufacture products, e.g. recombinant proteins of interest by these algae.