Algevir: An Expression System for Microalgae Based on Viral Vectors.

Bernardo Bañuelos-Hernández

doi:10.3389/fmicb.2017.01100

Abstract

The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus) and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin), which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture), which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins.

Highlights

The use of microalgae for the production of valuable proteins is acquiring relevance in the biotechnology field
Gene synthesis and molecular cloning techniques allowed the construction of the pAlgevir vector; whose physical map is shown in Figure 1 while the restriction profile and sequence are shown in Figures S1, S2, respectively
The Algevir system was developed as an attractive approach for the transient expression of recombinant proteins in microalgae using the replication elements from the begomovirus Ageratum enation virus

Summary

Introduction

The use of microalgae for the production of valuable proteins is acquiring relevance in the biotechnology field. Antibodies, and other therapeutic proteins; as well as enzymes for industrial use, have been produced in microalgae (Scranton et al, 2015; Rosales-Mendoza, 2016a). The highest yields, observed far, were achieved in photosynthetic microalgae transformed at the chloroplast genome; with yields up to 3 mg/L culture (1.86% of Total Soluble Protein (TSP); Gimpel et al, 2015). In the case of nuclear expression, genetic engineering faces two challenges: the improvement of yields and the genetic stability of the transformed clones (Rosales-Mendoza, 2016b). In an effort to overcome these obstacles, UV mutagenesis has been used to generate Chlamydomonas reinhardtii strains with improved transgene expression (showing yields up to 0.2% TSP; Neupert et al, 2009; Kurniasih et al, 2016). Secretion approaches have been implemented in these mutant strains, leading to yields up to 10 mg of secreted reporter protein per liter of culture (Lauersen et al, 2013)

Methods

Results

Conclusion