Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker.

Rouhollah Barahimipour,Ralph Bock,Juliane Neupert

doi:10.1007/s11103-015-0425-8

Abstract

The unicellular green alga Chlamydomonas reinhardtii has become an invaluable model system in plant biology. There is also considerable interest in developing this microalga into an efficient production platform for biofuels, pharmaceuticals, green chemicals and industrial enzymes. However, the production of foreign proteins in the nucleocytosolic compartment of Chlamydomonas is greatly hampered by the inefficiency of transgene expression from the nuclear genome. We have recently addressed this limitation by isolating mutant algal strains that permit high-level transgene expression and by determining the contributions of GC content and codon usage to gene expression efficiency. Here we have applied these new tools and explored the potential of Chlamydomonas to produce a recombinant biopharmaceutical, the HIV antigen P24. We show that a codon-optimized P24 gene variant introduced into our algal expression strains give rise to recombinant protein accumulation levels of up to 0.25 % of the total cellular protein. Moreover, in combination with an expression strain, a resynthesized nptII gene becomes a highly efficient selectable marker gene that facilitates the selection of transgenic algal clones at high frequency. By establishing simple principles of successful transgene expression, our data open up new possibilities for biotechnological research in Chlamydomonas.

Highlights

The unicellular green alga Chlamydomonas reinhardtii is widely used as a model organism for research on fundamental questions in cell and molecular biology, including chloroplast biology, photosynthesis, light perception and signaling as well as flagellar function and tactic movements (Harris 2001; Merchant et al 2007)
Expressing YFP gene variants that differ in GC content and/or codon usage, we have recently demonstrated that codon usage, rather than GC content, is the most important determinant of transgene expression efficiency in Chlamydomonas reinhardtii (Barahimipour et al 2015)
To identify factors involved in expression of the HIV antigen P24 in Chlamydomonas reinhardtii, we synthesized three variants of the P24 gene that encode the identical amino acid sequence but differ in codon usage and GC content (Fig. 1a, b; Table 1): (i) the wild-type sequence from HIV-1 subsequently referred to us P24w, (ii) a version with the codon usage optimized for the AT-rich chloroplast genome (CpP24), and (iii) a version with the codon usage optimized for the GC-rich nuclear genome of Chlamydomonas (CrP24)

Summary

Introduction

The unicellular green alga Chlamydomonas reinhardtii is widely used as a model organism for research on fundamental questions in cell and molecular biology, including chloroplast biology, photosynthesis, light perception and signaling as well as flagellar function and tactic movements (Harris 2001; Merchant et al 2007). The ease with which Chlamydomonas can be cultured in large volumes and the ease with which it can be genetically engineered have made the alga a preferred model organism in biotechnological research, especially for metabolic engineering, biofuel production and the synthesis of proteinaceous biopharmaceuticals and industrial enzymes, an area commonly referred to as molecular farming (Scaife et al 2015; Scranton et al 2015). Introduction of transgenes into the nuclear genome of the alga is straightforward, the isolation of transgenic strains that express the foreign gene

Methods

Results

Conclusion