Review

Biotechnology Advances 28 (2010) 910–918

Contents lists available at ScienceDirect

Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Strategies for high-level recombinant protein expression in transgenic
microalgae: A review
Gabriel Potvin, Zisheng Zhang
Department of Chemical andBiological Engineering, University of Ottawa, Ottawa, ON K1N 6N5, Canada

article

info

Article history:
Received 14 June 2010
Received in revised form 3 August 2010
Accepted 13 August 2010
Available online 20 August 2010
Keywords:
Chloroplast
High protein yields
Microalgae
Proteases
Stable expression
Transformation

abstract
Microalgae represent the ‘best of both worlds’, combiningthe high growth rate and ease of cultivation of
microorganisms with the ability to perform post-transcriptional and post-translational modi cations of
plants. The development of economically viable microalgal expression systems is, however, hindered by low
recombinant protein yields. Although there are still many obstacles to overcome before microalgae become
standard expression systems,considerable progress has been made in recent years in regards to elucidating
the causes for these low yields and in the development of strategies to improve them. Transgenes have
successfully been expressed in both nuclear and chloroplast microalgal genomes, although at economically
viable levels only in the latter. The present review describes recent progress in genetic manipulation of
microalgae,outlines strategies to increase protein yields and presents some interesting avenues of research
that remain to be explored.
© 2010 Elsevier Inc. All rights reserved.

Contents
1.
2.
3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
Current state of bioreactor technology . . . . . . . . . . . . . .
Genetic transformation methods . . . . . . . . . . . . . . . . .
3.1.Cell wall-de cient strains . . . . . . . . . . . . . . . . .
3.2.
Particle bombardment . . . . . . . . . . . . . . . . . .
3.3.
Glass beads method . . . . . . . . . . . . . . . . . . .
3.4.
Electroporation . . . . . . . . . . . . . . . . . . . . .
3.5.
Agrobacterium tumefaciens-mediated transformation . . . .
3.6.
Gene copy number . . . . . . . . . . . . . . . . . . . .
4.
Selectionmarkers . . . . . . . . . . . . . . . . . . . . . . . .
5.
Nuclear versus chloroplast genomes . . . . . . . . . . . . . . .
6.
Factors affecting protein expression and strategies for its increase .
6.1.
Codon optimization . . . . . . . . . . . . . . . . . . .
6.2.
Transformation-associated genotypic modi cations . . . .
6.3.
Endogenous enhancer and regulatory elements . . . . . .
6.4.Sensitivity to proteases . . . . . . . . . . . . . . . . . .
7.
Conclusions and future prospects . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Harnessing the bene ts of microalgae is by no means a novel
concept. Historical records indicate that Chinesecivilizations used
Corresponding author. Tel.: +1 613 562 5800x6110.
E-mail address: zzhang@uottawa.ca (Z. Zhang).
0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2010.08.006

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
..
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
....