Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas
Laure Michelet1, Linnka Legendre1, Sarah Burr2, Jean-David Rochaix1 and Michel Goldschmidt-Clermont1
1) Departments of Plant Biology and of Molecular Biology, University of Geneva, Quai E. Ansermet 30, 1211 Genève 4, Switzerland
2) Institute for Veterinary Bacteriology, University of Berne, Länggasstrasse 122, 3012 Berne, Switzerland
Chloroplast transformation in microalgae offers great promise for the production of proteins of pharmaceutical interest or for the development of novel biofuels. For many applications, high level expression of transgenes is desirable. We have transformed the chloroplast of Chlamydomonas reinhardtii with two genes, acrV and vapA, which encode antigens from the fish pathogen Aeromonas salmonicida. The promoters and 5' untranslated regions of four chloroplast genes were compared for their ability to drive expression of the bacterial genes. The highest levels of expression were obtained when they were placed under the control of the cis-acting elements from the psaA-exon1 gene. The expression of these chimeric genes was further increased when a nuclear mutation that affects a factor involved in psaA splicing was introduced in the genetic background of the chloroplast transformants. Both the accumulations of the chimeric mRNAs and of the recombinant proteins were dramatically increased, indicating that negative feedback loops limit the expression of chloroplast transgenes. Our results demonstrate the potential of manipulating anterograde signaling and negative regulatory feedback loops in the chloroplast to improve transgene expression.This work was supported by grants from the Swiss NRP59 program (Benefits and Risks of the Deliberate Release of Genetically Modified Plants) and from the Swiss National Science Foundation (3100AO-117712).
e-mail address of presenting author: laure.michelet@cea.fr