Light and heat-shock mediated TDA1 overexpression as a tool for controlled high-yield recombinant protein production in Chlamydomonas reinhardtii chloroplasts

Abstract

The microalga Chlamydomonas reinhardtii offers a rapid, scalable and low-cost platform for recombinant protein production. Its chloroplast provides a particularly robust expression system for high yield production of complex proteins requiring challenging post-transcriptional modifications. Controlled transgene expression provides an important advantage supporting many applications. Currently, however, only three inducible systems exist for algal chloroplast expression regulation. Here, we present the nuclear encoded translation enhancer TDA1 that regulates transgene expression in the chloroplast, as a component of an inducible system that is designed for high-yield production. Specifically, the C-terminus of TDA1 (cTDA1) promotes translation of the atpA transcript by interacting with its 5′-UTR, which has been shown to support high levels of recombinant protein expression. cTDA1 under the control of the inducible promoter HSP70A-RBCS2 was introduced into a chloroplast mutant expressing GFP under the control of the atpA 5′-UTR. These cTDA1/GFP mutants were grown under optimised illumination regimes and subjected to specific heat-shock treatments, after which cTDA1 and GFP expression levels were measured. A ~1.9-fold increase was detected in induced samples grown under optimised production conditions (4 days cultivation period under 6 h·d−1 illumination at 200 μE·m−2·s−1 followed by 2 h light exposure (200 μE·m−2·s−1) after the end of the 4 day period; 30 min heat-shock at 40 °C with subsequent 5 h incubation at RT (200 μE·m−2·s−1 illumination)). Western Blot analysis confirmed that after induction, the cTDA1 and GFP accumulation correlated. This demonstrates proof-of-concept that expression of the nuclear cTDA1 regulates atpA 5′-UTR mediated chloroplast transgene expression and thus provides the basis for a next generation nuclear inducible chloroplast regulation systems.