A METHOD FOR EXPRESSING MULTIPLE TRANSGENIC PROTEINS IN THE CHLAMYDOMONAS REINHARDTII CHLOROPLAST FOR BIOTECHNOLOGY APPLICATIONS

Abstract

Microalgae are a promising platform for producing valuable commercial products in various biotechnological applications. In particular, the simultaneous expression of distinct proteins has emerged as a desirable strategy for large-scale production but is not easily achieved. Here, I describe a synthetic operon strategy to express transgenic proteins in the chloroplast genome of Chlamydomonas reinhardtii. I generated chloroplast expression vectors for expressing up to three proteins (FBPase, FBA1, VHH or AtpB), using either a cyanobacterial operon spacer or a tobacco chloroplast operon spacer, or both. In all, nine operon expression vectors were integrated into the chloroplast genome and transformants were obtained for each of them. All operons containing FBP1 and atpB coding sequences expressed the products of those genes, however, neither FBA1 nor VHH proteins were expressed from the operons that contained the FBA1 or VHH genes. These results indicated that the synthetic operon strategy can work, but that not all proteins can be expressed using the vectors I generated. In my second project, I generated a chloroplast vector to express a single-chain, streptavidin-tagged VHH antibody against the food-poisoning agent Campylobacter jejuni and integrated it into the chloroplast genome of C. reinhardtii. I obtained transgenic C. reinhardtii strains that produced the VHH antibody. These transgenic strains ideally could be fed to chickens to reduce C. jejuni colonization levels or motility in target populations. However, the VHH antibody unfortunately did not work as intended in inhibiting C. jejuni mobility or colonization, possibly because the streptavidin tag interferes with antibody function. Although the strategies explored in these two projects encountered barriers to practical application, they should serve as a basis for future efforts to express valuable transgenic proteins in green algae.