Chlamydomonas offers advantages as a recombinant protein production system most importantly because it possesses the machinery required for post-translational modification of proteins. However transgene expression from the nuclear genome of Chlamydomonas is not yet adequate for industrial applications and requires optimization. With the objective of optimizing transgene expression in Chlamydomonas, we analyzed the effect of seven promoters and three transcription terminator elements on the expression of the reporter gene luciferase. We found that in addition to promoters, terminators can have a strong influence on transgene expression. In fact the psaD terminator was much more influential in improving transgene expression than the best promoter identified in our studies: psaD promoter (psaDP). Overall the psaDP-psaD terminator expression cassette was found to be the best expression cassette for transgene expression in Chlamydomonas. It was also established that the state of the plasmid used for Chlamydomonas transformation has a significant impact on transformation efficiency and the percentage of clones expressing the transgene. Furthermore, transgene expression in Chlamydomonas was found to be fairly stable under selection conditions. The findings of this study can be applied to significantly enhance transgene expression in Chlamydomonas and contribute towards developing Chlamydomonas as a biofactory.
The potential biotechnological applications of Chlamydomonas as a biofactory, a mode for oral delivery of vaccines and as a system to control insect-vectors were also demonstrated. A difficult to express human therapeutic protein, human paraoxonase1 (huPON1) and a bacterial protein, acyl homoserine lactone (AHL) lactonase were successfully expressed in Chlamydomonas and were found to be functional. Expression of AHL lactonase in Chlamydomonas resulted in significant reduction in the activity of AHL mimic compounds secreted by Chlamydomonas, indicating that these compounds were inactivated by AHL lactonase, implying the existence of structural similarity between the mimics secreted by Chlamydomonas and AHLs and their potential pharmaceutical applications in inhibiting bacterial growth. The possible application of Chlamydomonas as a system to deliver vaccines orally to fish, animals and possibly humans was demonstrated by expressing luciferase-infectious hematopoietic necrosis virus (IHNV) glycoprotein (G) fusion protein and subsequent feeding of these transgenic algae to rabbits. Feeding of transgenic algae expressing luciferase-G protein fusion to rabbits led to successful induction of immune response in rabbits against the G protein. These transgenic Chlamydomonas will now be evaluated for their ability to vaccinate trout against IHNV.
Chlamydomonas was also successfully developed into a mosquito larvicide, demonstrating its potential in controlling insect-vectors that feed on micro-organisms including microalgae in aquatic bodies. Feeding transgenic algae expressing dsRNA targeting the 3-hydroxy kynurenine (3HKT) enzyme involved in tryptophan metabolism resulted in up to 58% mortality in Anopheles stephensi larvae.
The studies presented in this thesis emphasize the role of regulatory elements like the transcription terminator in enhancing gene expression in Chlamydomonas. This is expected to help further develop Chlamydomonas as an organism of choice for expression of eukaryotic proteins. Moreover the research presented here in underlines the potential applications of transgenic Chlamydomonas as an agent for viral, bacterial and insect control.