Comparative protein analysis to investigate Chlamydomonas reinhardtii as a cell biofactory
- Publication Type:
- Thesis
- Issue Date:
- 2020
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Chlamydomonas reinhardtii is a eukaryotic unicellular green microalga historically used as a model organism to describe and analyse fundamental biological processes including photosynthesis. Recently, this species has been utilised as a biofactory to successfully produce recombinant therapeutic proteins. C. reinhardtii has many advantages over traditional biofactories, such as E. coli (bacteria) and ChineseHamster Ovary cells (CHO, mammalian). It has high growth rates at low production costs, it cannot be contaminated by human pathogens, it can effectively secrete recombinant proteins, and it possess a eukaryotic post-translational modification (PTM) machinery. Unfortunately, C. reinhardtii also displays two major disadvantages: recombinant protein yields can be low and glycosylation (a fundamental PTM) can be incorrect. Low yields and potential low quality of products are the only drawbacks that kept C. reinhardtii out of the recombinant biopharmaceutical market, now worth 140 billion US$.
The low recombinant protein yield issue was partially overcome in 2009 with the generation of a UV mutated strain called UVM4. This strain is now well-established and broadly-used for secreted recombinant protein production in C. reinhardtii, and is capable of yields up to 15 mg/L (3-fold higher than the non-mutated strain 137c). However, these yields are still far from the extensive ones obtained with CHO cells (up to 5 g/L). Interestingly, as frequently happens for strains generated by mutagenesis, the pathways altered by the mutation were not investigated. Therefore, the reasons for these higher recombinant protein yields produced by strain UVM4 are still unknown. Characterising the modified protein pathways in this strain might help to understand the causes for the general lower yields in C. reinhardtii, to subsequently optimise and finally completely overcome the issue. In addition, to fully validate strain UVM4 as a cell biofactory, it is also necessary to analyse recombinant protein quality, namely glycosylation. Incorrect glycosylation can lead to immunogenic biopharmaceuticals, therefore a complete glycosylation profiling of strain UVM4 is also required.
With the results obtained in my thesis, I provide a detailed analysis of the two major drawbacks in recombinant protein production in C. reinhardtii, unravel possible causes, provide potential solutions, and overall I corroborate this species as a future industrial cell biofactory.
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