Abstract
Photosynthetic microorganism like microalgae and cyanobacteria are considered as emerging biotechnology platforms for production of recombinant proteins and other high-value biomolecules with a wide range of applications. Moreover, microalgae offer significant advantages compared with other biotechnology hosts including safety, metabolic diversity, scalability, sustainability and low production cost. Over the past decades, considerable improvement has been made to express and secrete recombinant proteins in high levels: however current yields are still low.
The first research project presented in this thesis is concerned with establishing new genetic tools that allow the secretion of recombinant proteins from the model organism Chalmydomonas reinhardtii in high yields. First, we demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus in the culture media, however yields remained low. Therefore, in order to boost the secretion yields we implemented synthetic glycomodules used in plants cells cultures. These glycomodules are comprised of tandem serine (Ser) and proline (Pro) repeats. When Venus was fused to the glycomodules, accumulation of a fusion protein was dramatically increased by up to 12 folds, with the maximum yield of 15 mg L-1. Characterization of the secreted Venus showed the presence of glycosylations and increased resistance to proteolytic degradation. The results from this thesis demonstrate the potential of microalgae as a cell factory for secretion of recombinant proteins.
The second research project presented in this thesis aimed to establish a new robust method to allow in vivo measurements of metabolic enzyme activities in cyanobacteria, with a hope that the method would facilitate further optimization of metabolic facilities towards enhanced product yields. We have shown that by permeabilizing cyanobacteria cells with a commercially available B-PER reagent, cells can be directly used for measurement of intracellular enzymes in an easy, efficient and scalable manner.
The first research project presented in this thesis is concerned with establishing new genetic tools that allow the secretion of recombinant proteins from the model organism Chalmydomonas reinhardtii in high yields. First, we demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus in the culture media, however yields remained low. Therefore, in order to boost the secretion yields we implemented synthetic glycomodules used in plants cells cultures. These glycomodules are comprised of tandem serine (Ser) and proline (Pro) repeats. When Venus was fused to the glycomodules, accumulation of a fusion protein was dramatically increased by up to 12 folds, with the maximum yield of 15 mg L-1. Characterization of the secreted Venus showed the presence of glycosylations and increased resistance to proteolytic degradation. The results from this thesis demonstrate the potential of microalgae as a cell factory for secretion of recombinant proteins.
The second research project presented in this thesis aimed to establish a new robust method to allow in vivo measurements of metabolic enzyme activities in cyanobacteria, with a hope that the method would facilitate further optimization of metabolic facilities towards enhanced product yields. We have shown that by permeabilizing cyanobacteria cells with a commercially available B-PER reagent, cells can be directly used for measurement of intracellular enzymes in an easy, efficient and scalable manner.
| Originalsprog | Engelsk |
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| Forlag | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Antal sider | 189 |
| Status | Udgivet - 2016 |