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Synthesis of recombinant antibacterial proteins in the Chlamydomonas reinhardtii chloroplast

Blanshard, Maximilian Edward Alfred; (2018) Synthesis of recombinant antibacterial proteins in the Chlamydomonas reinhardtii chloroplast. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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The rise of antibiotic resistance and the decline in antibiotic discovery have been well publicised. These issues, in combination with a growing global reliance upon antibiotics for everyday modern life, urgently require the discovery of novel antibacterial drugs. Endolysins are one potential candidate to support, or replace, conventional antibiotics. Endolysins are lytic enzymes produced by bacteriophage in natura to enable the release of viral progeny from inside the host bacterium. When applied exogenously, endolysins can lyse Gram-positive bacteria, and thus could be used as a novel antibacterial for this group of pathogens. Biologically active endolysins have been successfully expressed as recombinants in the chloroplast of the green alga, Chlamydomonas reinhardtii. C. reinhardtii, and in particular the chloroplast, has several features as a cell factory which make it an attractive alternative to the traditional recombinant protein production platforms. C. reinhardtii is free of endotoxins, can be cultivated at low cost in photobioreactors, has GRAS status, and is genetically tractable. This study initially focuses upon improving the accumulation and activity of one endolysin, Cpl-1, targeting Streptococcus pneumoniae. Recombinant Cpl-1 has been shown previously in the Purton lab to accumulate to moderate levels in the C. reinhardtii chloroplast. Here we present two transgenic lines of C. reinhardtii that appear to accumulate recombinant Cpl-1 to higher levels – one through the incorporation of multiple expression cassettes, and one through codon pair optimization. To improve the activity of Cpl-1 as an enzyme, Cpl-1 binding site mutagenesis, Cpl-1 dimerization, and the production of a potentially synergistic holin protein were all attempted. Finally, an endolysin against Clostridium difficile, CD27L, was successfully produced in the C. reinhardtii chloroplast and shown to be active in vitro. Another endolysin, this time targeting Propionibacterium acnes, failed to express in C. reinhardtii, but was expressed in E. coli, albeit without obvious lytic activity.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Synthesis of recombinant antibacterial proteins in the Chlamydomonas reinhardtii chloroplast
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
URI: http://discovery.ucl.ac.uk/id/eprint/10045326
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