Taunt, HN; (2014) The synthesis of novel antibacterial proteins in the Chlamydomonas reinhardtii chloroplast. Doctoral thesis , UCL (University College London).

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Abstract

The Chlamydomonas reinhardtii chloroplast represents an attractive platform for therapeutic protein production, not least due to a robust molecular toolkit, low cost of cultivation, and the lack of endotoxins or potentially infectious agents in the algal host. The primary focus of this thesis has been the expression of bacteriophage endolysins in the C. reinhardtii chloroplast. Endolysins hold great promise as antibacterials since they can induce lysis of specific bacterial pathogens without affecting the body’s natural flora, do not result in acquired resistance in the pathogen, and can kill pathogens that colonize mucosal surfaces and biofilms. The expression of the lysin cpl-1 specific to the human pathogen Streptococcus pneumoniae has been confirmed in the C. reinhardtii chloroplast. The enzyme has subsequently been purified, and its lytic activity against culture collection and clinical strains of S. pneumoniae demonstrated, both for crude and enriched extracts. Two further endolysins, gp20 (specific to Propionibacterium acnes, strongly associated with clinical acne vulgaris) and lys16 (specific to Staphylococcus aureus, a common hospital acquired infection), have failed to express to detectable levels. This has instigated new investigations into the various factors affecting foreign gene expression in the C. reinhardtii chloroplast. Research has been conducted both in a wet lab context (with the use of modified leader sequences, full protein fusions and overhauled gene design) and in silico (looking particularly at codon- and codon pair usage in a wide panel of endogenous and recombinant genes). A defined codon pair bias has been shown to be present in the C. reinhardtii chloroplast, the first such bias to be reported in any organelle. The codon preferences observed have been related to a panel of transgenes that have previously been introduced into the chloroplast in the Purton lab, although no correlation has been found between codon pair usage and transgene expression.

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