The synthesis of novel antibacterial proteins in the Chlamydomonas reinhardtii chloroplast

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

Synthesis of bacteriophage lytic proteins against Streptococcus pneumoniae in the chloroplast of Chlamydomonas reinhardtii.

There is a pressing need to develop novel antibacterial agents given the widespread antibiotic resistance among pathogenic bacteria and the low specificity of the drugs available. Endolysins are antibacterial proteins that are produced by bacteriophage-infected cells to digest the bacterial cell wall for phage progeny release at the end of the lytic cycle. These highly efficient enzymes show a considerable degree of specificity for the target bacterium of the phage. Furthermore, the emergence of resistance against endolysins appears to be rare as the enzymes have evolved to target molecules in the cell wall that are essential for bacterial viability. Taken together, these factors make recombinant endolysins promising novel antibacterial agents. The chloroplast of the green unicellular alga Chlamydomonas reinhardtii represents an attractive platform for production of therapeutic proteins in general, not least due to the availability of established techniques for foreign gene expression, a lack of endotoxins or potentially infectious agents in the algal host, and low cost of cultivation. The chloroplast is particularly well suited to the production of endolysins as it mimics the native bacterial expression environment of these proteins while being devoid of their cell wall target. In this study the endolysins Cpl-1 and Pal, specific to the major human pathogen Streptococcus pneumoniae, were produced in the C. reinhardtii chloroplast. The antibacterial activity of cell lysates and the isolated endolysins was demonstrated against different serotypes of S. pneumoniae, including clinical isolates and total recombinant protein yield was quantified at ~1.3 mg/g algal dry weight. This article is protected by copyright. All rights reserved

Green biologics: the algal chloroplast as a platform for making biopharmaceuticals

Most commercial production of recombinant pharmaceutical proteins involves the use of mammalian cell lines, E. coli or yeast as the expression host. However, recent work has demonstrated the potential of eukaryotic microalgae as platforms for light-driven synthesis of such proteins. Expression in the algal chloroplast is particularly attractive since this organelle contains a minimal genome suitable for rapid engineering using synthetic biology approaches; with transgenes precisely targeted to specific genomic loci and amenable to high-level, regulated and stable expression. Furthermore, proteins can be tightly contained and bio-encapsulated in the chloroplast allowing accumulation of proteins otherwise toxic to the host, and opening up possibilities for low-cost, oral delivery of biologics. In this commentary we illustrate the technology with recent examples of hormones, protein antibiotics and immunotoxins successfully produced in the algal chloroplast, and highlight possible future applications

The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism

Sustainable and economically viable support for an ever-increasing global population requires a paradigm shift in agricultural productivity, including the application of biotechnology to generate future crop plants. Current genetic engineering approaches aimed at enhancing the photosynthetic efficiency or composition of the harvested tissues involve relatively simple manipulations of endogenous metabolism. However, radical rewiring of central metabolism using new-to-nature pathways, so-called "synthetic metabolism", may be needed to really bring about significant step changes. In many cases, this will require re-programming the metabolism of the chloroplast, or other plastids in non-green tissues, through a combination of chloroplast and nuclear engineering. However, current technologies for sophisticated chloroplast engineering ("transplastomics") of plants are limited to just a handful of species. Moreover, the testing of metabolic rewiring in the chloroplast of plant models is often impractical given their obligate phototrophy, the extended time needed to create stable non-chimeric transplastomic lines, and the technical challenges associated with regeneration of whole plants. In contrast, the unicellular green alga, Chlamydomonas reinhardtii is a facultative heterotroph that allows for extensive modification of chloroplast function, including non-photosynthetic designs. Moreover, chloroplast engineering in C. reinhardtii is facile, with the ability to generate novel lines in a matter of weeks, and a well-defined molecular toolbox allows for rapid iterations of the "Design-Build-Test-Learn" (DBTL) cycle of modern synthetic biology approaches. The recent development of combinatorial DNA assembly pipelines for designing and building transgene clusters, simple methods for marker-free delivery of these clusters into the chloroplast genome, and the pre-existing wealth of knowledge regarding chloroplast gene expression and regulation in C. reinhardtii further adds to the versatility of transplastomics using this organism. Herein, we review the inherent advantages of the algal chloroplast as a simple and tractable testbed for metabolic engineering designs, which could then be implemented in higher plants