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

Abstract

SummaryThere 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.

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

“…The reported yields are generally in the range of 0.1 to 5 % total soluble protein (TSP), although caution should be exercised when making comparisons, since the preparation of soluble extracts and the assay used for quantification differ between groups. A better measure is perhaps protein yield per gramme of dried biomass [31]. Whilst such levels are below those of established recombinant platforms, new synthetic biology approaches (see below) and moleculargenetics strategies based on our understanding of chloroplast gene regulation in C. reinhardtii are now leading to significant improvements in yield.…”

“…These include hormones such as human growth hormone [28], anti-hypertensive peptides [45], cancer therapeutics [46], antibody mimics [47], autoantigens [48], wound-healing factors [47] and anti-bacterial enzymes [31]. These examples serve to illustrate the potential of the chloroplast as a platform for a wide variety of recombinants.…”

Multigenic engineering of the chloroplast genome in the green alga Chlamydomonas reinhardtii

“…Having demonstrated that three foreign proteins could be synthesized in a single transgenic line, we then sought to further engineer this line by addition of two more transgenes at a second locus. We therefore built a second transformation plasmid (named pAP) that was designed to target the aadA cassette that confers spectinomycin-resistance [35] and a cassette for expression of the endolysin gene cpl-1 [29] into the middle of the non-essential chloroplast gene, chlL. This gene encodes a subunit of the dark-operative protochlorophyllide reductase and a knockout of chlL gives rise to a 'yellow-in-the-dark' phenotype [36].…”

“…1. Since the cpl-1 gene was designed to encode an HA-tagged version of Cpl-1 [29], we anticipated that Western analysis of the ICS3-AP transformants using the anti-HA antibodies would demonstrate the synthesis of four different recombinant proteins: namely, Cpl-1, together with IBV-CTB, CodA and SplB. However, only Cpl-1 and IBV-CTB could be detected in the three transformant lines despite CodA and SplB being readily detectable in the ICS3 recipient line (Fig.…”

Processos de produção da proteína heteróloga m<I>Cherry</i> por<i> Chlamydomonas reinhardtii</i>

“…. É importante notar que a maioria das proteínas foram clonadas no cloroplasto e apenas três delas no núcleo.Tabela 2.2: Resumo das biomoléculas expressas em Chlamydomonas reinhardtii, organela geneticamente transformada, percentual de expressão (%) da proteína solúvel total, concentração em miligramas por litro (mg / L) e vetor de clonagem STOFFELS et al, 2017(RASALA et al, 2012).…”