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

Abstract

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.

Keywords: biopharmaceuticals; chlamydomonas; chloroplast; microalgae; synthetic biology.

Figure 1.

A low-cost, single-use photobioreactor system for commercial production of algal biomass. This ‘hanging bag’ system was developed by the Cawthron Institute, New Zealand for production of microalgae as aquaculture feed and for cultivation of Haematococcus pluvialis – a natural source of the high-value nutraceutical astaxanthin. We have successfully adapted the system for endolysin and vaccine production in C. reinhardtii (L. Stoffels, B. Parker and S. Purton, submitted). The 40 litre bags are optimally illuminated and sterile 5% CO₂/95% air supplied at the base of each bag for phototrophic growth and for mixing. Both batch and continuous operation is possible. ©Supreme Health, New Zealand. Reproduced by permission of Supreme Health, New Zealand. Permission to reuse must be obtained from the rightsholder.

Figure 2.

The chloroplast genome of Chlamydomonas reinhardtii. Generated from Genbank entry BK000554 using OGDRAW (ogdraw.mpimp-golm.mpg.de). Genes are coloured according to function (e.g. photosystem II genes in dark green), with genes transcribed anticlockwise on the outer side of the circle; those transcribed clockwise on the inner side. Examples of verified neutral sites for transgene insertion are indicated by arrowheads, with those within the inverted repeat (IR) regions that therefore give rise to two transgene copies per genome shown in light or dark blue.

Figure 3.

A synbio strategy for creating marker-free transgenic lines that also incorporate a biocontainment feature. Standardised DNA parts are assembled in order using Golden Gate to create the transgene device, with left (L) and right (R) flanking plastome elements (shown as bold lines) added for homologous recombination in the chloroplast. One element carries a wild-type copy of an essential photosynthetic (p/s) gene allowing phototrophic selection in the recipient chassis that lacks this gene. The synthetic gene-of-interest is codon-optimised and fused to promoter and untranslated region (UTR) parts. Biocontainment can be incorporated into the transgene by replacing one or more tryptophan codons with the UGA stop codon (*), thereby preventing function transfer of the gene to other microorganisms. Correct translation in the chloroplast is achieved by inclusion of a part carrying trnW^UCA. This gene encodes an orthogonal variant of the chloroplast's tryptophan tRNA that recognises UGA.

Figure 4.

Illustration of a designer immunotoxin produced in the C. reinhardtii chloroplast showing the multi-domain structure. The human CD22-scFv domain was fused to the hinge and constant domains of a human IgG1 and to exotoxin A from Pseudomonas aeruginosa lacking domain 1a. This created an immunotoxin that formed a homodimer through disulphide bonds between the hinge regions. Redrawn from.