Harnessing the Algal Chloroplast for Heterologous Protein Production

Abstract

Photosynthetic microbes are gaining increasing attention as heterologous hosts for the light-driven, low-cost production of high-value recombinant proteins. Recent advances in the manipulation of unicellular algal genomes offer the opportunity to establish engineered strains as safe and viable alternatives to conventional heterotrophic expression systems, including for their use in the feed, food, and biopharmaceutical industries. Due to the relatively small size of their genomes, algal chloroplasts are excellent targets for synthetic biology approaches, and are convenient subcellular sites for the compartmentalized accumulation and storage of products. Different classes of recombinant proteins, including enzymes and peptides with therapeutical applications, have been successfully expressed in the plastid of the model organism Chlamydomonas reinhardtii, and of a few other species, highlighting the emerging potential of transplastomic algal biotechnology. In this review, we provide a unified view on the state-of-the-art tools that are available to introduce protein-encoding transgenes in microalgal plastids, and discuss the main (bio)technological bottlenecks that still need to be addressed to develop robust and sustainable green cell biofactories.

Keywords: Chlamydomonas reinhardtii; chloroplast; green cell factories; heterologous expression systems; microalgae; molecular pharming; plastome engineering; recombinant protein production; synthetic biology; transplastomic biotechnology.

Figure 1

Graphical summary of emerging tools for chloroplast engineering in microalgae. (1) Typical quadripartite structure of a plastid chromosome with two inverted repeats (IRs) and transgene integration enabled by recombination between homology regions (HR1-2). The gene(s) of interest (GOI) and the selectable marker (SM) are connected by a linking element (*), or individually equipped with cis-regulatory sequences: promoter (P), 5′- and 3′-UTRs (untranslated regions). (2) Under constant selection, the plastome is enriched in transformed chromosomes (blue–red circles), although untransformed copies (blue circle) may persist and expose the system to the risk of genetic instability. In addition, spontaneous inter- or intrachromosomal recombination events may lead to transgene loss. (3) The PTXD enzyme performs the conversion of phosphite ions (PO₃³⁻) into phosphate (PO₄³⁻) and serves as a metabolic selectable marker, also enabling axenic algal cultivation in non-sterile media [77]. (4) Transgene expression can be finely controlled via chemical- or temperature-inducible, nucleus-encoded, trans-acting factors that (5) bind the 5′-UTRs of plastid mRNAs and promote their translation [109]. (6) The CITRIC (cold-inducible translational readthrough in chloroplasts) system requires plastome manipulation only and exploits a temperature-sensitive suppressor tRNA to regulate translation [110]. (7) Polycistronic constructs, in which multiple open reading frames (ORFs) are connected via native intercistronic expression elements (IEEs), are processed by endogenous trans-acting factors into separate mono-cistronic transcripts that are independently translated [111]. (8) As pioneered in plant plastids, transgene expression can potentially be achieved in microalgae via episomal vectors that do not require integration into the circular chromosome, but are stably maintained by the host.