New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii

Abstract

In recent years, there has been an increasing interest in the exploitation of microalgae in industrial biotechnology. Potentially, these phototrophic eukaryotes could be used for the low-cost synthesis of valuable recombinant products such as bioactive metabolites and therapeutic proteins. The algal chloroplast in particular represents an attractive target for such genetic engineering, both because it houses major metabolic pathways and because foreign genes can be targeted to specific loci within the chloroplast genome, resulting in high-level, stable expression. However, routine methods for chloroplast genetic engineering are currently available only for one species—Chlamydomonas reinhardtii—and even here, there are limitations to the existing technology, including the need for an expensive biolistic device for DNA delivery, the lack of robust expression vectors, and the undesirable use of antibiotic resistance markers. Here, we describe a new strain and vectors for targeted insertion of transgenes into a neutral chloroplast locus that (i) allow scar-less fusion of a transgenic coding sequence to the promoter/5′UTR element of the highly expressed endogenous genes psaA or atpA, (ii) employ the endogenous gene psbH as an effective but benign selectable marker, and (iii) ensure the successful integration of the transgene construct in all transformant lines. Transformation is achieved by a simple and cheap method of agitation of a DNA/cell suspension with glass beads, with selection based on the phototrophic rescue of a cell wall-deficient ΔpsbH strain. We demonstrate the utility of these tools in the creation of a transgenic line that produces high levels of functional human growth hormone.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7354-6) contains supplementary material, which is available to authorized users.

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

“…As illustrated in Fig. 1, this results in marker-free transformants in which the only foreign DNA in the plastome is the gene of interest [27,28]. Expression of the gene is achieved by fusing the coding sequence to promoters and untranslated regions from highly expressed endogenous genes, such as the photosynthesis genes psaA and psbA.…”

“…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.…”

Engineering microalgae through chloroplast transformation to produce high‐value industrial products

“…Wannathong et al. used glass beads for the transformation of algal chloroplast and reported that recombinant human growth hormone could be stably expressed up to 25 μg/mL. In another study, Castellanos‐Huerta et al.…”

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

“…Introduction of three gene cassettes into the C. reinhardtii plastome using a combinatorial approach. Design of the cassettes, chloroplast transformation and analysis of transformant lines were carried out using the methods described previously [24,30]. (a) The transformation plasmid pICS is derived from the expression vector pASapI and was assembled using three transgene cassettes in which each coding sequence (ibv-ctb, codA and splB) was codon-optimised and extended to encode a C-terminal HA tag.…”

“…As shown in Fig. 1a, the first three genes were assembled into a single plasmid (pICS) that is based on the chloroplast expression vector pASapI [30]. Details of this vector and the cloning strategy used are given in the supplementary data (available in the online version of this article).…”