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 availability of routine techniques for the genetic manipulation of the chloroplast genome of Chlamydomonas reinhardtii has allowed a plethora of reverse-genetic studies of chloroplast biology using this alga as a model organism. These studies range from fundamental investigations of chloroplast gene function and regulation to sophisticated metabolic engineering programs and to the development of the algal chloroplast as a platform for producing high-value recombinant proteins. The established method for delivering transforming DNA into the Chlamydomonas chloroplast involves microparticle bombardment, with the selection of transformant lines most commonly involving the use of antibiotic resistance markers. In this chapter we describe a simpler and cheaper delivery method in which cell/DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. Furthermore, we highlight the use of an expression vector (pASapI) that employs an endogenous gene as a selectable marker, thereby avoiding the contentious issue of antibiotic resistance determinants in transgenic lines.

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