Fusion Proteins Specific regions within the coding regions of chloroplast genes enhance efficient expression of foreign genes in plants and algae (Anthonisen et al. 2002; Gray et al. 2009; Kuroda and Maliga 2001b). Genetic fusion of endogenous regions to an exogenous protein of interest may represent another effective strategy for high-level transgene expression (Gray et al. 2011; Kasai et al. 2003). A dis­advantage of the fusion protein approach may be reduced industrial or clinical values or increased cost of purifying the protein of interest from its fusion partner.

Codon Optimisation Codon optimisation has been shown to be an important factor of heterologous gene expression in algae (Heitzer et al. 2007). It has been dem­onstrated that protein expression levels can be improved in the chloroplast by up to * 80-fold (Franklin et al. 2002a) by adjusting the codon bias of the transgene to the AT-rich chloroplast codon bias. Despite this, the effects of codon bias on expression levels are largely heuristic and still not well understood at a theoretical level.

Replacement of Highly Expressed Photosynthesis Genes The highest protein expression levels in Chlamydomonas have so far been demonstrated in transfor­mants carrying the psbA promoter and 5′ UTR for transcription and translation initiation in a psbA knockout background, which leads to a non-photosynthetic strain (Manuell et al. 2007; Surzycki et al. 2009). PsbA encodes for the D1 protein in the photosystem II reaction centre and is the most rapidly synthesised protein at high light intensity in higher plants and algal cells (Trebitsh et al. 2000). Although photosynthesis was restored by introducing the psbA gene at a different location, the presence of the psbA protein decreased the yield of foreign protein production (Manuell et al. 2007). This may reflect competition between the two genes, but could also be due to primary energetic or biosynthetic limitations.

Open Reading Frame Orientation In the chloroplast, 3′ UTR of the RNA often shows the potential for stem-loop formation, which serves for transcript stabilisa­tion rather than transcription termination (Rott et al. 1998). Therefore, a degree of ‘read through’ from the upstream gene is possible (Oey et al. 2009), which in parallel can increase the amount of translatable RNA and thus potentially increase the amount of protein (Stern and Gruissem 1987).

Heterologous and Hybrid Regulatory Systems Rasala et al. (2011) have recently shown that an increase in protein production can be achieved by the fusion of the 16S ribosomal promoter, which does not contain translation initiation signals such as Shine-Dalgarno sequences, to the endogenous atpA 5′ UTR containing the translation initiation signal. This, and the demonstration that heterologous regula­tory elements can significantly induce the expression rate (Kuroda and Maliga 2001a; Oey et al. 2009; Ruhlman et al. 2010), suggests that designed regulatory elements could serve to improve expression.

Inducible Systems Environmental changes or developmental factors naturally influence the up — and down-regulation of genes. Understanding those regulatory mechanisms provides a valuable genetic tool, for example to switch protein expression automatically or under the control of specific circumstances. Examples of inducible algal promoters include light responsive genes (Falciatore et al. 1999), nitrogen starvation (Poulsen et al. 2006; Poulsen and Kroger 2005), a sulphur — regulated arylsulfatase gene and an ISG glycoprotein. Tightly controlled expression of toxic proteins (e. g. the growth factor DILP-2) is also often desirable (Surzycki et al. 2007). The expression of psbD (D2 component of PSII) is dependent on the Nac2 gene fused to the copper-sensitive cytochrome c6 promoter (cyc6) and induced in copper deficiency and repressed in the presence of copper.

Riboswitches Riboswitches that can be used to regulate protein expression at the translational level have been shown to be functional in C. reinhardtii and Volvox carteri (Croft et al. 2007) suggesting that it may be a useful technique for other algae species as well.