Two adjacent nuclear genes are required for functional complementation of a chloroplast trans-splicing mutant from Chlamydomonas reinhardtii

The chloroplast tscA gene from Chlamydomonas reinhardtii encodes a co-factor RNA that is involved in trans-splicing of exons 1 and 2 of the psaA mRNA encoding a core polypeptide of photosystem I. Here we provide molecular and genetic characterization of the trans-splicing mutant TR72, which is defective in the 3'-end processing of the tscA RNA and consequently defective in splicing exons 1 and 2 of the psaA mRNA. Using genomic complementation, two adjacent nuclear genes were identified, Rat1 and Rat2, that are able to restore the photosynthetic growth of mutant TR72. Restoration of the photosynthesis phenotype, however, was successful only with a DNA fragment containing both genes, while separate use of the two genes did not rescue the wild-type phenotype. This was further confirmed by using a set of 10 gene derivatives in complementation tests. The deduced amino acid sequence of Rat1 shows significant sequence homology to the conserved NAD+-binding domain of poly(ADP-ribose) polymerases of eukaryotic organisms. However, mutagenesis of conserved residues in this putative NAD+-binding domain did not reveal any effect on restoration efficiency. Immunodetection analyses with enriched fractions of chloroplast proteins indicated that Rat1 is associated with chloroplast membranes. Using the yeast three-hybrid system, we were able to demonstrate the specific binding of tscA RNA by the Rat1 polypeptide. We propose that the two nuclear factors Rat1 and Rat2 are involved in processing of chloroplast tscA RNA and in subsequent splicing of psaA exons 1 and 2.     

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene （apcA and apcB） into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.     

Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii

There is a growing interest in the use of microalgae as low‐cost hosts for the synthesis of recombinant products such as therapeutic proteins and bioactive metabolites. In particular, the chloroplast, with its small, genetically tractable genome (plastome) and elaborate metabolism, represents an attractive platform for genetic engineering. In Chlamydomonas reinhardtii, none of the 69 protein‐coding genes in the plastome uses the stop codon UGA, therefore this spare codon can be exploited as a useful synthetic biology tool. Here, we report the assignment of the codon to one for tryptophan and show that this can be used as an effective strategy for addressing a key problem in chloroplast engineering: namely, the assembly of expression cassettes in Escherichia coli when the gene product is toxic to the bacterium. This problem arises because the prokaryotic nature of chloroplast promoters and ribosome‐binding sites used in such cassettes often results in transgene expression in E. coli, and is a potential issue when cloning genes for metabolic enzymes, antibacterial proteins and integral membrane proteins. We show that replacement of tryptophan codons with the spare codon (UGG→UGA) within a transgene prevents functional expression in E. coli and in the chloroplast, and that co‐introduction of a plastidial trnW gene carrying a modified anticodon restores function only in the latter by allowing UGA readthrough. We demonstrate the utility of this system by expressing two genes known to be highly toxic to E. coli and discuss its value in providing an enhanced level of biocontainment for transplastomic microalgae.     

Outlook in the application of Chlamydomonas reinhardtii chloroplast as a platform for recombinant protein production

Microalgae, also called microphytes, are a vast group of microscopic photosynthetic organisms living in aquatic ecosystems. Microalgae have attracted the attention of biotechnology industry as a platform for extracting natural products with high commercial value. During last decades, microalgae have been also used as cost-effective and easily scalable platform for the production of recombinant proteins with medical and industrial applications. Most progress in this field has been made with Chlamydomonas reinhardtii as a model organism mainly because of its simple life cycle, well-established genetics and ease of cultivation. However, due to the scarcity of existing infrastructure for commercial production and processing together with relatively low product yields, no recombinant products from C. reinhardtii have gained approval for commercial production and most of them are still in research and development. In this review, we focus on the chloroplast of C. reinhardtii as an algal recombinant expression platform and compare its advantages and disadvantages to other currently used expression systems. We then discuss the strategies for engineering the chloroplast of C. reinhardtii to produce recombinant cells and present a comprehensive overview of works that have used this platform for the expression of high-value products.     

Method to assemble and integrate biochemical pathways into the chloroplast genome of Chlamydomonas reinhardtii

Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. Here, we show that a modified DNA Assembler approach can be used to rapidly assemble multiple‐gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site‐specific location in the chloroplast genome of the microalgal species Chlamydomonas reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C. reinhardtii. An analysis of the relative gene expression of the engineered strains showed significant differences in the mRNA expression levels of the reporter genes and thus highlights the importance of proper promoter/untranslated region selection when constructing a target pathway. This new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast genome of microalgae and should aid current efforts to engineer algae for biofuels production and other desirable natural products. Biotechnol. Bioeng. 2012; 109: 2896–2903. © 2012 Wiley Periodicals, Inc.