Analysis of heterologous regulatory and coding regions in algal chloroplasts

Abstract

The basic photosynthetic apparatus is highly conserved across all photosynthetic organisms, and this conservation can be seen in both protein composition and amino acid sequence. Conservation of regulatory elements also seems possible in chloroplast genes, as many mRNA untranslated regions (UTRs) appear to have similar structural elements. The D1 protein of Photosystem II (psbA gene) is a highly conserved core reaction center protein that shows very similar regulation from cyanobacteria through higher plants. We engineered full and partial psbA genes from a diverse set of photosynthetic organisms into a psbA deficient strain of Chlamydomonas reinhardtii. Analysis of D1 protein accumulation and photosynthetic growth revealed that coding sequences and promoters are interchangeable even between anciently diverged species. On the other hand functional recognition of 5' UTRs is limited to closely related organisms. Furthermore transformation of heterologous promoters and 5' UTRs from the atpA, tufA and psbD genes conferred psbA mRNA accumulation but not translation. Overall, our results show that heterologous D1 proteins can be expressed and complement Photosystem II function in green algae, while RNA regulatory elements appear to be very specific and function only from closely related species. Nonetheless, there is great potential for the expression of heterologous photosynthetic coding sequences for studying and modifying photosynthesis in C. reinhardtii chloroplasts.

Production of recombinant proteins in microalgae at pilot greenhouse scale

“…Additionally, the expression of the transgene is constitutive and not dependent upon induction by shifting the light conditions, as a previously developed system (see Introduction) (Manuell et al, ). To achieve this we tested different combinations of 5′ promoters/UTRs and psbA coding sequences from various organisms to restore psbA expression (Gimpel and Mayfield, ), without abolishing translation of the gene of interest. The key for achieving this seems to rely on the specific 5′ regulatory elements, because the expression of different heterologous psbA coding sequences resulted in low MAA accumulation (strains TS1 and Syne1Opt).…”

“…All strains were previously described in Gimpel and Mayfield (2013). We preserved the original names for the strains in this paper.…”

“…Fifty milliliter of cultures were spun down by centrifugation and resuspended in exactly the same volume (10 mL) of PBS. Sample processing, quantitation, and Western blots were performed as in Gimpel and Mayfield (2013). Monoclonal rat anti-MAA antibody was obtained from Dr. McDonald laboratory (McDonald et al, 1991).…”

“…In a previous work we have been able to reestablish photosynthesis in a psbA deficient strain by complementing the strain with different combinations of endogenous and heterologous promoters and 5′ UTRs driving different psbA coding sequences (Fig. ) (Gimpel and Mayfield, ). In the present work we show that the constitutive accumulation of the psbA ‐driven MAA transgene can be optimized in an autotrophic strain by selecting the right combination of regulatory regions and psbA coding sequences used to reestablish photosynthesis.…”

Refactoring the Six-Gene Photosystem II Core in the Chloroplast of the Green Algae Chlamydomonas reinhardtii

“…41 The length of the regulatory regions used in our expression vectors and the references reporting their transcription start sites are shown in Supporting Information Table S3.…”