Expression of a Synthetic Gene for the Major Cytotoxin (Cyt1Aa) of Bacillus thuringiensis subsp. israelensis in the Chloroplast of Wild-Type Chlamydomonas

Abstract

Chlamydomonas reinhardtii (Chlamydomonas) strains that are toxic to mosquito larvae because they express chloroplast transgenes that are based on the mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis (Bti) could be very useful in mosquito control. Chlamydomonas has several advantages for this approach, including genetic controls not generally available with industrial algae. The Bti toxin is produced by sporulating bacteria and has been used for mosquito control for >30 years without creating highly resistant mosquito populations. The suite of toxins is four main proteins: three Cry proteins and the cytotoxic Cyt1Aa (27 kDa). Cyt1Aa is not very toxic to mosquitoes by itself, but it prevents the development of resistance. The production of Cyt1Aa in other microbes, however, has been challenging due to its affinity for certain membrane phospholipids. Here we report on the production of recombinant Cyt1Aa (rCyt1A) in the chloroplast of photosynthetic Chlamydomonas at levels of at least 0.3% total protein. Live cell bioassays demonstrated toxicity of the rCyt1Aa Chlamydomonas to larvae of Aedes aegypti. We also expressed the chloroplast cyt1Aa gene in a wild-type Chlamydomonas strain (21 gr) that can grow on nitrate. These results have implications for developing a Chlamydomonas strain that will be toxic to mosquito larvae but will not induce strongly resistant populations.

Overcoming Poor Transgene Expression in the Wild-Type Chlamydomonas Chloroplast: Creation of Highly Mosquitocidal Strains of Chlamydomonas reinhardtii

“…With the latter strain under the inducing condition, rCry11A is 0.35-0.4% of total protein [12] and this is our benchmark strain. These results indicate that the cry11A coding region is recalcitrant to expression in a wild-type setting, because the cyt1A coding region, also from the Bti toxin [13], was highly expressed in wild-type using these same 5 and 3 elements (i.e., psbD m and psbA, respectively) [29].…”

“…We chose the unique BamH1 site in the intergenic region between psbA and 5S as the site for insertion of the cry11A transgene, but first a second BamHI site in the MCS of p322 was removed as previously described to give p322.1 [12]. Next, the recyclable selectable marker 483-atpA-aadA-rbcL-483 [28] was inserted into p322 as previously described [29]; this allows selection of transformants by spectinomycin resistance. For insertion of the cry11A transgene into the BamHI site of p322.1, they were excised from the pET-16b constructs by cutting with BamHI, purified by GTG agarose gel electrophoresis, and then ligated into BamHI-cut, Antarctic phosphatase-treated p322.1 (containing the 483-atpA-aadA-rbcL-483 marker) with T4 DNA ligase.…”

“…Transformants were grown in TAP medium (50 mL) to early stationary phase (5-7 × 10 6 cells/mL) and then for another 24 h before harvesting (cell density was rechecked just before harvesting but had increased <10% from the day before). Total protein was calculated from total chlorophyll measurements using the approximate conversion, 1 µg chlorophyll = 10 µg total protein, which we had previously determined for these strains using the Bradford assay for protein determination [12,29]. Cells were then pelleted at room temperature for 5 min at 2600× g and resuspended in 0.6 mL of 100 mM Tris-HCl pH 8.5, 100 mM DTT, 7 mM benzamidine, 5 mM EDTA (Resuspension Buffer or RB), and then transferred to 1.7 mL microfuge tubes.…”

Selectable Markers and Reporter Genes for Engineering the Chloroplast of Chlamydomonas reinhardtii

“…Chloroplast engineering has also been used to produce other therapeutically-valuable proteins, such as antibodies [9], a class of antibody–drug conjugates known as immunotoxins that have promise in cancer treatment [10], and recombinant allergens for the treatment of peanut and birch pollen allergies [19,20]. Furthermore, recent research has shown the viability of expressing a toxin known to kill mosquito larvae in the C. reinhardtii chloroplast, an anti-mosquito strategy that could prove particularly useful due to the cohabitation of C. reinhardtii and mosquito larvae in certain habitats [21]. Additionally, microalgae are being explored as industrial biotechnology platforms for the production of metabolites that have applications ranging from biofuels to bioactives [22].…”

CITRIC: cold-inducible translational readthrough in the chloroplast of Chlamydomonas reinhardtii using a novel temperature-sensitive transfer RNA

“…A wide range of proteins have been expressed in the C. reinhardtii chloroplast, as recently reviewed [ 4 , 6 – 8 ]. These include complex multi-domain immunotoxins for cancer therapy [ 9 ], oral vaccine candidates for aquaculture [ 10 ], mosquitocidal proteins [ 11 ] and a cellulose-hydrolyzing enzyme [ 12 ]. This expression platform is capable of producing proteins with disulphide bonds or phosphorylation [ 13 ].…”