Expression of multiple antihypertensive peptides as a fusion protein in the chloroplast of Chlamydomonas reinhardtii

Hypertension affects about a quarter of the global population and is a major risk factor in cardiovascular diseases. Current pharmacological treatments are considered useful but lead to secondary effects that end in low compliance. In this work, a transplastomic Chlamydomonas reinhardtii strain producing three antihypertensive peptides (AHP) as a fusion protein has been developed. The chimeric protein was coded by a synthetic gene and transferred to the plastome in a site-specific integration approach. Expression was mediated by the rbcL promoter. Molecular analysis confirmed the presence of the transgene inserted in the chloroplast genome. An ELISA quantification assay indicated that the amount of recombinant protein is 34.4 ng per mg of freeze-dried biomass. The RPLKPW and AINPSK peptides were identified by HPLC after in vitro digestion form biomass of the transplastomic C. reinhardtii strain. The antihypertensive effect of the recombinant protein was demonstrated after intragastric administration of the genetically modified strain to spontaneously hypertensive rats (SHR) at a dose of 10 mg of recombinantAHP3 protein per kg of body weight with the maximal decrease in blood pressure 6 h post-administration. These results suggested that this transplastomic strain can be used to obtain a large quantity of antihypertensive peptides which could be useful for the production of functional foods.