Introduction

Anemia affects one third of the world's population, mostly in relation to iron deficiencies. Anemia and similar mineral deficiency diseases are primarily widespread in developing countries like India, partly resulting from the local diet that is mainly made up of cereal grains and seeds such as rice . In these types of food, iron bioavailability is substantially reduced by the presence of phytic acid (C₆H₁₈O₂₄P₆), which chelates minerals and forms insoluble salts which precludes their absorption in the gastrointestinal tract.
Current research on increasing the bioavailability of iron or zinc involves the bioengineering of crop plants which not only poses challenges in terms of the production of efficient genetically modified crops but also requires extensive research for drawing any conclusions on strain sustainability.
We propose an alternative strategy that focuses on the bioengineering of microorganisms involved in the fermentation of idli, a dish widely used as primary food source throughout much of India. Indeed, the lab model organism Saccharomyces cerevisiae is a strain present in the idli microbiome that naturally produces phytases. Phytases are enzymes that are able to hydrolyze phytic acid even when complexed with minerals, resulting in a greater mineral bioavailability.
However the production of phytases in Saccharomyces cerevisiae is down-regulated by two genes: PHO80, present on chromosome 15 and PHO85, found on chromosome 16. The knockout of these genes would likely increase the yield of phytase production and therefore increase the general bioavailability of minerals in fermentation-based dishes such as idli.

We used the yeast Saccharomyces cerevisiae, more precisly the strain SK1, a gift of the INSERM unit U1001. We designed two primers to replace the two phytase synthesis repressor genes PHO80 and PHO85 on chromosomes 15 and 16. The plasmid used here was pSB1C3 with the geneticin resistance gene or the gene of RFP that we take and put between two CRE-lox to replace PHO80 and between two FRT or the other localisation.

After the transformation of our yeast, we attempted to measure whether the level of phytase production was increased. This was done through the use of a quantification colorimetry kit, which measures the OD655 to determine how much phytic acid is found in different growth conditions. To do that, we used a kit to quantify, using colorimetry and OD655, how much phytic acid we can find in different conditions of growth of ours strains (the final one, the pho80 replace, the pho85 replace, the pho80 avoid and the pho85 avoid). The different conditions was initially like the kit told us to use : food sample. Here it was rice, rice after fermentation, and with or without our different strains.

In the table nearby, we can see the typical data that we obtain after usage of the kit. The values are very low in comparison with what we normally have to observe (for rice is egal to 6 mg.g^-1, and here we observe a concentration of 5 µg.g^-1, 10^-3 less).