Team:Paris Bettencourt/Project/Phytase

Ferment It Yourself

iGEM Paris-Bettencourt 2O15

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Phytase

Background	Aims	Results
The yeast Saccharomyces cerevisiae naturally produces phytase.	We engineered Saccharomyces cerevisiae to produce higher amounts of phytase during the fermentation of idli batter to increase iron bioavailability.	Two repressor genes of the phytase biosynthesis pathway in Saccharomyces cerevisiae were successfully deleted to increase phytase production.

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.

Figure 1: Phytic acid in complex with calcium, magnesium, zinc and iron

Figure 2:Phytase hydrolyzes phytic acid.

Experimental design

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.

Results

The different tranformations were successful, as demonstrated through gel electrophoresis and by growth of the yeast on geneticin or by the color of the colony (red cause the RFP), or both. The kit that we used to see the presence or not of phytate by titration was inefficient. For all the usage of this kit on our sample it didn't work at all. It worked however for the test sample give with the kit

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).

Figure 3: Titration of phytic acid for multiple conditions.

Here we can see the histogram of the optic density post treatment with the phytic acid kit. Bigger is the free phosphorus agents and higher is the concentration of phytate in the media. As we observe, the difference between rice alone and with micro-organisms is not very high. Moreover there are no real difference between wild type S. cerevisiae and our S. cerevisiae.

We studied the diffences of growth between the wild typ S. cerevisiae and our three strains without pho80, pho85 or both, on YPD. The growth is not a lot affected by the suppression of this two genes

Bibliography

Veide, J. & Andlid, T. Improved extracellular phytase activity in Saccharomyces cerevisiae by modifications in the PHO system. International Journal of Food Microbiology 108, 60-67 (2006).