Motivation

Vitamin A deficiency is a crucial issue in India, affecting millions of people.
+ numbers and consequences of deficiency
The government developed different programs to provide people with vitamin A supplements, but they are not very convenient (people need to go to a center everyday to receive it), only helps a small portion of the population, and the retinol present in the supplements is not as healthy as the ß-carotene found in food. Another solution which has been proposed is Golden Rice, a rice that have been genetically engineered to synthesize vitamin A. However, the Golden Rice is the subject of many controversies, and has not been implemented in India.
Our idea is to have the vitamin A produced by the microbiome of fermented foods, and not by the cereal itself. It is much more easier, cheaper and faster to genetically engineer micro-organisms than plants. And for the consumer, it is much less intrusive and constraining to have a starter of yeast and bacteria which they can chose to add or not in their food at anytime, than to have to change their entire crops as proposed by the Golden Rice project.

Design

To produce vitamin A in idli, a popular indian rice cake that is fermented, we chose to use the yeast Saccharomyces cerevisiae since it is commonly found in idli batter (Soni and Sandhu, 1989 and Nout, 2009). So it has a better chance to grow well and not affect the taste of idli than a yeast that isn’t normally present in the batter. Though S. cerevisiae doesn’t naturally produces ß-carotene, it has been shown that with the introduction of two carotenogenic genes from the carotenoid-producing ascomycete Xanthophyllomyces dendrorhous, S. cerevisiae could synthesize ß-carotene (Verwaal et al., 2007). These two genes are crtYB which codes for phytoene synthase and lycopene cyclase, and crtI, which encodes phytoene desaturase.

Additional overexpression of crtE (GGPP synthase) from X. dendrorhous, and an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) from S. cerevisiae were both reported to increase the carotenoid production levels in S. cerevisiae (Verwaal et al., 2007). A more recent study also showed that ß-carotene synthesis in this yeast could also be increased with codon-optimization of crtI and crtYB, and by introducing the HMG-CoA reductase (mva) from Staphyloccocus aureus rather than the truncated HMG-CoA reductase (tHMG1) from S. cerevisiae (Li, 2013).

The polycistron

In 2014, Beekwilder & als. assembled the crtE, crtYB and crtI genes into a polycistronic construct where the individual Crt proteins were separated by the T2A sequences of the Thosea asigna virus.
They were able to show that the addition of those genes to Saccharomyces cerevisiae was enough to make it produce beta-carotene.

2A sequences or cis-acting hydrolase element:
2A like sequences are able to force the ribosome to "skip" a codon. The ribosome releases the part that it has already translated and to keep translating the mRNA. It allows transcription of multiple proteins from only 1 mRNA with 1 promoter, like bacterial polycistronic elements, but also with only one kozac sequence (yeast RBS) which ensure that the quantities of all the translated product are the same.

The construct we designed is very similar to theirs, except that we moved the crtE gene to the first place of the polycistron, in order to increase the carotenoid yield. Indeed, it has been shown that the efficiency of translation decreases after every 2A sequence (de Felipe et al. 2006), and that an increase of CrtE may improve the ß-carotene production (Verwaal et al. 2007). We kept the same 2a sequences between the cistrons, as well as the same strong promoter TDH3 and the same terminator TEF1.