Motivation

Vitamin A deficiency is a crucial issue in India, which affects 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.

Design

To produce vitamin A in idli, a popular fermented rice cake, 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 crtE, crtYB and crtI genes were designed in a single polycistronic construct, and synthesized as gBlocks. The gBlocks were then assembled with Gibson Assembly in an integrative vector that replicates in E. Coli, and integrates in the genome of S. cerevisiae at the HO locus. We then added an additional copy of the crtI gene, as well as the HMG-CoA reductase from S. aureus, also in the genome of S. cerevisiae, because both strategies have been reported to increase the vitamin A production. We wanted to produce as much ß-carotene as possible, so all the genes were codon-optimized for S. cerevisiae.

The pathway:

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.

The backbone:

To integrate our construction into the yeast chromosome, we used the HO-Poly-KanMX4-HO plasmid (AddGene plasmid #51662), which is a yeast plasmid for sequence integration into the HO locus, with a selection marker for yeast (KanMX4). This plasmid also has an origin of replication for E. coli and a selection marker for bacteria (Ampicillin).

Design and assembly:

All our genes (crtE, crtYB, crtI and HMG) were codon-optimized for S. cerevisiae using the IDT Codon Optimization tool, then synthesized as gBlocks by IDT. The polycistron, along with the promoter and terminator (6 kb), couldn’t fit into one gBlock, so we cut the whole sequence into 4 parts of ~1.5 kb each, with 30 bp of overlap between each part. We called these gBlocks vA-1, vA-2, vA-3 and vA-4, as they were coding for the pathway of vitamin A production.

IDT wasn’t able to synthesize our first gBlock though, because of the too low GC content and multiple repetitive sequences in the promoter. So we had to cut gBlock vA-1 into two smaller pieces (vA-1.1 and vA-1.2, which had an overlap of 30 bp) and we also had to change the TDH3 promoter to the ADH1 promoter, which is also a strong promoter for yeast. The sequence of the ADH1 promoter was found on AddGene in the plasmid p406ADH1. The GC content was still too low, so we had to introduce two single substitutions in the promoter sequence (250A>G and 256A>G) so that the gBlock could finally be synthesized.

Oligos were designed to amplify each gBlock. The oligos had tails to increase the overlap region between each gBlock. We also designed oligo to linearize and amplify the HO-Poly-KanMX4-HO plasmid (AddGene plasmid #51662), in order to assemble all the gBlocks together in this plasmid with Gibson Assembly. We named the plasmid thus obtained HO-E/YB/I-Poly-KanMX4-HO.

Bibliography

• Li, Q., Sun, Z., Li, J. & Zhang, Y. Enhancing beta-carotene production in Saccharomyces cerevisiae by metabolic engineering. FEMS Microbiology Letters 345, 94-101 (2013).
• Beekwilder, J. et al. Polycistronic expression of a ß-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to ß-ionone production. Journal of Biotechnology (2014).
• Voth, W.P., Richards, J.D., Shaw, J.M. & Stillman, D.J. Yeast vectors for integration at the HO locus. Nucleic acids research 29, E59-E59 (2001).
• Gietz, R.D. & Schiestl, R.H. High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method. Nature Protocols 2, 31-35 (2008).