Motivation

Saccharomyces cerevisiae is a yeast commonly found in idli batter (Soni and Sandhu, 1989 and Nout, 2009). Though it 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: