1. We successfully completed the biosynthesis of exogenous products in eukaryotes under polycistronic gene expression system, and achieve the ABA synthesis using 2A system in Saccharomyces cerevisiae. In addition, we explored the new application of CRISPR-Csy4 system in the construction of eukaryotic polycistronic gene expression system, and verified that Csy4 can efficiently cleave CRISPR hairpin on mRNA in Saccharomyces cerevisiae nucleus. The end users of this part of project are biologists and future iGEMers. We hope this idea will help them further explore this field.
There are two main challenges: On the one hand, Csy4's CRISPR cleavage of eukaryotic mRNAs will lead to the loss of 5’Cap of downstream mRNAs; On the other hand, the upstream protein expression efficiency in the 2A system is higher than that in the downstream protein, but the residual peptide of the 2A sequence in the C-terminal of the upstream protein will affect its structure and function, which makes it difficult to efficiently express the rate-limiting enzyme in the enzymatic reaction.
For the first challenge, we hope to to construct a secondary 5’capping system in the cytoplasm of Saccharomyces cerescenae, or consider using dCas13b-Capping enzyme fusion protein to achieve the direct capping after cleavage in the nucleus.
For the second challenge, we used the protein structure model to predict the impact of residual peptides of 10 efficient 2A sequence on the upstream protein structure and function, and uploaded improvement Parts, hoping to be useful for future iGEMers.
2. We use Saccharomyces cerevisiae in ABA biosynthesis, mainly hoping that farmers can save our engineered yeast in the form of sour dough, which would be used in the production of ABA before the LSC. In terms of biosecurity, ABA is non-toxic to humans, while Saccharomyces cerevisiae is a strain with high nutrient requirements and extremely weak competitiveness in natural soil. But in order to avoid genetic contamination, we should also consider introducing a suicide system downstream of the inducible promoter into S. cerevisiae later in the project. For example, pGAL1 is a promoter induced by lactose, and milk contains natural lactose. Therefore, it is a good idea for farmers to add lactose themselves and start the yeast to commit suicide.
We still facing two main challenges: On the one hand, dough fermentation overnight using our engineered yeast might lead to a lack of ABA content through water elution; On the other hand, laboratory yeast strains are less efficient than market ones during fermenting (e.g. Angel yeast) and less competitive when LSC doesn't happen.
For the first challenge, we used HPLC to determine ABA synthesis efficiency under the laboratory conditions, showing that the product concentration is higher than spraying concentration required. At the same time, we can also consider to use "full version" CRISPR - Csy4 system in the future, which has more advantages than 2A system. Knocking out endogenous metabolic pathways downstream of FPP and raising FPP upstream gene expression are also great ideas in order to increase yields of ABA.
For the second challenge, we hope to transfer the phaseic acid (PA) biosynthesis pathway into our engineered yeast (using ABA as a substrate, induced express under pGAL1), giving it certain value when LSC doesn't happen and improving market competitiveness. This idea is consistent with the results of our questionnaire HP. In addition, knockout or up-regulation of some genes related to respiration can also be considered to improve fermentation efficiency.