The main purpose of our project is to develop a farmer-kindly Saccharomyces cerevisiae for LSC prevention, so our design consists of four part. Learn more.
1. Main part: Four genes of bcABA family from Botrytis cinerea were transformed into S. cerevisiae for ABA biosynthesis.
2. Main part: The efficiency of 2A sequence in S. cerevisiae was verified, and the mRNA cleavage efficiency of CRISPR-Csy4 system in eukaryotes was preliminarily explored.
3. Future work to expand the function of our S. cerevisiae strain: Enrich the nutrients to the dough by introduced PA synthesis pathway.
4. Future work as a exploration: Explore another strategy based on CRISPR-Csy4 as a polycistronic gene expression system to overcome the shortage of 2A polypeptide.
1. The bcABA genes was introduced into Saccharomyces cerevisiae to achieve ABA biosynthesis
Botrytis cinerea is a plant pathogenic fungus, which can infest plant fruits with the help of ABA synthesis. Previous studies have identified the bcABA gene family in the genome of B. cinerea and confirmed that the four enzymes expressed could achieve fungal biosynthesis from FPP to ABA [1]. Therefore, we would like to introduce this pathway into S. cerevisiae. It has been shown that FPP can be used as a substrate for exogenous product biosynthesis to achieve biosynthesis of carotenoids[2].
Fig 1. bcABA gene family enables biosynthesis from FPP to ABA
Because of the natural signle cistron expression system in eukaryotes, the transformation of four genes in S. cerevisiae requires high-cost ORFs cascade construction in one plasmid Due to COVID-19, we started the experiment in August. This process may prevent us from completing the experimental verification. Therefore, we hope to express 4 bcABA genes on a Y26 plasmid vector using the T2A sequence from the virus. ABA modeling also provides guidance for the plasmid building part of this idea.
Learn more.
Fig 2. Plasmid construction method for achieving co-expression of four bcABA family genes on a Y26 plasmid
2. Construction of polycistronic expression system of exogenous genes in eukaryotes
It is well known that eukaryotes and prokaryotes have distinct gene expression systems, and polycistronic expression system is naturally in prokaryotes. Although eukaryotes do not exist this structure, they have complex post-transcriptional and post-translational process and modification capabilities[3]. These two expression systems give eukaryotes and prokaryotes different advantages in biosynthesis. For eukaryotic systems, biosynthesis of most natural products is advantageous. On the one hand, since most natural products are derived from eukaryotes, the expression system of eukaryotes can ensure the correct processing of target proteins. On the other hand, unlike de novo biosynthesis in prokaryotes, the intermediate products of some biochemical reactions in eukaryotes can be directly used as the reaction substrate of the synthesis pathway, thus greatly reducing the number of exogenous genes required for exogenous production biosynthesis. For example, we can use Saccharomyces cerevisiae to achieve the biosynthesis of important natural products, such as artemisinic acid, geranyl acetate, and trichodermin, by using intermediate or final products of the MVA pathway as a substrate in yeast[4,5,6].
At the same time, the natural monocistronic gene expression system of eukaryotes has serveral drawbacks. For example, when introducing complex pathway, it required multi-step plasmid construction and transforming verification, which causes complex experimental process. In addition, the current understanding of eukaryotes' promoters is not as thorough as that prokaryotic ones, so the construction of multigene vectors under multiple promoters makes the system poorly predictable [7]. Therefore, we hope to design a method for the construction of eukaryotic polycistronic gene expression system, which can make full use of the eukaryotic chassis, simplify the expression system, and improve its predictability.
Fig 3. Comparison of monocistronic and polycistronic plasmid construction
Currently, researchers have used 2A systems from viruses to achieve polycistronic gene expression in eukaryotes, and some 2A sequences with high cleavage efficiency have been found [8]. However, our modeling and experiment results indicate that the use of 2A systems has the following problems for the construction of eukaryotic polycistronic gene expression systems:
(i) 2A peptide residual at the C-terminus of upstream proteins may result in the impact of their structure and function.
(ii) Downstream protein expression efficiency of 2A sequences is lower than upstream, making it difficult to perform two or more 2A sequences.
(iii) The 2A system has a probability of leading to fusions of upstream and downstream proteins, and the 2A sequence itself is not a qualified protein linker.
(iv) The mechanism of multiple protein expression in a single ORF in 2A system is not fully explored.
Therefore, we think that there are still some difficult problems in the 2A system, which makes it difficult to realize the expression of polycistrons at the translation level of eukaryotes. For this, we designed a set of CRISPR-Csy4 system named “RNAlpha” based on Csy4's ability to effectively recognize and cleave the structure of ssRNA CRISPR hairpins. Which multiple genes are linked using the CRISPR sequence to simultaneously present multiple genes on a single mRNA. Csy4 protein was then expressed in S. cerevisiae and transported into the nucleus to make Csy4 cleave CRISPR hairpins on mRNA to achieve the co-expression of polycistrons in an ORF. In this way, polycistrons can be co-expressed in an ORF with only one pair of promoter and terminator in S. cerevisiae. At present, studies have shown that Csy4 can achieve gRNAs cleavage in the nucleus of S. cerevisiae [9]. However, we found that this plasmid construction method would cause the loss of 5 'cap in downstream mRNAs due to Csy4 cleavage, thus unable to translate. Therefore, we have explored the solution to this problem and discussed it later (see details 4).
Fig 4. Construction of the Csy4-CRISPR system
3. Saccharomyces cerevisiae synthesis of PA (ABA as substrate)
Through analyzing the results of the questionnaire, we know that the population has great acceptance of genetically modified (GM) yeast, but has extra demands on it at the same time, such as containing certain nutrients. GM yeast that synthesize only ABA has great seasonal and regional use restrictions and clearly does not cater to these needs. Therefore, we hope to add a synthetic pathway of phaseic acid to the project in order to give our GM yeast a better marketability.
Fig 5. Phaseic acid[10]
Phaseic acid (PA) is a metabolite of ABA. Among plants, PA acts as a phytohormone to regulate the stress response [11]. It has potential therapeutic effects on chronic diseases, diabetes and stroke. In addition, PA can be used as sweetener and is one of the main components of maple syrup [10,12,13]. In plants, ABA can be oxidated to 8'-OH-ABA by ABA 8'-hydroxylase. 8′-OH-ABA is unstable and can spontaneously isomerize to PA [14]. Therefore, we considered the conversion from ABA to PA by expressing ABA 8'-hydroxylase after ABA synthesis to enhance the value in use of our GM yeast during the un-LSC period.
Fig 6. Expression of ABA 8'-hydroxylase through an inducible promoter
4. The solution of 5 'Cap loss of downstream mRNA after cutting by CRISPR-Csy4 system in Saccharomyces cerevisiae
It is known that eukaryotic mRNA without a 5 'cap would be difficult to translate into protein. However, the first step to mRNA capping in S. cerevisiae is at the same time with transcription, and therefore 5 'cap cannot be obtained through endogenous way after Csy4 cleavage [15]. Hence, we hope to explore some methods of eukaryotic "mRNA secondary capping" and provide some ideas for future iGEMers and researchers.
Currently, the mechanism of mRNA capping in eukaryotes has been clearly studied (including CEG1, CET1, ABD1, et al.), and the structure and function of those enzymes have been analyzed [16,17]. Therefore, it is worthwhile to add a modular target sequence at the N-terminal of downstream uncapped mRNA and target a dCas13b-Capping enzyme fusion protein to this location for a secondary capping in the cytoplasm. Meanwhile, dCas13b can also be fused with some translation initiation factors, such as eIF4E, for directly starting translation without 5'cap [18].
(Some researchers also found that the D1-D12 complex enzyme of vaccinia virus could directly realize extracellular mRNA 5 'capping. The characterization and functional verification of this complex enzyme in the cytoplasm of S. cerevisiae was also valuable [19,20,21].
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