Team:SCUT-ChinaA/Experiments
Construction
- 1. Add overlap region to adjacent DNA fragments via PCR;
- 2. Combine the to-be-assembled DNA to a total volume of 5 μl;
- 3. On ice, add 15μl Master Mix to the DNA, mix well and briefly centrifuge;
- 4. Incubate at 50℃ for 1 hour;
- 5. Store reactions at -20℃ or proceed to transformation.
1. Design
In our project, for obtaining various engineered strains, we applied module assembly technology like gibson assembly and overlap extension PCR to construct gene expression modules in E. coli. In the process of pathway construction, we successfully constructed pUC19-NDPS1-LS, pUC19-Catcher-NDPS1-Tag-LS, pUC19-tHMG1, pUC19-ERG8-ERG12-ERG19, pCAS1yl-ku70, pCAS1yl-pox5, respectively. The upstream and downstream regions are homologous to the target integration site and prepared for homologous recombination in further gene editing. The LEU2 and URA3 was used for selection in Y. lipolytica and the AmpR was used in E.coli. NotI restriction sites was added between the homology regions. These plasmids will be further linearized in the subsequent steps.
2.Method
Gibson assembly
The method can simultaneously combine up to 15 DNA fragments based on sequence identity. It requires that the DNA fragments contain ~20-40 base pair overlap with adjacent DNA fragments. These DNA fragments are mixed with a cocktail of three enzymes, along with other buffer components. The three required enzyme activities are: exonuclease, DNA polymerase, and DNA ligase. The exonuclease chews back DNA from the 5' end, thus not inhibiting polymerase activity and allowing the reaction to occur in one single process. The resulting single-stranded regions on adjacent DNA fragments can anneal. The DNA polymerase incorporates nucleotides to fill in any gaps. The DNA ligase covalently joins the DNA of adjacent segments, thereby removing any nicks in the DNA.
The detailed protocol is as follows:
. CnaB2 contains an internal isopeptide bond between amino acid residue Lys31 and residue Asp117. CnaB2 can be split into two fragments: An N-terminal protein fragment containing Lys31, named SpyCatcher and a C-terminal peptide containing Asp117, named SpyTag. Reaction occurred in high yield simply in diverse conditions of pH, temperature, and buffer. As the isopeptide bond is covalent, the SpyTag/SpyCatcher complex forms irreversibly and has great stability. The SpyTag can be placed at the N-terminus, at the C-terminus and at internal positions of a protein. Thus, the SpyTag/SpyCatcher system has versatile potential and is suitable for generally use. In our pathway, for the IPP accumulation caused by overexpression, applying the SpyTag/SpyCatcher system to attach NDPS1 and LS together, we developed a high-performance enzyme self-assembling system (HESS) to pull the metabolic flux to NPP and limonene instead of GPP.
</p>
<h3>3. CRISPR/Cas9</h3>
<p>
Clustered regularly interspaced short palindromic repeats (CRISPR) is an RNA guided adaptive immunological mechanism in bacteria and archaea during its long-term evolution. CRISPR functions in fighting against the invasive virus and foreign DNA. The class 2 type II DNA-targeting CRISPR/Cas9 system has been developed as a powerful genome-editing tool for its high specificity basing on the nucleic acid sequence.
According to its principle, the mechanism of CRISPR/Cas9 system is divided into three distinct stages: adaptation, expression and interference. During the adaptation stage, the complex forming by Cas1 and Cas2 nuclease recognizes foreign target element and integrates it into the CRISPR array. In the following expression stage, transactivating CRISPR RNA (tracrRNA) and CRISPR RNAs (crRNAs) is processed and matured. In addition, the Cas9 gene is expressed. The dual crRNA-tracrRNA can be fused into a chimeric single-guide RNA (sgRNA), and a two-component system composed of Cas9 and its sgRNA is created. Cas9 effector nuclease assembles with sgRNA forming the crRNA-effector complex. Finally, in the stage termed interference, through the sequence-targeting binding of crRNA, the RuvC domain and HNH domain of Cas9 endonuclease cut the top and bottom strand of the invading elements at the target site. The double stranded break (DSB) generating by the cleavage would be repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ).
CRISPR/Cas9 system also has a lot of new developments in <em>Y. lipolytica</em>. In a new research, Cory M. Schwartz et al modified the CRISPR/Cas9 system to develop a highly efficient pCRISPRyl system, and the modified system was successfully used to perform gene knockout and knockin in <em>Y. lipolytica</em>. Professor Sheng Yang, from Institute of Plant Physiology and Ecology of the Chinese Academy of Science, also developed the CRISPR/Cas9 gene editing system for Yarrowia lipolytica and applied it to the transformation of carotene synthesis.
</p>
<p>
NHEJ preforms the repair for DSB without the need for a homologous template. Ku heterodimer does the recognition and binding of the DSB during the NHEJ repairing. Ku heterodimer recruits multiple subunits, including KU70 and KU80. HR replies on homology based repair process to repair DSB with a homologous template. However, the efficiency of NHEJ to repair DSB is very high, which has been widely mentioned in previous studies. From the mechanism, obviously, the deletion of ku70/ku80 can improve the efficiency of homologous recombination and create conditions for subsequent gene editing in <em>Y. lipolytica</em>. Based on strains with deletion of ku70/ku80, we used the CRISPR/Cas9 system to introduce exogenous and endogenous fragments into specific sites in the yeast genome.
</p>
<div class=)
Figure 2: Cartoon of SpyTag/SpyCatcher system. Reactive residues are highlighted in red.
Design
1. Pathway Construction
As mentioned before, to construct a complete limonene synthesis pathway, NDPS1and LS are supposed to be introduced into Y. lipolytica. Here we optimized two key synthetase genes (NDPS1 and LS) and integrated them into the the chromosome of Y. lipolytica Po1f to obtain the engineered strain Po1f/lim. The gene encoding d-limonene synthase (LS, GenBank ID: AY055214.1) from Agastache rugosa and gene encoding neryl diphosphate synthase 1 (NDPS1, GenBank ID: NM_001247704.1) from Solanum lycopersicum were codon-optimized and synthesized by Sangon Biotech. Using gibson assembly technology, the NDPS1 and LS gene were cloned into pUC19, to construct the gene expression cassettes (P-NDPS1-T, P-LS-T), respectively. Based on the plasmid pUC19-rDNAu-HUH-rDNAd (which represents the plasmid pUC19-rDNAupstream-HisG-URA3-HisG-rDNAdownstream), the expression cassettes were cloned into it to construct a co-expression module, as a donor DNA vector.
Meanwhile, in order to obtain a high efficient biological chassis for HR, we first constructed pCAS1yl-ku70 plasmid targeting ku70 gene in Y. lipolytica. And then we introduced the plasmids, pCAS1yl-ku70upstream-HUH-ku70downstream into Y. lipolytica, thereby knocking the ku70 gene out, improving the efficiency of homologous recombination repairing. Based on the ku70 deletion strain, we introduced the co-expression module with the plasmid pCAS1yl-rDNA, so the module would be integrated into the rDNA site of the chromosome by CRISPR/Cas9 system, to get a engineered strain named Po1f/lim. The same method would be applied in the subsequent experiments.
Figure 3: the pathway design of limonene synthesis. The yellow arrow represents the rate-limiting enzymes (tHMG1 and ERG12). The orange arrow represents the enzymes (NDPS1 and LS) heterologously expressed in Y. lipolytica.
2. Overexpression
Enhancing the gene copy number of the enzyme on the synthetic pathway, might lead to improvements of the limonene production. For further optimizing the flow of metabolic flux, we decided to overexpress the rate limiting enzymes involved in the limonene synthesis pathway, including tHMG1, ERG8, ERG12 and ERG19. The tHMG1 gene was cloned from the chromosome of the Y. lipolytica, while the ERG8, ERG12 and ERG19 with their terminators were cloned from the genome. Moreover, to increase the application scope of overexpression modules, we assembled the expression cassettes of ERG8/12/19 together for the overexpression of other metabolic pathway. Homologous tHMG1 gene from Y. lipolytica was overexpressed under the control of GPD2 promoter in the limonene-producing strain Po1f/lim. ERG8/12/19 was fused with EXP1, TEF1 and GPD2 promotor, respectively. Then the tHMG1 expression cassettes fused with ku80upstream/downstream was transformed into Po1f/lim with the plasmid pCAS1yl-ku80, simultaneously achieve the purpose of knocking out ku80 gene and introducing tHMG1 gene. The ERG8/12/19 module was integrated into the pox5 site of the genome synchronously, obtaining engineered strain named Po1f/lim-tE.
3. Scaffold Application
The SpyTag/SpyCatcher system has a wide range of applications in enhancing the expression of metabolic pathways and rerouting pathways. We applied SpyTag/SpyCatcher tagging system to develop a high-performance enzyme self-assembling system (HESS) to pull the metabolic flux to NPP and limonene instead of GPP. Considering the size of the proteins structure, we attached SpyCatcher to NDPS1 and SpyTag to LS. On the other hand, we fused SpyCatcher to the C terminal of NDPS1 and SpyTag to the N terminal of LS for ensuring the catalytic activity of the enzymes. So, based on the co-expression module, which contents the expression cassettes of NDPS1 and LS, we developed the HESS to enhance the flow of metabolic pathway. The SpyCatcher gene was cloned into the module between the NDPS1 and XPR terminator, while the SpyTag gene was cloned into the module between the EXP promoter and LS gene. The HESS module would be integrated into the rDNA site of the chromosome in the Po1f/lim-tE to get the strain Po1f/lim-tESS for fermentation.
Reference