Team:SCUT-ChinaA/Experiments

Construction

    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:

    • 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.

Integration

    1.Design

    After the successfully construction of various expression modules, we linearized them with NotI and then were transformed into competent Po1f cells together, using the kit, Frozen-EZ yeast transformation II. After transformation, cells were cultured on SD medium plates without uracil or leucine. The URA3 marker was integrated into the cells along with the expression cassettes, which would affect the insertion of subsequent gene expression modules. Therefore, YPD media supplemented with 1 mg/mL 5-FOA was used for URA3 marker plasmid removal. Screening for integration was accomplished using colony PCR of single colonies. Furthermore, we would further verify it by extracting the genome of the yeast, using the kit from TIANGEN Biotech (Beijing, China). At the transcriptional level, we verified the transcription of the integrated gene modules by quantitative PCR with a kit from XXXXX.

    2. Method

    Transformation

    First, prepare the yeast competent cells. Grow yeast cells at 30℃ in 10ml YPD broth until mid-log phase (OD600 of 0.8-1.0). The following steps are accomplished at room temperature.

    • 1. Pellet the cells at 500 x g for 4 minutes and discard the supernatant.
    • 2. Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.
    • 3. Add 1 ml EZ 2 solution to resuspend the pellet.
    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