Part:BBa_K4937018
OAZ1_upstream-OAZ1_downstream
OAZ1_upstream-OAZ1_downstream:
This composite part was created by fusion PCR and used to construct the oaz1Δ strain by CRISPR/Cas9. The results strain has a greater flux of polyamine synthesis, especially putrescine. We investigate whether this part can confer thermo-tolerance to resulted strain.
According to literature reports, the presence of the endogenous OAZ1 gene in S. cerevisiae restricts the synthesis of polyamines[1], thereby limiting the direct precursor spermidine's production. To enhance the metabolic flux of spermidine synthesis in our strain, we initiated the knockout experiment of the endogenous OAZ1 gene. Our experiment consisted of three main phases: fragment construction, yeast transformation, and validation.
We obtained the plasmid backbone through PCR amplification, as shown in Figure 1A, and its size was as expected. Subsequently, we annealed to obtain the gRNA sequence. Following the extraction of yeast genomic DNA, we amplified the upstream and downstream sequences of the repair template by PCR, as depicted in Figure 1B, and their sizes were as expected. Finally, we generated the complete repair template through fusion-PCR, as illustrated in Figure 1C, and its size was as expected.
Figure 1
Following the acquisition of the aforementioned fragments, we proceeded with yeast competent cell preparation and transformation onto Delft agar plates. Subsequently, we randomly selected 10 individual clones from the plates for further expansion culture and genomic DNA extraction for preliminary validation. The length of PCR at the original locus was 1949bp, as shown in the Positive Control (PC) group in Figure 2A. The theoretically expected length after successful knockout should be 1011bp. Gel electrophoresis results indicated that colony 1-5 might be yeast strains with the OAZ1 gene knockout, as shown in Figure 3A. Therefore, we sequenced the PCR products from these colonies, and the results aligned with our expectations (Figure 2B and 2C).
Figure 2
Then we employed 5-FOA for counter-selection to obtain strains that had lost the gRNA editing plasmid.
And we performed HPLC test to ensure that our engineered strains have improved production of putrescine and/or spermidine (Figure 3, 4, 5). As shown in Figure 6, the OAZ1Δ strain produces 5.70 mg/L putrescine and 7.15 mg/L spermidine. Compared to wild type strain produces 1.3 mg/L putrescine and 2.9 mg/L spermidine, the engineered strain has a higher production of polyamines, which means that we successfully increased the flux of polyamine synthesis.
Figure 3
Figure 4
Figure 5
Figure 6
Subsequently, we conducted a thermo-tolerance growth test at 35°C for the aforementioned strains, as shown in Figure 7.
Figure 7
The experimental results indicated that the knockout of the OAZ1 gene did not have a significant impact on the thermo-tolerance of the strains.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 924
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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