Difference between revisions of "Part:BBa K4937019"

Latest revision as of 11:57, 3 October 2023

OAZ1_upstream-TEF1p-SPE1-PRM9t-OAZ1_downstream

OAZ1_upstream-TEF1p-SPE1-PRM9t-OAZ1_downstream:

This composite part was created by fusion PCR and used to construct the oaz1Δ::spe1 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.

SPE1 encodes an ornithine decarboxylase, catalyzing the decarboxylation of ornithine to form putrescine, and its activity is inhibited by the antizyme enzyme OAZ1. To enhance putrescine expression and consequently increase the supply of spermidine precursors, we constructed yeast strains with OAZ1 knockout and SPE1 knock-in for putrescine (and subsequently spermidine) overexpression. Our experiment consisted of three main phases: fragment construction, yeast transformation, and validation.

After extracting yeast genomic DNA, we amplified the upstream and downstream sequences of the repair template by PCR, as well as the TEF1p, PRM9t (Figure 1A) and SPE1 (Figure 1B) fragments, and their sizes were as expected. Subsequently, we successfully prepared the repair template using a two-round fusion-PCR strategy. In the first round, we fused the first two fragments (upstream-2 and TEF1p) and the last two fragments (PRM9t and downstream-2), as shown in Figure 1C. In the second round, we fused three fragments (upstream-2-TEF1p, SPE1, and PRM9t-downstream-2), as shown in Figure 1D.

Figure 1

Then we tried to transform the fragments and validate the colonies. The length at the original locus was 1949bp, as shown in the Positive Control (PC) group in Figure 5A. The theoretically expected length after successful editing should be 3080bp. Gel electrophoresis results indicated that Colony2-1, Colony2-3, Colony2-4, and Colony2-7 might be successfully edited strains (Figure 2A). Therefore, we selected the PCR product from Colony2-1 for sequencing, and the results matched our expectations (Figure 2B).

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. As shown in Figure 6, the OAZ1::SPE1 strain produces 9.5 mg/L putrescine and 15.2 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 and the overexpression of SPE1 did not have a significant impact on the thermo-tolerance of the strains.

Sequence and Features