Part:BBa_K4937022

TDH3p-AtACL5-DIT1t

TDH3p-AtACL5-DIT1t:

This composite part was created by fusion PCR and used to exogenously express AtACL5 gene and results in the overexpression of thermospermine. This part was used in the oaz1Δ strain, in which the polyamine synthesis flux is stronger. We investigate whether this part can confer thermo-tolerance to resulted strain.

Using the OAZ1 knockout strain we previously constructed, we generated yeast expression plasmids to overexpress AtACL5, to achieve overexpression of thermospermine. We initially obtained the plasmid backbone through PCR (Figure 1: 7-1 to 7-4) and the insert fragments (Figure 1: 7-6 is TDH3p-AtACL5-DIT1t). Subsequently, we used homologous recombination to construct the complete plasmids and validated their successful construction through sequencing (Figure 2).

Figure 1

Figure 2

And we performed HPLC test to ensure that our engineered strains have improved production of putrescine, spermidine (Figure 3,4,5) and thermospermine. As shown in figure 6, the strain contains plasmid that overexpress AtACL5 produces 5.8 mg/L putrescine and 6.15 mg/L spermidine, respectively, compared to 5.00 mg/L and 6.7 mg/L in strain contains plasmid vector. As we didn’t get the standard of thermospermine, we didn’t get a directly evidence of thermospermine prodution. However, according to the decreased spermidine, we think it may indicated the improvement production of thermospermine.

Figure 3

Figure 4

Figure 5

Figure 6

Then we tested the growth of these strains under 35°C conditions (Figure 7). The results indicate that the expression of the AtACL5 gene did not have a significant impact on the yeast's thermo-tolerance capabilities.

Figure 7

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]