Part:BBa_K4937023

TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t

TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t:

Using the OAZ1 knockout strain we previously constructed, we generated yeast expression plasmids to overexpress SPE1 and AtACL5, to achieve overexpression of putrescine (and subsequently spermidine) and thermospermine. We initially obtained the plasmid backbone through PCR (Figure 1: 7-1 to 7-4) and the insert fragments (Figure 1: 7-7 is TEF1p-SPE1-PRM9t-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 SPE1 and AtACL5 produce less putrescine(7.0 mg/L) and spermidine(5.2 mg/L), compared to 8.1 mg/L and 6.7 mg/L in strain contains plasmid that overexpress SPE1 only. 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 SPE1-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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1871
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1607
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 167