Part:BBa_K4761222

adh6-linker3-ARO10

adh6-linker3-ARO10 fusion expression unit for tyrosol production. Designed based on BBa_K4761001 and BBa_K4761000 linked with BBa_K4761032, a flexible linker. Because the reaction catalyzed by yeast-derived pyruvate decarboxylase ARO10 and endogenous alcohol dehydrogenase ADH6 is an adjacent reaction in salidroside metabolism, the adh6 gene and ARO10 gene are linked through the linker designed by 2023 HiZJU-China in attempt to increase its generating efficiency.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2895
  Illegal SpeI site found at 1846
  Illegal PstI site found at 1724
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2895
  Illegal SpeI site found at 1846
  Illegal PstI site found at 1724
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2895
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2895
  Illegal SpeI site found at 1846
  Illegal PstI site found at 1724
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2895
  Illegal SpeI site found at 1846
  Illegal PstI site found at 1724
  Illegal NgoMIV site found at 991
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

In 2023 HiZJU-China, fusion tyrosol generator units are used with UGT85A1(BBa_K4761002), to produce salidroside. We suppose that by fusing the two proteins, the reaction for tyrosol production would get enhanced, thus elevating the salidroside yield.

The link was made using a fusion PCR method (see Guidelines for Fusion Gene and its Primer Design below), in which the stop codon of the adh6 gene was replaced with a linker sequence.

Fig.1 Successful preparation for fusion gene 1 to 5(Lane 1 to 5 respectively)

The successfully constructed fusion expression units are then installed on pET vector(BBa_K4761101), transferred into chassis and went through verification:

Fig.2 Successful verification for fusion gene transformed strains. Lane 1: fusion gene 1; Lane 4: fusion gene 3; Lanes 8 & 10: fusion gene 4; Lane 12: fusion gene 5.

After further sequencing, fusion gene 3 and 4, the representatives for flexible and rigid connection linkers, transformed into chassis to construct fermentation strain. Strains then went under fermentation after inoculation into M9 broth.

Fig.3 Fermentation strain pACYCDuet-UGT85A1+pET-adh6-linker3-ARO10 in a shake flask. 18h after inoculation into M9 broth.

Fermentation results after fusion expression are shown in Table.1 below.

Table.1 Detailed data of HPLC analysis(Fusion express)

After comparison, we could conclude that the flexible connector Linker3 can indeed increase the yield of salidroside, which is speculated to be the reason of optimizing the metabolic reaction. The comparison with Linker4 shows that flexible joints show better optimization results than rigid joints.

Guideline for Fusion Gene and its Primer Design

To fuse two desired genes together with a linker, you need to delete the end codon of the first protein coding gene, and then install the linker sequence. Fusion expression genes are synthesized by fusion PCR. To perform fusion PCR, the four types of primers are needed. Give adh6-linker-ARO10 fusion expression for example as shown in Fig.4:

Fig.4 Primers for using adh6-linker-ARO10 fusion gene synthesis. For its detailed sequence, see https://2023.igem.wiki/hizju-china/primer.

P1: The forward primer for adh6. Contains the sequence at the left end of adh6 with an appropriate Tm.
P2: The reverse primer for adh6. Contains not only the sequence at the right end(end codon deleted of course), but also the linker sequence right after it.
P3: The forward primer for ARO10. Contains not only the sequence of the left end, but also the linker sequence before it.
P4: The reverse primer for ARO10. Contains the sequence at the right end of ARO10.

Fig.5 Process of fusion PCR[1].

Fusion PCR consists of two rows of PCR. At the first row, two reactions are made (preparation of adh6-linker and linker-ARO10) using primers P1-P2, P3-P4 respectively. Then the second row is performed with the products of two reactions as the template using primers only P1 and P4. The product would have both the adh6 and ARO10 sequence, and with linker in the middle.

References

[1]Cha-aim, K., Hoshida, H., Fukunaga, T., Akada, R. (2012). Fusion PCR via Novel Overlap Sequences. In: Peccoud, J. (eds) Gene Synthesis. Methods in Molecular Biology, vol 852. Humana Press. https://doi.org/10.1007/978-1-61779-564-0_8