Part:BBa_K3909015

pYLXP-ylPOX4

A composite part consist of pTEF promoter (BBa_K3909008 ), ylPOX4 (BBa_K3909003) and XPR2_terminator (BBa_K3909009).

Our goal is to improve the cell growth of Y. lipolytica and convert a large amount of gutter oil to γ-linolenic acid when Y. lipolytica grows with gutter oil as the sole carbon source . We plan to enhance the oli degradation pathway by expressing three endogenous fatty acid degradation genes ylMEF1 (BBa_K3909006), ylPOT1 (BBa_K3909007), and ylPOXn (from BBa_K3909000 to BBa_K3909005), which are related to the metabolim of transforming acyl-CoA into acetyl-CoA in peroxisome (β-oxidation). Specifically, the β-oxidation includes three steps: i) oxidation, that catalyzed by six acyl-CoA oxidases (translated from ylPOX1 to ylPOX6); ii) hydration and dehydration, that catalyzed by multifunctional enzyme (translated from ylMFE1); and iii) thiolysis, that catalyzed by 3-ketoacyl-CoA thiolase (translated from ylPOT1)[1].

Firstly,we constructed the single gene-overexpressed plasmids by the aforementioned method, including pYLXP’-ylPOT1(BBa_K3909010), pYLXP’-ylMEF1 (BBa_K3909011) and pYLXP’-ylPOXn (from BBa_K3909012 to BBa_K3909017) , which are shown in Figure 1.

Fig.1 Single fragment plasmid construction.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 726
  Illegal BamHI site found at 1322
  Illegal BamHI site found at 2201
  Illegal XhoI site found at 164
  Illegal XhoI site found at 2238
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1759
  Illegal NgoMIV site found at 1927
  Illegal NgoMIV site found at 2092
  Illegal AgeI site found at 2754
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2
  

1. PCR amplification of genes

Firstly, we amplified the sequences of genes ylPOT1, ylMFE1, ylPOX1, ylPOX2, ylPOX3, ylPOX4, ylPOX5 , and ylPOX6 with using the Y. lipolytica as the template by PCR method. The result of genes amplification has been showed in Figure 2.

Fig.2 Amplifying the fragments of genes ylPOX1 (a), ylPOX2 (b), ylPOX3 (c), ylPOX4 (d), ylPOX5 (e), ylPOX6 (f), ylPOT1 (g), and ylMFE1 (h).

2. Linearizing plasmid pYLXP’

The YaliBrick plasmid pYLXP’ was used as the expression vector in this project. Plasmid constructions were performed by using preciously reported methods[2]. For linearizing plasmid, we used the nuclease SnaBI and KpnI to digest plasmid pYLXP’. The result of plasmid pYLXP’ digestion has been showed in Figure 3.

Fig.3 plasmid pYLXP’ digested by the nuclease SnaBI and KpnI.

3. Construction of recombinant plasmids (the single-gene expression plasmids)

The recombinant plasmids for the single-gene expression (Table 1) were assembled by Gibson Assembly method with using linearized pYLXP’ (digested by SnaBI and KpnI) and the PCR-amplified fragments of genes, which were transformed into Escherichia coli DH5α. The selected marker is AMPr in E.coli, and the positive transformants were determined by colony PCR. The results of E. coli transformation plates and colony PCR have been showed in Figure 4 and Figure 5. The modified DNA fragments and plasmids were sequenced by Sangon Biotech (Shanghai, China).

Table 1 The single-gene expression plasmids.

Fig.4 The plates of E. coli DH5α transformation.

Fig.5 Colony PCR of the transformants. (a) The design of primers for colony PCR; (b) The results of colony PCR.

References：

[1] Green A , Silver P , Collins J , et al. Toehold switches: de-novo-designed regulators of gene expression.[J]. Cell, 2014, 159(4):925-939.

[2] Lv, Y., Edwards, H., Zhou, J., Xu, P. 2019. Combining 26s rDNA and the Cre-loxP system for iterative gene integration and efficient marker curation in Yarrowia lipolytica. ACS Synth Biol.