Part:BBa_K3909010
pYLXP-ylPOT1
A composite part consist of pTEF promoter (BBa_K3909008 ), ylPOT1 (BBa_K3909007) 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1343
Illegal XhoI site found at 164
Illegal XhoI site found at 565 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 832
Illegal NgoMIV site found at 925
Illegal NgoMIV site found at 1159
Illegal AgeI site found at 1657
Illegal AgeI site found at 1741
Illegal AgeI site found at 1893 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2
Illegal BsaI.rc site found at 844
Usage and Biology
Results: Constructing recombinant plasmids for overexpressing the β-oxidation pathway
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.
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.
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