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.

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 BglII site found at 1343
  Illegal XhoI site found at 164
  Illegal XhoI site found at 565
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2
  Illegal BsaI.rc site found at 844