Part:BBa_K1027002

Delta 12 Desaturase, optimised for expression in Escherichia coli

Introduction

This enzyme is obtained from the gene Oleic acid desaturase (ode). The gene is obtained from Synechocystis sp. PCC 6803, a cyanobacterium and introduced in E. coli. It is a trans-membrane enzyme, involved in the biosynthesis of C18:2 from C18:1. C18:2 corresponds to linoleic acid. The delta 12 desaturase introduces a second cis carbon double bond on the 12th carbon in C18:1.

Characterisation

In order to characterise our biobrick, we made use of the standard parts found in the registry. By inserting the created biobrick and BBa_K1027002 in to BBa_K608002 (a biobrick consisting of a ribosomal binding site and a constitutive promoter), we were able to create a new construct that expressed the delta 12 desaturase proteins.

LC-MS Data The delta 12 desaturase enzyme, when expressed in its host organism (Synechocystis sp. PCC 6803), converts oleic acid into linoleic acid. Therefore, we fed batches of transformed DH5-alpha with 2 different concentrations of exogenous fatty acid (0.1% and 0.5% oleic acid fed to the delta 12 desaturase batch), left the cultures growing overnight and then harvested the cells.

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To analyse the metabolites extracted from both wild-type DH5-alpha and DH5-alpha expressing our delta 9 desaturase and delta 12 desaturase enzymes, we made use of the Manchester Institute of Biotechnology’s in-house Orbitrap Liquid Chromatography - Mass Spectrometry (LC-MS). This technique was chosen because of its high mass accuracy and sensitivity. Upon analysing the most abundant metabolites extracted from our expression strains and comparing this data with the most abundant metabolites extracted from wild-type, it is apparent that a massive increase in linoleic acid (incorporated in phosphatidylethanolamines, PE) has occurred (shown above). The chromatograms produced for the delta 12 desaturase expression strains are shown below. There is a clear difference between the wild-type E. coli fed with exogenous substrate compared with the E. coli strains expressing delta 12 desaturase. It is probable that the peak appearing around 7.9 min in the delta 12 desaturase strains is due to phospholipid incorporating 18:2 (9Z, 12Z) - linoleic acid. <img src="" />

The data obtained from the LC-MS was enormous. 2721 metabolites were detected in the samples, and so we filtered this down to the 43 fatty acids and phospholipids incorporating the compounds we were interested in (oleic acid and linoleic acid). A heat map was generated (seen below) showing the abundances of these 43 phospholipids and fatty acids in the 17 samples we run on the LC-MS. From this heatmap the diversity of detected compounds and high dynamic range can be seen. For the characterisation of our delta 12 desaturase BioBrick construct, a focus on the individual compounds of high intensity was necessary. An example of this can be seen in the bar chart below. Here you can see that, when fed with oleic acid (to a total w/v concentration of 0.1% and 0.5%), the amount of linoleic acid found within this representative phospholipid is much higher in the delta 12 desaturase expression strain than in the wild-type DH5-alpha strain.

Heatmap <img src="" />

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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 66
  Illegal BamHI site found at 426
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 247
  Illegal AgeI site found at 511
  Illegal AgeI site found at 649
  
• 1000
  COMPATIBLE WITH RFC[1000]