Part:BBa_K1660003

d-limonene synthase (J23100 + B0032 + d-limonene synthase)

The length of this part is 1883bp. d-limonene synthase is from Citrus unshiu. Limonene is a kind of valuable terpenoid normally expressed in plants and has two enantiomers in natural source, d-limonene and l-limonene. The precursor of limonene is geranyl pyrophosphate (GPP), GPP is synthesized by Isopentenyl diphosphate (IPP) and IPP’s isomer dimethylallyl diphosphate (DMAPP), which are the two essential building blocks to synthesize all terpenoids^[1]. The synthesis pathways of IPP and DMAPP in most prokaryotes is MEP pathway (Fig.1).

Fig.1 Engineered pathway for l-limonene biosynthesis in E. coli^[1].

Baced on the production of IPP and DMAPP by the above pathways, GPP synthase (GPPS) catalyzes the condensation between IPP and DMAPP to synthesize GPP, and then Limonene synthase (LS) catalyzes the intramolecular cyclization of GPP to stnthesize limonene. So in our research, we transferred LS gene into the E.coli BL21 (DE3) to make the E.coli synthesize limonene.

</body> </html> Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 57
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1596
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1593
  Illegal SapI.rc site found at 315
  Illegal SapI.rc site found at 1568
  

Design

The DNA sequence of d-limonene synthase gene is from NCBI(GenBank: AB110636.1). A constitutive promoter (BBa_J23100) and a RBS(BBa_B0032) are added upstream the functional gene to produce this synthase. In order to change the promoter and RBS to control the expression of the synthase, a XhoI restriction site is added between RBS and the start codon. d-limonene synthase-pSB1C3 plasmid is transformed into E. coli strain BL21 (DE3) to express the protein. d-limonene synthase can catalyst the expression of d-limonene.

Results

In Fig. 2，d-limonene synthase gene connected with the backbone plasmid-pSB1C3 was digested by PstI and EcoRI. The length of target band which contains this part is 1924bp, and the length of the backbone is 2624bp. In Fig. 2, the target genes were marked with arrows, and the figure shows that we transferred the limonene synthase gene into the E.coli BL21 (DE3) successfully. In Fig. 3, the target proteins (89 kDa) are marked with arrows, and the figure shows that we expressed the d-imonene synthase successfully.

Fig.2 The agarose gel electrophoresis of plasmid d-limonene synthase.

Lane M, DNA marker DL2000; Lane 1 and 2, d-limonene synthase gene digested by EcoRI and PstI.

Fig. 3 SDS-PAGE of d-limonene synthase, the two bands d-1&d-2 represent the expression of d-limonene synthase.

Comparing the difference of TIC between standard samples and test samples, we confirm the time of standard limonene samples' peak appearance is at 4.934 minute. And in the figure of our d-limonene sample extracted from bacterium solution there is a peak at 4.934 minute, which means that our test samples may contain limonene. Then we analyzed the test samples at this time point by mass spectrometric detection. The MS was operated in SIM mode using ions of 136, 68, and 93 m/z. Compared with Fig. 4a, there are similar peaks at 136, 68, and 93 m/z in Fig. 4b. The result shows that the E.coli we designed express the d-limonene successfully.

Fig. 4A The result of GC-MS:Control group of Pure d-limonene compound.

(The MS is operated in SIM mode using ions of 136, 68, and 93 m/z)^[1]

Fig. 4B The result of GC-MS : The peaks of d-limonene extracted by us are marked by arrows.

(The MS is operated in SIM mode using ions of 136, 68, and 93 m/z)^[1]

Reference

• [1]Du et al.: Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E. coli. Bioresources and Bioprocessing 2014 1:10.