4-coumarate-CoA ligase (4CL) under pTET constitutive promoter
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1147
- 21Illegal BglII site found at 1714
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI.rc site found at 1346
The 4-coumarate-CoA ligase (4CL) enzyme catalyzes the ligation of a CoA group to the end of the molecule (2). The addition of this CoA provides better kinetics for a variety of downstream reactions. 4CL is also a member of the phenylpropanoid pathway and is found in a wide variety of organisms that partake in the degradation of lignin (2,3). Here we harness the 4CL to provide better kinetics for reduction of cinnamic acid to cinnamaldehyde.
The genes encoding PAL (BBa_K1129003), 4-Courmarate CoA ligase(BBa_K1129042) and a Cinnamoyl Co-A reductase(BBa_1129005) were assembled into one PSB1C3 plasmid under constitutive promoters and transformed into E. coli. E.coli 10G cells were grown over night before being incubated with L-Phenylalanine for 7 hours, the sample was extracted before it was run on GC-MS.
The first two panels show experimental data for our PAL (Enc-P) expressed from PSB1C3, under constitutive promoters alone. The last panel shows experimental data for the entire biosynthetic pathway for the generation of cinnamaldehyde.
Figure 1. Compound generation identification by GC-MS. Chromatograms and mass spectra for select peaks are shown. Structures represent predictions based on library matching or comparison to standards. Controls represent plasmids missing the gene of interest. Top Internal control using cinnamic acid. Middle cinnamic acid detection from culture containing phenylalanine and strains harbouring constitutive EncP. Bottom Cinnamic acid and cinnamaldehyde detection from culture containing phenylalanine and strains harbouring constitutive EncP, 4CH and ATRCC1.