Team:NPU-China/BasicParts
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K2347000 | Coding | DHAP/G3P->acrylic acid | 1719bp |
| BBa_K2347001 | Coding | glycerol->DHA | 1113bp |
| BBa_K2347002 | Coding | DHA->DHAP | 1827bp |
| BBa_K2347003 | Coding | H2O2->O2 | 1584bp |
| BBa_K2347004 | Coding | NADH->NAD+ | 618bp |
Our best basic part, ceaS2
CEAS, which is N2- (2-carboxyethyl) arginine synthase, is an enzyme in Streptomyces clavuligerus, EC
2.5.1.66. CEAS is a TPP-related enzyme, can catalyze the condensation of D-G3P and L-Arg with the
involvement of TPP and magnesium ions (thiamine pyrophosphate) to produce N2- (2-carboxyethyl) arginine,
which will continue to participate in the biosynthesis of clavulanic acid as the first intermediate.
According to the earlier literature, CEAS (N2-(2-carboxyethyl) arginine synthase) was a synergistic
effect of Ceas1 and Ceas, namely, N2- (2-carboxyethyl) arginine came from the condensation of G3P
and L-Arg, which was catalyzed by Ceas1 and Ceas2. The process would be accompanied by the formation
of acrylic acid[1]. But with the deepening of the study, Matthew E.C. Caines found that Ceas2 played
the main role in the catalytic process. And they speculated the catalytic mechanism of Ceas2, as
shown below [2].
How we use these basic parts?
Fig.5:DAK;6:NOX;7,ceaS2;8:gld;10:CAT
We designed a GDC (GlyDH-DAK-Ceas2) pathway that could produce acrylic acid using glycerol. In this pathway,
GlyDH (Glycerol dehydrogenase，BBa_K2347001) can efficiently convert glycerol to DHA (1,3-Dihydroxyacetone), and
then DAK（Dihydroxyacetone kinase，BBa_K2347002） enzyme can phosphorylate DHA to DHAP, and then the ceaS2 Enzyme
BBa_K2347000）can make it into acrylic acid.
In addition, since GlyDH is NAD + dependent, in order to increase the supply of reducing force, we also
added the NOX (NADH dehydrogenase, BBa_K2347003) and CAT (Catalase, BBa_K2347004) to this pathway, providing
the reduction force for GlyDH through two layers of substrate. Finally, we created the new combination of GNCDC
(GlyDH-NOX-CAT-DAK-ceaS2) pathway using five enzymes from BBa_K2347000 to BBa_K2347004 to produce acrylic acid.
[1] MERSKI M, TOWNSEND C A. Observation of an Acryloyl–Thiamin Diphosphate Adduct in the First Step of
Clavulanic Acid Biosynthesis [J]. Journal of the American Chemical Society, 2007, 129(51): 15750-1.
[2] CAINES M E, ELKINS J M, HEWITSON K S, et al. Crystal structure and mechanistic implications
of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway
[J]. Journal of Biological Chemistry, 2004, 279(7): 5685-92.
In addition, since GlyDH is NAD + dependent, in order to increase the supply of reducing force, we also added the NOX (NADH dehydrogenase, BBa_K2347003) and CAT (Catalase, BBa_K2347004) to this pathway, providing the reduction force for GlyDH through two layers of substrate. Finally, we created the new combination of GNCDC (GlyDH-NOX-CAT-DAK-ceaS2) pathway using five enzymes from BBa_K2347000 to BBa_K2347004 to produce acrylic acid.
[2] CAINES M E, ELKINS J M, HEWITSON K S, et al. Crystal structure and mechanistic implications of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway [J]. Journal of Biological Chemistry, 2004, 279(7): 5685-92.
