Part:BBa_K3742001
EC WS2
Abstract
There are two main enzymes involved in the metabolic pathway of hydroxy fatty acid polymerization, namely coenzyme A ligase and acyl transferase in this part.We selected a new Coenzyme A ligase and cooperated with the T7 promoter to polymerize hydroxy fatty acids. We set 1 group of control substrates nonanoic acid and hydroxydodecanoic acid (brown line) and 3 groups of experimental group substrates were 1. Dodecanoic acid and isopropanol (blue line) 2. Nonanoic acid and hydroxydodecanoic acid (purple line) 3. Nonanoic acid and isopropanol (black line). By adding 3~4% DMSO to increase the solubility of the substrate hydroxy fatty acid and the permeability of the cell membrane, after constructing the engineered bacteria, protein expression and induction fermentation are carried out. The fermentation product was extracted with chloroform for further gas phase product analysis. The data showed that, except for the experimental group 3, the other two groups showed dimerized hydroxydodecanoic acid products by comparison with the control group, indicating that the polymerization pathway of the engineered bacteria had a positive effect on polymerization. Hydroxydodecanoic acid is feasible; in addition, experimental group 2 has nonanoic acid hydroxydodecanoate, which proves that the hydroxyl group of hydroxy fatty acid can be used as the catalytic site of WS2 acyltransferase, and the carboxyl group at the other end does not affect the acyltransferase.Our research has proved that the polymerization process of EC coenzyme A ligase and WS2 acyltransferase can play a role in two identical or different monomers and form at least two degree of polymerization. Due to the limitation of the experimental time and the experimental equipment caused by the epidemic in summer vacation, we have not been able to provide more evidence of terpolymers and above polymers. The vaporization temperature of the gas-phase analyzer we use is 380℃, which is such a temperature. It may not be possible to vaporize the higher poly hydroxy fatty acids and enter the analytical instrument, so we did not get the relevant data.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2532
Illegal EcoRI site found at 3171
Illegal PstI site found at 79
Illegal PstI site found at 604
Illegal PstI site found at 1003
Illegal PstI site found at 1045
Illegal PstI site found at 1108
Illegal PstI site found at 1773
Illegal PstI site found at 1833 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2532
Illegal EcoRI site found at 3171
Illegal PstI site found at 79
Illegal PstI site found at 604
Illegal PstI site found at 1003
Illegal PstI site found at 1045
Illegal PstI site found at 1108
Illegal PstI site found at 1773
Illegal PstI site found at 1833
Illegal NotI site found at 1430 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2532
Illegal EcoRI site found at 3171
Illegal BglII site found at 83
Illegal BglII site found at 947 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2532
Illegal EcoRI site found at 3171
Illegal PstI site found at 79
Illegal PstI site found at 604
Illegal PstI site found at 1003
Illegal PstI site found at 1045
Illegal PstI site found at 1108
Illegal PstI site found at 1773
Illegal PstI site found at 1833 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2532
Illegal EcoRI site found at 3171
Illegal PstI site found at 79
Illegal PstI site found at 604
Illegal PstI site found at 1003
Illegal PstI site found at 1045
Illegal PstI site found at 1108
Illegal PstI site found at 1773
Illegal PstI site found at 1833
Illegal NgoMIV site found at 1697
Illegal AgeI site found at 697
Illegal AgeI site found at 1306
Illegal AgeI site found at 1354
Illegal AgeI site found at 2220
Illegal AgeI site found at 2232
Illegal AgeI site found at 2490
Illegal AgeI site found at 2625 - 1000COMPATIBLE WITH RFC[1000]
EC+WS2->gene sequence
The gene sequence contains two target genes: EC (long chain acyl CoA synthase) and WS2 (wax ester synthase).It could express two proteins, CoA ligase and acyltransferase. The function of the two proteins as follows (EC for CoA ligase; WS2 for acyltransferase) At the same time, in our design, it contained a T7 promoter of EC between the two segments (EC / WS2)
Purpose of the experiment
In order to verify their functions, we constructed the plasmid pETDuet (WS2/EC). In the verification experiment, we used hydroxydodecanoic acid and nonanoic acid as substrates. We expressed pETDuet (WS2/EC) into E. coli BL21 (DE3). The functions of EC and WS2 are shown below. During the polymerization process, EC acts as a CoA ligase to remove H in CoA-SH and -OH in the carboxyl group of hydroxy fatty acid through dehydration and condensation, and connect CoA and hydroxy fatty acid at the same time; WS2 As an acyltransferase, the acyl group in the hydroxy fatty acid CoA replaces the H in hydroxyl group of the hydroxy fatty acid, and CoA-SH is generated at the same
Experimental results and analysis
1.SDS protein electrophoresis
Molecular weight of target protein
EC 62 kDa
WS2 52.5 kDa
ACS2 61.9 kDa
Through SDS protein electrophoresis, we can confirm that our genes EC and WS2 are expressed, and the E.coli has the enzymes we need by comparing with marker. Which allowe us to conduct further experiments.
2.Results of GC-MS with Hydroxydodecanoic acid and nonanoic acid as substrates
After the engineering bacteria were successfully constructed, we added an exogenous substrate and added 3~4% DMSO to increase the solubility of the substrate hydroxy fatty acid and the permeability of the cell membrane. After the induction of fermentation with the IPTG inducer, the cells were broken and used Trichloromethane extracts the fermentation broth and prepares samples for gas phase detection. Set 1 group of control substrates nonanoic acid and hydroxydodecanoic acid (brown line) and 3 groups of experimental group substrates are 1. hydroxydodecanoic acid and isopropanol (blue line) 2. Nonanoic acid and hydroxydodecanoic acid (Purple line) 3. Nonanoic acid and isopropanol (black line).
According to the GC-MS spectrum, in addition to experimental group 3, the other two groups of experimental group 1 hydroxylauric acid and isopropanol (blue line) and experimental group 2 nonanoic acid and hydroxylauric acid (purple line) pass and control Dimeric hydroxy dodecanoic acid was found in the comparison of group nonanoic acid and hydroxydodecanoic acid (brown line), indicating that the polymerization pathway of engineered bacteria is feasible to polymerize hydroxydodecanoic acid; in addition, experimental group 2 nonanoic acid and hydroxydodecanoic acid ( Purple line) The product Nonanoic acid hydroxydodecanoate appears, which proves that the hydroxyl group of the hydroxy fatty acid can be used as the catalytic site of WS2 acyltransferase, and the carboxyl group at the other end does not affect the catalytic reaction of the acyltransferase. Our research has proved that the polymerization process of EC coenzyme A ligase and WS2 acyltransferase can play a role in two identical or different monomers and form at least two degree of polymerization. Due to the limitation of the experimental time and the experimental equipment caused by the epidemic, we have not been able to provide more evidence of terpolymers and above polymers. The vaporization temperature of the gas-phase analyzer we use is 380℃, which is such a temperature. It may not be possible to vaporize the higher poly hydroxy fatty acids and enter the analytical instrument, so we did not get the relevant data. In the next experiment, we plan to further increase the intracellular substrate concentration during fermentation and use a gas phase instrument with a higher vaporization temperature for product detection.
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