The synthesis and decolorization system of curcumin without signal peptide
As a composite part, it consists of Panb1 -4CL-AOX1 Terminator-Panb1 -ACC-AOX1 Terminator-Panb1-CUS-AOX1 Terminator-Pynr071c-curA-AOX1 Terminator- Pynr071c-ROX1-AOX1 Terminator. Panb1, as a constitutive promoter, will express 4CL, ACC, and CUS under any circumstances and keep them inside of the cell without the signal peptide. When the substrate, ferulic acid, is added, these three enzymes react in order and catalyze ferulic acid into curcumin. Pynr071c promoter, as a xylose-inducible promoter, translates and expresses curA in the presence of xylose. CurA reduces lycopene to colorless substances. At the same time, it also induces the expression of ROX1. ROX1 inhibits the Panb1 promoter and blocks the expression of its downstream proteins, thus blocking the synthesis of pigment.
Usage and Biology
In the curcumin biosynthesis pathway, 4CL is located in the upstream of the metabolic pathway and plays a key role in the synthesis of phenylpropane derivatives. 4CL is the branching enzyme that connects the lignin synthesis pathway and flavonoid pathway, controls the metabolic synthesis direction of phenylpropane derivatives, and is the key enzyme in the phenylpropane synthesis pathway. 4CL acts on different substrates to produce acyl CoA thiolipids for subsequent reactions. Through the synthesis of these phenylpropane derivatives CoA lipids (e.g. p-gumaroyl CoA, feruloyl CoA, p-coumaryl CoA), downstream enzymes use them as substrates to form different phenylpropane metabolites. Therefore, 4CL enzyme plays a switching role in the biosynthesis of curcumin. In the curcumin biosynthesis pathway, the role of 4CL in dipeptide-CoA synthase DCS curcumin synthase CURS is to catalyze cinnamic acid to produce cinnamyl-CoA and make the reaction to the direction of curcumin production. In the curcumin synthase CUS pathway, ferulic acid is used to catalyze the formation of gumaroyl-CoA in order to facilitate the following reaction.
Acetyl-CoA carboxylase (ACC) is a biotin enzyme that can catalyze the reaction of "acetyl-CoA+ATP+HCO3→malonyl-CoA+ADP+Pi". It exists widely in nature. ACC is a rate-limiting enzyme for ab initio synthesis of fatty acids, which catalyzes acetyl-CoA to malonyl-CoA, which eventually forms C16 acyl-CoA. ACC can be divided into multi-subunit ACC and multi-functional ACC. Polysubunit ACC exists in plants and bacteria and consists of four subunits, namely, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and two subunits of carboxyltransferase (CT), α-CT and β-CT. Multifunctional ACC mostly exists in eukaryotes. ACC has been used in the drug design of obesity, diabetes and plant herbicides, and is also a target gene for some crops.
Curcumin is synthesized sequentially by two different type III polyketone synthase (PKS) in curcumin rhizome, which is named dipeptide CoA synthase (DCS) and curcumin synthase (CURS). In addition to the DCS/CURS biosynthesis system in curcuma rhizome, we also found and characterized another type III PKS in rice plant Oryza sativa, curcumin synthase (CUS). The synthesis of curcumin catalyzed by CUS is as follows: firstly, p-gumaryl-CoA and malonyl-CoA are condensed to form dipeptide-CoA intermediate. The synthesized dipeptide CoA condensed with another p-coumaryl CoA to synthesize didemethoxycurcumin. CUS itself catalyzes both reactions of DCS/CUS, so the CUS system is simpler than the DCS/CURS system. In this respect, CUS is a better enzyme than DCS/CURS and is used in the metabolic engineering of curcumin in microorganisms. Besides. CUS can produce cinnamyl methane and curcumin from cinnamyl CoAn and ferulyl CoA.
curA is derived from curcumin-transforming microorganisms isolated from human faeces with a molecular weight of about 82 kDa and consists of two identical subunits. During the purification of the expressed curA enzyme, the activity of curA enzyme was lost during dialysis. However, we found that the addition of NADPH increased the activity, but the addition of NADH did not increase the activity. These findings indicate that the enzyme catalyzes the NADPH-dependent transformation of curcumin. In the curA catalytic reaction, two steps of curcumin metabolism pathway (curcumin→dihydrocurcumin→tetrahydrocurcumin) were found. Under the catalysis of CurA, dihydrocurcumin is first the product, then the substrate. These products are produced from curcumin by reducing the diarylheptatrienone chain which could devastate the color of the hair. The curA does not produce more reduction products than tetrahydrocurcumin, which indicates that curA only catalyzes the reduction of compounds with C=C. In the reaction process, the optimum reaction temperature of curA is 35 ℃. The enzyme showed maximum activity at pH 5.9. Although a large number of different compounds have been tested as potential substrates of curA, curA seems to have a narrow substrate spectrum and preferentially acts on curcumin. Considering substrate specificity and NADPH dependence, curA was named NADPH-dependent curcumin/dihydrocurcumin reductase.
As a natural compound, curcumin is good at fighting against inflammatory and cancer. Derived from the rhizomes of some plants in the family Curphinae, Ceraceae, curcumin is a diketone compound existing in rhizoma curcumae longae for about 3% to 6%. Rarely, there is little botanic pigment with diketone structure like it. The outward appearance of it is an orange-yellow crystal powder, tastes slightly bitter and insoluble in water. In food production, it is mainly used for intestinal products, canned products, sauced products and others. Aside from cancer, curcumin could also decrease the blood fat, benefit the gallbladder, be against oxidation and according to some reports, contribute to the treatment of drug-resistant tuberculosis.
Hair dyeing experiment
We measured the standard curves of three pigments before using them for hair dyeing experiment. We also found that the amount of melanin contained in hair can have a significant effect on hair dyeing outcomes. Therefore, we define different colors of hair based on bleaching.
We have gained the best dye conditions of three kinds of hair dye(indigo, curcumin and lycopene) at a certain concentration. Under optimal conditions, we dyed 4-9 degrees of hair to get a series of dyeing discs. And we found that as for the three colors selected for the experiment, bleach the hair to 8 degrees could achieve a bright coloring effect.
Chart of the best condition of hair dye
|Dye/Condition||time||temperature||Dyeing aid ingredients||concentration（g/L）||comment|
Problem: The coloration rate of the curcumin aqueduct solution is low Solution: We carried out the same dye addition and elution experiments as lycopene, and found that alum, potassium tartarate and citric acid cannot improve the coloring effect of curcumin. The data showed that curcumin is soluble in ethanol, and the optimal coloring temperature is 50 degree, so we adjusted the solvent to 33% ethanol solution. The process of coloring was conducted in the oven at 50 degrees, and the results showed that the coloring effect significantly improved.
After finishing the solution experiment, we try to mix the natural pigment into a dye that can be applied directly to the hair. At present, lycopene dye and curcumin dye with NO.1 cream matrix as carrier are obtained, and natural essence is added to improve the odor of dye paste. Indigo is an oxidizing dye with special properties, so we designed a timely fermenter. In this way, we can use our product right now when indigo is produced and reduced to indigo white.
EDTA is added to the curcumin paste to prevent metal ions from interfering with the effect -- for example, Fe3 + producing a reddish-brown tint.
Curcumin dye cream
|pH 6.8 phosphate buffer||1ml|
|Solid paraffin||1 drop or not|
Color fastness test Color fastness is an important aspect to measure the effect of dye, so we design a set of elution scheme and test the color fastness of three kinds of natural pigment dye products and the same color traditional dye paste. The results showed that the color fastness of the natural pigment dyes was better than that of the traditional dyes.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 4001
Illegal NheI site found at 4499
Illegal NheI site found at 4541
Illegal NotI site found at 11686
- 21Illegal XhoI site found at 124
Illegal XhoI site found at 2496
Illegal XhoI site found at 4964
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 3459
Illegal NgoMIV site found at 4219
- 1000Illegal BsaI site found at 1210
Illegal BsaI site found at 5318
Illegal BsaI site found at 10605
Illegal BsaI.rc site found at 1345
Illegal BsaI.rc site found at 3121
Illegal BsaI.rc site found at 4119
Illegal BsaI.rc site found at 4423
Illegal SapI site found at 10864