Designed by: Jiacheng Shi   Group: iGEM21_HUST-China   (2021-10-03)

AOX1-α factor-Curcumin-AOX1 Terminator


As a composite part, it consists of AOX1-α factor-4CL-AOX1 Terminator-AOX1-α factor-ACC-AOX1 Terminator-AOX1-α factor-CUS-AOX1 Terminator. AOX1, as a methanol induced strong promoter, will express 4CL, ACC, and CUS in the presence of methanol and secret them out of the cell through the signal peptide, α-factor. When the substrate, ferulic acid, is added, these three enzymes react in order and catalyze ferulic acid into curcumin.

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.

Background related to curcumin

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
curcumin 30min 50℃ Ethyl alcohol 0.5

Under the best conditions, we dyed the hair from 4 degree to 9 degree, and got a series of colors. It is found that it only needed to be bleached to 8 degree so that the hair would show a bright color for all three kinds of dye.

The dyeing results of curcumin(room temperature, ethanol added,0.5g/L). From left to right: 10min(9-4°),30min(9-4°)8°

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.

Dyeing result of curcumin made in different solvents(30 min, 50°C). Left: water solution, Right: 33% ethanol alcohol solution

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

Ingredient Content
Cream matrix 100g
Sodium sulfite 0.2g
Absolute ethanol 1ml
Isopropyl alcohol 2ml
pH 6.8 phosphate buffer 1ml
Solid paraffin 1 drop or not
Essence 1 drop
curcumin 0.5g
The result of curcumin staining cream. 9 ° from left to right. Hair was dyed at room temperature for 30min, 60min and 0min; Dyeing at 50 ℃ for 10min and 30min

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

Assembly Compatibility:
  • 10
  • 12
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    Illegal NheI site found at 6121
    Illegal NheI site found at 6163
  • 21
    Illegal XhoI site found at 1187
    Illegal XhoI site found at 4370
    Illegal XhoI site found at 7649
  • 23
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    Illegal NgoMIV site found at 5081
    Illegal NgoMIV site found at 5841
  • 1000
    Illegal BsaI site found at 2021
    Illegal BsaI site found at 7751
    Illegal BsaI.rc site found at 2156
    Illegal BsaI.rc site found at 4743
    Illegal BsaI.rc site found at 5741
    Illegal BsaI.rc site found at 6045