Revision as of 10:30, 21 October 2021

CUS

Sequence and Features

Usage and Biology

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.

Molecular cloning

Fig1. Colony PCR results of AOX1-α factor-CUS-AOX1 Terminator, AOX1-α factor-ACC-AOX1 Terminator, AOX1-α factor-4CL-AOX1 Terminator and AOX1-α factor-LOX2-AOX1 Terminator transformed E.coli

The bands of AOX1-α factor-CUS-AOX1 Terminator (3000bp) , AOX1-α factor-ACC-AOX1 Terminator (3000+bp), AOX1-α factor-4CL-AOX1 Terminator (3000+bp) and AOX1-α factor-LOX2-AOX1 Terminator (almost 5000bp) from colony PCR are identical to the theoretical lengths of 3046bp, 3619bp, 3523bp and 4528bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli

Fig2. Plasmid construction and colony PCR results of Panb1-α factor-4CL-AOX1 Terminator, Panb1-α factor-crtI-AOX1 Terminator, Panb1-α factor-crtB-AOX1 Terminator and Panb1-α factor-crtE-AOX1 Terminator transformed E.coli

The bands of Panb1-α factor-4CL-AOX1 Terminator (3000+bp), Panb1-α factor-crtI-AOX1 Terminator (3000bp), Panb1-α factor-crtB-AOX1 Terminator (2000+bp) and Panb1-α factor-crtE-AOX1 Terminator (2000+bp) from colony PCR are identical to the theoretical lengths of 3185bp, 3046bp, 2198bp and 2177bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed.
Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.

Fig3. Colony PCR result of yeast after electroporation through electrophoresis

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.

SDS-PAGE

After confirmation from colony PCR and sequencing, we using the successfully integrated yeast for expression. At first, we try to detect our target protein in the supernatant since there is signal peptide.

Fig4. SDS-PAGE result of Laccase GS115 4CL LOX2 ACC pepACS DsbC+pepACS detecetion in the supernatant

Due to glycosylation modification of yeast expression, the molecular weight exhibited on SDS-PAGE will be larger than theoretical. Primary detection shows that we have laccase, 4CL and ACC bands of about 75kDa, LOX2 band of 100+kDa and DsbC+pepACS of about 40kDa, all of which is a bit larger(Laccase：57.01 kDa; 4CL：61.88 kDa; ACC：63.40 kDa; LOX2：102.88 kDa; DsbC+pepACS：31.72 kDa) but still within explainable and acceptable range, which could be evidence of successful expression.

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

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 indigo（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 dying cream

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.