Efficient madecassoside preparation method based on yeast metabolic engineering and madecassoside compound cosmetic composition

Abstract

The invention discloses a madecassoside efficient preparation method based on yeast metabolic engineering and a madecassoside compound cosmetic composition. Three glycosyl transferases CaGlcT1, CaGlcT2 and CaRhaT are screened from a centella asiatica transcriptome, after the catalytic activity of the glycosyl transferases is verified, polygene co-expression is performed in saccharomyces cerevisiae, and madecassic acid can be catalyzed to synthesize madecassoside. After fermentation condition optimization and fed-batch fermentation, the conversion yield of madecassoside reaches 582 mg / L from 165 mg / L, 3.5 times of yield increase is achieved, the purity of obtained madecassoside reaches 95% or above, and commercial application and scientific research requirements are met. The obtained madecassoside is compounded with the chrysanthellum indicum extract and the purslane herb extract, so that the synergistic anti-oxidation and whitening effects are achieved. A new scheme is provided for green and intelligent manufacturing of cosmetic raw materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical fields of synthetic biology and cosmetic raw material preparation, in particular to a high-efficiency preparation method of hydroxyl asiaticoside based on yeast metabolic engineering and a complex cosmetic composition thereof. BACKGROUND

[0002] Madecassoside is one of the main active ingredients in Centella asiatica, with significant anti-inflammatory, antioxidant and wound repair-promoting pharmacological activities, and is widely used in the fields of medicine and cosmetics. Currently, the industrial production of madecassoside mainly relies on plant extraction methods (such as ethanol-ultrasonic assisted extraction, water vapor distillation, etc.), but there are the following bottlenecks: ① high dependence on raw materials: the growth cycle of Centella asiatica is as long as 6-8 months, and the content of effective components is affected by environmental fluctuations, resulting in unstable supply and high cost of raw materials; ② low extraction efficiency: ultrasonic-assisted enzymatic hydrolysis (such as cellulase treatment) can improve the extraction rate to 1.92%, but still requires multiple purification steps, complex process and high energy consumption; ③ difficulty in chemical synthesis: madecassoside contains multiple glycosylation sites, and the chemical synthesis path is complex, with low purity and complex process.

[0003] The development of synthetic biology provides an innovative path for the heterologous synthesis of plant-derived natural products. By using microorganisms (such as Saccharomyces cerevisiae) as hosts and through modular assembly and optimization of metabolic pathways, high-efficiency synthesis of natural products such as madecassoside can be achieved. Compared with traditional extraction processes, the synthetic biology technology for preparing madecassoside not only significantly reduces production costs, but also strictly guarantees product quality and safety. Through optimization of the metabolic pathway, high-purity madecassoside can be produced on a large scale to meet the needs of commercial applications and scientific research experiments. This innovative synthesis strategy based on gene editing and metabolic engineering is reshaping the production mode of plant-derived active ingredients and providing sustainable solutions for the modern biological pharmaceutical industry.

[0004] In the field of pharmacology and cosmetics, hydroxy asiatic acid has been proved to have multiple biological functions. In the Centella asiatica, multiple key genes involved in triterpenoid biosynthesis have been identified, including farnesyl pyrophosphate synthase (CaFPS), squalene synthase (CaSQS), β-amyrin synthase (CaβAS) and dammarane synthase (CaDDS). Modifications of the triterpenoid skeleton include oxidation, substitution or glycosylation by P450 monooxygenase, UDP-Glc glycosyltransferase and other enzymes. Glycosyltransferases are key enzymes in plant glycosylation process and are essential for the biological activity of saponins. In existing research, glycosyltransferases such as UGT73AD1, UGT94M2 and CaUGT4 have been individually verified to have hydroxy asiatic acid monoglycoside activity (such as UGT73AD1 catalyzing the generation of hydroxy asiatic acid monoglycoside), but the existing technology has not yet reported the co-expression of multiple glycosyltransferases in series to realize the multi-step glycosylation reaction from hydroxy asiatic acid to hydroxy asiatic acid.

[0005] Most of the products on the market for whitening and antioxidant have a single action path, and single plant extracts often have difficulty in comprehensively solving the problem of antioxidant, so it is necessary to develop a composition with diverse pathways, which can effectively and instantly whiten and brighten with few side effects. The planting cycle of plants is usually long, the supply of raw materials is unstable, and the cost is high. Natural plant active ingredients generally have problems such as poor solubility, low permeability and low absorption rate. Therefore, it is of great practical significance to break through the existing enzyme combination catalytic efficiency bottleneck by applying synthetic biology, combine new glycosyltransferase genes, produce and develop an active raw material, and apply it to an efficient and industrialized biological manufacturing system to realize the low-cost and high-yield conversion of cheap substrates to high-value hydroxy asiatic acid. SUMMARY

[0006] The first object of the present application is to provide a multi-gene tandem recombinant protein, which is a glycosyltransferase encoding gene with nucleotide sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 inserted in series in the same expression vector, formed by simultaneously expressing the three glycosyltransferases (UGTs) in host cells.

[0007] Through transcriptome sequencing, different tissues (roots, stems, leaves) of Centella asiatica were analyzed, and 3 UGTs with functions were screened by bioinformatics methods such as local Blast, sequence alignment and phylogenetic tree analysis, and were named as CaGlcT1, CaGlcT2 and CaRhaT, respectively. The nucleotide sequences correspond to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the amino acid sequences correspond to SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0008] A second object of the present application is to provide a recombinant expression vector containing a glycosyltransferase-encoding gene with a nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0009] The vector of the present application is well known to those skilled in the art, including but not limited to plasmids, bacteriophages, viral vectors, etc. In an embodiment of the present application, the vector is pY26TEF-GPD vector.

[0010] A third object of the present application is to provide an engineered bacterium obtained after transformation of the recombinant expression vector described above.

[0011] Preferably, the host bacterium of the engineered bacterium includes but is not limited to yeast.

[0012] A fourth object of the present application is to provide the use of the multi-gene tandem recombinant protein, the recombinant expression vector or the engineered bacterium described above as an enzyme catalyst in one or more of the following reactions:

[0013] (D1) catalyzing the generation of hydroxyl aescin monosaccharide from hydroxyl aescin;

[0014] (D2) catalyzing the generation of hydroxyl aescin disaccharide from hydroxyl aescin monosaccharide;

[0015] (D3) catalyzing the generation of hydroxyl aescin from hydroxyl aescin disaccharide.

[0016] A fifth object of the present application is to provide the use of the multi-gene tandem recombinant protein, the recombinant expression vector or the engineered bacterium described above in the preparation of hydroxyl aescin.

[0017] Preferably, the preparation of hydroxyl aescin is based on hydroxyl aescin as a starting material.

[0018] A sixth object of the present application is to provide the use of a composition comprising hydroxyl aescin, golden chamomile extract and spinach extract in the preparation of an antioxidant whitening product, wherein the hydroxyl aescin is a hydroxyl aescin with a purity of ≥ 95% produced by fermentation of the engineered bacterium described above, and the mass ratio of the hydroxyl aescin, the golden chamomile extract and the spinach extract is preferably 3:5:2.

[0019] Preferably, the antioxidant whitening product includes but is not limited to a mask, a moisturizing cream, a lotion or a cleanser.

[0020] A seventh object of the present application is to provide a method for preparing hydroxyl aescin, comprising the following steps: culturing the engineered bacterium described above using YPD liquid medium to obtain seed liquid; and inoculating the seed liquid into YPG liquid medium containing hydroxyl aescin for culturing.

[0021] Preferably, the formula of the YPD liquid medium is: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, and the solvent is deionized water.

[0022] Preferably, the formula of the YPG liquid medium is: D-galactose 25 g / L, peptone 20 g / L, yeast extract 15 g / L, and the solvent is deionized water.

[0023] The application screens and identifies three glycosyltransferases (CaGlcT1, CaGlcT2 and CaRhaT) involved in the biosynthesis of hydroxyl asiaticoside by combining genomes and transcriptomes, constructs an expression vector by multi-gene tandem recombination, realizes the heterologous expression in Saccharomyces cerevisiae, and greatly improves the yield of hydroxyl asiaticoside in Saccharomyces cerevisiae by optimizing the fermentation conditions. Compared with the three-gene tandem expression of UGT73AD1, UGT94M2 and CaUGT4 in the comparative experiment, the yield of hydroxyl asiaticoside is only 118 mg / L, which is significantly lower than the combination of genes (165 mg / L) in the application. Through the synergistic effect of the compounded composition (hydroxyl asiaticoside: golden yellow chamomile extract: portulaca oleracea extract = 3:5:2), excellent performance is shown in antioxidant (DPPH clearance rate 88.24%), inhibition of melanin production (relative content reduction 54.58%) and anti-inflammatory trans-epidermal water loss (TEWL value reduction 31.42%), which is significantly better than single component. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The synthesis path of hydroxyl asiatic acid to hydroxyl asiaticoside.

[0025] Figure 2 It is a SDS-PAGE electrophoresis map of multi-gene tandem recombinant proteins. Wherein 1 is CaGlcT1, CaGlcT2 and CaRhaT multi-gene tandem recombinant protein; 2 is UGT73AD1, UGT94M2 and CaUGT4 multi-gene tandem recombinant protein; M is Marker.

[0026] Figure 3 It is the HPLC detection results of hydroxyl asiatic acid standard, hydroxyl asiaticoside standard and hydroxyl asiatic acid standard + multi-gene (CaGlcT1, CaGlcT2 and CaRhaT) tandem recombinant protein.

[0027] Figure 4 It is the MS / MS detection results of hydroxyl asiaticoside synthesized in Saccharomyces cerevisiae. DETAILED DESCRIPTION

[0028] The following examples are further illustrations of the application and are not intended to limit the application.

[0029] The related experimental methods involved in the technical solution are general technical means in the field of biochemistry unless otherwise specified. The materials, reagents and instruments required for the experiment can be purchased through conventional commercial channels except for special marking.

[0030] Example 1: Mining and characterization of glycosyltransferases CaGlcT1, CaGlcT2 and CaRhaT in Centella asiatica

[0031] (1) 143 potential glycosylation modification related genes were obtained from the measured Centella asiatica transcriptome by local Blast using glycosyltransferase genes with identified functions. Through phylogenetic tree construction technology, the screening results were analyzed for evolutionary correlation with known pentacyclic triterpenoid glycosyltransferases.

[0032] (2) The transcriptome expression profile data was integrated to analyze the specific expression pattern of each candidate gene in root, stem and leaf tissues. Functional verification experiments were carried out on the leaf tissue high expression target, and finally the glycosyltransferase genes CaGlcT1, CaGlcT2 and CaRhaT with catalytic function were screened out.

[0033] The CaGlcT1 gene sequence is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 4.

[0034] The CaGlcT2 gene sequence is shown as SEQ ID NO: 2, and the amino acid sequence is shown as SEQ ID NO: 5.

[0035] The CaRhaT gene sequence is shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 6.

[0036] Example 2: Cloning of target genes and construction of expression vector

[0037] Fresh Centella asiatica leaf tissue was taken, total RNA was separated by EASYspin plant RNA rapid extraction kit (Aidlab), and cDNA was reverse transcribed by reverse transcription kit (Novozyme). Specific amplification primers were designed:

[0038] Table 1 primer sequences

[0039]

[0040] PCR amplification was performed using KOD plus high-fidelity DNA polymerase. The reaction volume was 50 μL, containing 1.0 μL (10 ng / μL) of forward and reverse primers, 5 μL of dNTP mixture, 3.5 μL of MgSO4, 5 μL of buffer, 0.5 μL of polymerase, 1.0 μL of template, and water to 50 μL. The thermal cycling parameters were as follows: pre-denaturation at 98°C for 2 min; cycle stage (30 cycles) at 98°C for 10 s, 60°C for 30 s, and 68°C for 2 min; and final extension at 72°C for 5 min. After 1% agarose electrophoresis verification, the amplification product was purified using an Axygen gel recovery kit.

[0041] Enzymatic digestion and ligation: The pY26TEF-GPD vector was subjected to double enzyme digestion to obtain a linearized pY26TEF-GPD fragment. The linearized vector was amplified based on PrimeSTAR GXL high-fidelity DNA polymerase, and the enzyme digestion product was mixed with CaGlcT1 at a 1:3 molar ratio after column purification. BsaI-HFv2 and T4 DNA ligase (NEB) were added, and ligation was performed at 16°C overnight.

[0042] Transformation and screening: The reaction solution was transformed into E. coli DH5α competent cells (Thermo Fisher), and plated on LA plates containing 100 μg / mL ampicillin and incubated at 37°C for 16 h. After preliminary screening by colony PCR, 10 positive clones were randomly selected for Sanger sequencing to confirm correct gene insertion. Sequencing verification was completed, and recombinant plasmid 1 was obtained.

[0043] Recombinant plasmid 1 was subjected to linearization as above, and the enzyme digestion product was mixed with CaGlcT2 at a 1:3 molar ratio after column purification. Recombinant plasmid 2 was obtained after positive ligation verification as above.

[0044] Recombinant plasmid 2 was subjected to linearization as above, and the enzyme digestion product was mixed with CaRhaT at a 1:3 molar ratio after column purification. The multi-gene tandem recombinant protein was obtained after positive ligation verification as above.

[0045] Example 3: Saccharomyces cerevisiae transformation, induction of expression, and product synthesis

[0046] The logarithmic phase Saccharomyces cerevisiae (OD 600The cells were prepared by washing three times with 100 mM LiAc / 10 mM DTT after pretreatment. 5-10 μL of the recombinant plasmid was mixed with 80 μL of the competent cells, and then transferred to a pre-cooled electroporation cup (0.2 cm) in an ice bath for 5 min, followed by a 1.5 kV, 25 μF, 200 Ω electric pulse (5 ms). Immediately after transformation, 1 mL of pre-cooled 1 M sorbitol solution was added, and recovery culture was performed in two steps: after standing, 1 mL of YPD liquid medium (preparation method: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, dissolved in deionized water, sterilized at 115°C for 20 min) was added, and recovery culture was performed at 30°C, 150 rpm for 2 h.

[0047] After 72 h of culture at 30°C, single colonies were randomly picked for PCR verification. The positive clones confirmed by screening were inoculated into 5 mL of YPD liquid medium, and cultured at 30°C, 200 rpm for 24 h to an OD 600 The seed liquid was prepared at a ratio of 1:1.5. In the precursor addition stage, the seed liquid was mixed with YPG liquid medium containing 1 mM hydroxyl asiatic acid (preparation method: D-galactose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, dissolved in deionized water, sterilized at 115°C for 20 min) at a volume ratio of 50:1, and cultured at 20°C, 150 rpm for 72 h to promote the synthesis of hydroxyl asiatic acid.

[0048] Example 4: Optimization of fermentation conditions of the engineered strain

[0049] To achieve efficient operation of the fermentation system of the engineered strain, comprehensive and in-depth multi-dimensional optimization research was carried out, and the carbon and nitrogen source ratio and induction parameters were finely adjusted. Under the culture system of 50 mL of YPG liquid medium (preparation method: D-galactose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, dissolved in deionized water, sterilized at 115°C for 20 min), the medium composition and culture conditions of the engineered strain were optimized in all aspects, including the concentration of D-galactose, the concentration of yeast extract, the concentration of peptone, the OD 600 value during fermentation culture, and the induction temperature. The optimized culture conditions are as follows: the concentration of D-galactose is 25 g / L, the concentration of yeast extract is 15 g / L, the concentration of peptone is 20 g / L, the OD 600= 1.0, induction temperature 25°C. The conversion yield of hydroxyas- tragaloside A before optimization (initial process without fermentation condition optimization) was only 125 mg / L, while the conversion yield of hydroxyas- tragaloside A after optimization was 165 mg / L, which was 32% higher than that before optimization, confirming that the metabolic regulation strategy of the present application can significantly enhance the product synthesis efficiency.

[0050] Table 2 Comparison of fermentation condition optimization

[0051]

[0052]

[0053] Example 5: Fed-batch fermentation process of the engineered strain

[0054] The metabolic regulation fermentation of the engineered strain was carried out in a 5L fermenter containing 2L medium. After the strain was obtained by step-by-step expansion of YPD liquid medium to obtain secondary seed liquid, it was transferred to the fermenter at a 10% inoculation amount, and 5mM hydroxyas- tragaloside acid precursor was added according to the method of Example 3. Based on the previous optimization parameters, a two-stage dynamic feeding strategy was established:

[0055] Primary culture stage: the initial concentration of D-galactose was increased to 50g / L to strengthen the induction efficiency, and the dissolved oxygen was maintained at ≥30% by 400rpm stirring rate and 1.2vvm aeration, and the pH was stabilized at 5.5. When the carbon source was close to exhaustion after 18h of culture, constant flow addition (0.5g / L / h) of feeding medium I (formula: 120g / L D-galactose, 5g / L yeast extract, 2g / L KH2PO4, 1g / L MgSO4·7H2O, solvent: water) was started to precisely control the D-galactose concentration in the tank at 1.0g / L level. After 48h, the strain biomass tended to be stable, OD 600 169.51, completing the cell proliferation stage.

[0056] Secondary metabolism stage: switch to ethanol + D-galactose compound carbon source strategy, initially add ethanol at a low rate of 0.1g / L / h, while maintaining the D-galactose concentration at 1.0g / L to ensure continuous activation of the GAL1 promoter. At 48h, the ethanol concentration was high, so the ethanol was added at a lower rate, and after 90h, the ethanol concentration was detected to be reduced to 18.2g / L (lower than the preset lower limit of 20g / L), and then the ethanol addition rate was increased to stabilize the ethanol concentration in the range of 20-25g / L, which was determined to be beneficial to efficient synthesis of the target product in the previous optimization, until the end of fermentation. The whole process monitoring showed that after the strain concentration increased, the product synthesis rate increased rapidly, and the final yield reached 582mg / L, which was 3.5 times higher than that of the initial process (165mg / L) of Example 4, meeting the industrial production standard.

[0057] Example 6: Isolation and purification of hydroxyl asiaticoside and identification of metabolites

[0058] The fermentation broth in a 5L fermenter was treated by a disc centrifuge (8000xg, 10 min) to collect the bacterial cells, which were then resuspended with a pre-cooled lysis buffer (50mM Tris-HCl pH 8.0, 1mM EDTA), ultrasonically broken (parameters: 300W, 5 seconds on / 5 seconds off, total duration 10 min), and centrifuged again to take the supernatant. The supernatant was preliminarily adsorbed by HPD700 macroporous resin, and then further purified by a C18 reverse-phase chromatographic column. The purified concentrated solution was filtered by a 0.22μm filter membrane and used for HPLC and LC-MS analysis.

[0059] The chromatographic separation of the purified concentrated solution was performed by HPLC, in which the chromatographic column was Agilent Zorbax Extend C-18 (250x4.6mm, 5μm), the mobile phase A was acetonitrile, and the mobile phase B was a 2mM β-cyclodextrin aqueous solution containing 0.1% phosphoric acid. The initial ratio (A:B) was 20:80, and the gradient elution conditions were as follows: 0-8min acetonitrile ratio 20%-30%; 8-15min acetonitrile ratio 30%-40%; 15-25min acetonitrile ratio 40%-60%; 25-30min acetonitrile ratio 80%, flow rate 1mL / min, column temperature 30℃. Mass spectrometry detection was performed by a Q-TOF 6545 system, with the mass spectrometry parameters being ESI negative ion mode, scanning range m / z 100-1500, ion source temperature 150℃, and desolvation gas temperature 350℃.

[0060] The standard curve of hydroxyl asiaticoside determined by HPLC had good linearity (r=0.9995), and the limit of quantification was 0.2188μg. The purity of the purified concentrated solution determined by HPLC was as high as 95%. By comparing the fragment ion ratio of the compound with that of the standard product, the compound was identified as hydroxyl asiaticoside. Figure 1

[0061] Example 7: Construction of UGT73AD1, UGT94M2 and CaUGT4 tandem expression system and yield comparison

[0062] To verify the superiority of the glycosyltransferases (CaGlcT1, CaGlcT2, CaRhaT) selected in the application, a multi-gene tandem recombinant protein of the control gene combination UGT73AD1, UGT94M2 and CaUGT4 was constructed.

[0063] The UGT73AD1 gene sequence is shown as SEQ ID NO: 13, and the amino acid sequence is shown as SEQ ID NO: 16.

[0064] ​The UGT94M2 gene sequence is shown as SEQ ID NO: 14, and the amino acid sequence is shown as SEQ ID NO: 17.

[0065] The CaUGT4 gene sequence is shown as SEQ ID NO: 15, and the amino acid sequence is shown as SEQ ID NO: 18.

[0066] The specific steps are as follows:

[0067] Gene cloning and vector construction: referring to the method of Example 2, UGT73AD1, UGT94M2, CaUGT4 are sequentially inserted into pY26TEF-GPD vector to construct recombinant plasmid 3.

[0068] Saccharomyces cerevisiae transformation, induction and fermentation expression: the recombinant plasmid 3 is transformed into Saccharomyces cerevisiae for induction expression under the conditions of Example 3, and then induction fermentation expression is carried out under the same culture conditions (i.e. optimized fermentation conditions) of Example 4, and the yield of transformed hydroxyl asperosaponin is taken as the comparative yield.

[0069] Product detection and yield analysis: the HPLC method of Example 6 is used to detect the yield of hydroxyl asperosaponin. The results show that the combination of CaGlcT1, CaGlcT2 and CaRhaT genes used in the present application, by optimizing the enzyme sequence and regulatory elements, has a significantly higher yield of hydroxyl asperosaponin (165 mg / L) than the combination of UGT73AD1, UGT94M2 and CaUGT4 genes (118 mg / L), achieving an unexpected technical effect.

[0070] Example 8: combination of hydroxyl asperosaponin with gold chrysanthemum extract and purslane extract

[0071] The purified hydroxyl asperosaponin solution of Example 6 is combined with gold chrysanthemum extract and purslane extract to prepare an antioxidant whitening product. The composition, based on 100% total mass percentage, comprises the following components: hydroxyl asperosaponin, gold chrysanthemum extract, purslane extract, and water in excess.

[0072] The preparation method of the above composition comprises the following steps:

[0073] 1) Mix the gold chrysanthemum extract with water to obtain a gold chrysanthemum extract solution;

[0074] 2) Mix the purslane extract with the hydroxyl asperosaponin solution, and homogenize it with a high-pressure microfluidizer (using a Y-shaped alternating homogenizing cavity, operating pressure 800-1000 bar, flow rate 5 L / h, temperature 25℃±2℃, circulating 3 times) to obtain a plant extract mixture;

[0075] 3) Mix the mixture of step 2 with the solution of step 1, homogenize again, filter, and obtain the plant extract composition.

[0076] Subsequently, the madecassic acid, the chamomile extract, and the spilanthes acmella extract were set in several groups with different mass percentages for combination. The following is the mass combination of raw materials (the total mass of the composition is 10 g).

[0077] Table 3 Raw materials of the composition and their mass fractions

[0078]

[0079] To verify the best synergistic ratio of madecassic acid, chamomile extract, and spilanthes acmella extract among different compositions, and to verify the relationship between the components in the composition, the following performance tests were performed, as shown in Table 3.

[0080] Example 9: Antioxidant performance test-DPPH free radical scavenging

[0081] The determination principle of DPPH free radical scavenging capacity is based on the characteristic color reaction of its ethanol solution at the maximum absorption wavelength of 517 nm. The deep purple solution of this free radical will fade under the action of antioxidants, and the degree of absorbance attenuation can be quantitatively characterized by spectrophotometric method.

[0082] Test design: Dissolve DPPH powder in anhydrous ethanol to prepare a 20 mM stock solution (the working concentration needs to be adjusted according to the pre-experiment results). Take 1 mL of the sample to be tested and 1 mL of DPPH solution (final concentration 10 mM) and mix thoroughly in the dark. Measure the absorbance at 517 nm after standing at room temperature for 30 min (recorded as A1). Set two groups of controls: ① sample group (1 mL sample + 1 mL ethanol, absorbance A2); ② blank control group (1 mL DPPH solution + 1 mL ethanol, absorbance A3). Each group sets 3 parallels, and the data is taken as the mean to eliminate systematic error.

[0083] The DPPH free radical scavenging rate of each test sample is calculated according to the following formula and recorded in Table 4 below.

[0084] Scavenging rate (%) = (1-(A1-A2) / A3) x 100%

[0085] In the formula: A1 is the absorbance of 1 mL of test sample + 1 mL of DPPH solution mixture; A2 is the absorbance of 1 mL of test sample + 1 mL of anhydrous ethanol mixture; A3 is the absorbance of 1 mL of DPPH solution + 1 mL of anhydrous ethanol mixture.

[0086] Table 4 DPPH scavenging rate of different compositions

[0087]

[0088] Example 10: Melanin inhibition efficacy test

[0089] Test grouping: ① Blank control group: containing zebrafish embryos and standard dilution water, one blank control group was set for each test; ② Composition test group: different compositions and zebrafish embryos.

[0090] Composition treatment: The composition concentration range of the formal test was determined according to the results of the pre-test. Normal 6hpf zebrafish embryos were selected and placed in a six-well plate, 15 embryos per well. After removing the standard dilution water, 3mL of composition was added, mixed well and incubated in the dark for 45h (28.0℃±0.5℃) using a constant temperature incubator.

[0091] Observation, photography and image analysis: After incubation, 12 normal zebrafish were randomly selected and fixed with 3% methyl cellulose, and photographed under a stereomicroscope (head left, abdomen down, consistent conditions). After photography, image analysis software was used to analyze the melanin signal intensity (optical density value OD) of the zebrafish head, and the relative content of melanin was calculated: relative content of melanin = absorbance value of sample treatment group / absorbance value of blank control group.

[0092] Table 5 Relative content of melanin of different compositions

[0093]

[0094] Example 11: Trans-epidermal water loss effect test

[0095] (1) Test instrument: Cutommeter dual MPA580 skin tester

[0096] (2) Test method: 190 female volunteers were selected, who had no systemic diseases and other skin disorders, had not used other skin care products within 2-3 days, were not in pregnancy or lactation period, and voluntarily participated in the test throughout the process; the 190 volunteers were randomly divided into 19 groups, 10 people in each group, and used the product twice a day, once in the morning and once in the evening, for 28 consecutive days. The blank control group only used the base of the composition - water. The TEWL values of the volunteers in each group before and after 28 days of use were tested, and the test results are shown in Table 6.

[0097] Table 6 Change rate of trans-epidermal water loss TEWL value of different compositions

[0098]

[0099] The present application screens three glycosyltransferase genes CaGlcT1, CaGlcT2 and CaRhaT from the transcriptome of Centella asiatica, verifies the catalytic activity, and performs multi-gene co-expression in Saccharomyces cerevisiae. After adding the substrate hydroxyl asiatic acid, the hydroxyl asiatic acid glycoside can be heterogeneously catalyzed. In addition, after the fermentation condition optimization and batch feeding fermentation, the conversion yield of hydroxyl asiatic acid glycoside reaches 165 mg / L, which is significantly improved compared with the yield (118 mg / L) of hydroxyl asiatic acid glycoside converted by the three genes UGT73AD1, UGT94M2 and CaUGT4 in the comparative experiment. After separation and purification, the purity of hydroxyl asiatic acid glycoside reaches more than 95%, meeting the commercial application and scientific research needs. The purified hydroxyl asiatic acid glycoside is compounded with chamomile extract and purslane extract at a mass ratio of 3:5:2, and a synergistic effect is obtained. In the DPPH clearance rate in antioxidant, the inhibition of melanin generation and the test of anti-inflammatory trans-epidermal water loss, the performance is excellent, which is better than that of single component.

[0100] Since asiatic acid glycoside cannot be purchased and is difficult to extract or synthesize, we screen three glycosyltransferase genes CaGlcT1, CaGlcT2 and CaRhaT from the transcriptome of Centella asiatica, use multi-gene tandem recombination to construct an expression vector, transfer it into Saccharomyces cerevisiae, and synthesize hydroxyl asiatic acid glycoside in Saccharomyces cerevisiae by feeding the low-cost hydroxyl asiatic acid as a substrate. The multi-gene tandem recombinant protein catalytic step is as shown in Figure 1 The protein expression SDS-PAGE detection result is as shown in Figure 2 The liquid phase detection result is as shown in Figure 3 CaGlcT1 catalyzes the synthesis of hydroxyl asiatic acid monosaccharide glycoside from hydroxyl asiatic acid, CaGlcT2 catalyzes the synthesis of hydroxyl asiatic acid disaccharide glycoside from hydroxyl asiatic acid monosaccharide glycoside, and CaRhaT is responsible for catalyzing the formation of hydroxyl asiatic acid glycoside from hydroxyl asiatic acid disaccharide glycoside. The MS / MS detection result of the synthesis of hydroxyl asiatic acid glycoside in Saccharomyces cerevisiae is as shown in Figure 4 .

[0101] >SEQ ID NO:1

[0102]

[0103] >SEQ ID NO:2

[0104]

[0105] >SEQ ID NO:3

[0106]

[0107] >SEQ ID NO:4

[0108] MASNIQQLHFVLVPLMSQSHIIPLTDFGKLLAQRGVVVTMIKTPLNAVRCKPIIDGAQ NANLNIQLASLHFPTREVGLPEGVENLDELSMKSLKTLGINFFQANEMLREPLEKLLA EMVPRPSCIISTDALPWTNEVAWKFKIPRYSFNTISCFSLVLFHKLKISRVHESVTSD SESFVVPGLPDKIELKRSQLPEWVKRTSNDNNSHVMDKLKATEHLPRGVLVNTFEEM EPRYVEEFKKQEKLVFCVGSVSLCNTDQSDMSSRGNKASVEEDSCLKWLDSMKPCSVI YVCFGSLSNIPYPQLIELGLGLEASNRPFIWIIRKGDYSPRLEKWLEEYRFEEKVKGR GLIIRGWAPLVLILSHPSVGGFFTHCGWNSILEAVCEGMPMITWPMFAEQFYNERFV VHVVKIGVPVGVEEALTSVEDEEKFELLVTSSQVKEAIDELMYGEDAGERRKRAREL GEKAKKAIEDGGSSYLYITNLIEDVRQQNSSRF

[0109] >SEQ ID NO: 5 MVTNSKDNNALRILMFPWLGHGHISPYLELAKKLSRKDSKIYFRSTPINLKPIKNKILDYNSIELIEFPLPSSQELPPHYHKTTGLPPHLLPALKDAFEMASPQLSNILDTITPDLLIYDIYQPWVPKLASSYKIPAVHFQTTGATAISYFYRLSMNLKTAFPSTIIHLKDIELVRMLETGGGDESDPENEQDRDRVFDSILGSVEILLIKSSREIEGKYIDCLSDFIKKKIVSVGPLVQEFVGNDQENDDVITWLSKKEPFSTVYVSFGTESFLSKKDLEVLAHGLELSGVNFIWALKFPEAEKITKVEEALPQGFLERVGEKGLVLGGWVPQAKILNHSSIGGFVSHCGWSSVIESLSFGVPIIAMPLQNDQPLNARLVVEVGVGLEVEKDDKLEFGREEVARVVKEVVVEEKSRETFGKRVKELSEVLKVKADEDVDNAIKELKRLCENNLRKVE

[0110] >SEQ ID NO: 6

[0111] MAIKEHSNLRVLMFPWLAYGHISPYLELAKKLSKRNFHIYFCSTPVNLNSIKTRITPSNDDFNAKSLELVELHLPTFPNLPPHLHTTNGLPPELDSTLVKAADMARPNFEDIMKDLKPDLIIYDVMQSWVSDLASWYAIPAVHLQILPATALSYIRCMFGGKQFPSPAIFIRPAEIKIMTKGAPKYEEGEDPMTASFKRSCEIVLIQSSKNFENEYIDFLSDLVDKKVVPVGPLIQAAAPPKEEDDKIMEWLNRKEADSTVLVSFGSQYYLSKEELEELAYGLELSQVNFVWVIRFPKGEEVKAVEVLPQGFMDRVGDRGLLVEGWAPQTKILEHSSIGGFVSHCGWNSATESMSLGVPIIAIPMVIEQPLNCRRVVERGVGMEVMKDENSEFDREEIARVIREVVVEKSGEEIRRKAQELSENVRHTSDEEVNNVAQELRKLCNKKLDAHPF

[0112] SEQ ID NO: 7

[0113] ATGGCATCCAACATTCA

[0114] SEQ ID NO: 8

[0115] TCAAAATCGGCTTGAATT

[0116] SEQ ID NO: 9

[0117] ATGGTTACCAATAGCAAAG

[0118] SEQ ID NO: 10

[0119] TTATTCCACTTTCCTCA

[0120] SEQ ID NO: 11

[0121] ATGGCTATCAAAGAACAT

[0122] SEQ ID NO: 12

[0123] TTAAAAGGGGTGTGCGTCC

[0124] >SEQ ID NO: 13

[0125]

[0126] >SEQ ID NO: 14

[0127]

[0128] >SEQ ID NO: 15

[0129]

[0130] >SEQ ID NO: 16

[0131] MASNIQQLHFVLVPLMSQSHIIPLTDFGKLLAQRGVVVTMITTPLNAVRCKPIIDGAQ NANLNIQLASLHFPTREVGLPEGVENLDELSMKSLKTLGINFFQANEMLREPLEKLLA EMVPRPSCIISTDALPWTNEVAWKFKIPRYSFNTISCFSLVLFHKLKISRVHESVTSD SESFVVPGLPDKIELKRSQLPEWVKRTSNDNNSHVMDKLKATEHLPRGVLVNTFEEME PRYVEEFKKQEKLVFCVGSVSLCNTDQSDMSSRGNKASVEEDSCLKWLDSMKPCSVI YVCFGSLSNIPYPQLIELGLGLEASNRPFIWIIRKGDYSPRLEKWLEEYRFEEKVKGR GLIIRGWAPQVLILSHPSVGGFFTHCGWNSILEAVCEGMPMITWPMFAEQFYNERFVVD VVKIGVPVGVEEALTSVEDEEKFELLVTSSQVKEAIDELMDGEDAGERRKRARELGE KAKKAIEDGGSSYLYITNLIEDVRQQNSSRF

[0132] >SEQ ID NO: 17

[0133] MVTNSKDNNALRILMFPWLGHGHISPYLELAKKLSRKDSKIYFCSTPINLKPIKNKILDYNSIELIEFPLPSSQELPPHYHTTTGLPPHLLPALKDAFEMASPQLSNILDTITPDLLIYDIYQPWVPKLASSYKIPAVHFQTTGATAISYFYRLSMNLKTAFPSTIIHLKDIELVRMLETGGGDESDPENEQDRDRVFDSILGSVEILLIKSSREIEGKYIDCLSDFIKKKIVSVGPLVQEFVGNDQENDDVITWLSKKEPFSTVYVSFGTESFLSKKDLEELAHGLELSGVNFIWALKFPEAEKITKVEEALPQGFLERVGEKGLVLGGWVPQAKILNHSSIGGFVSHCGWSSVIESLSFGVPIIAMPLQNDQPLNARLVVEVGVGLEVEKDDKLEFGREEVARVVKEVVVEEKSRETFGKRVKELSEVLKVKADEDVDNAIKELKRLCENNLRKVE

[0134] >SEQ ID NO: 18

[0135] MAIKEHSNLRVLMFPWLAYGHISPYLELAKKLSKRNFHIYFCSTPVNLNSIKTRLTPSNDDFNAKSLELVELHLPTLPNLPPHLHTTNGLPPELDSTLVKAADMARPNFEDIMKDLKPDLIIYDVMQSWVSDLASWYAIPAVHLQILPATALSYIRCMFGGKQFPSPAIFIRPAEIKIMTKGAPKYEEGEDPMTASFKRSCEIVLIQSSKNFENEYIDFLSDLVDKKVVPVGPLIQAAAPPKEEDDKIMEWLNRKEADSTVLVSFGSQYYLSKEELEELAYGLELSQVNFVWVIRFPKGEEVKAVEVLPQGFMDRVGDRGLLVEGWAPQTKILEHSSIGGFVSHCGWNSATESMSLGVPIIAIPMVIEQPLNCRRVVELGVGMEVMKDENSEFDREEVARVIREVVVEKSGEEIRRKAKELSENVRHTSDEEVNNVAQELRKLCNKKLDAHPF

[0136] The above detailed description is a specific description for the embodiments of the present application, which is not used to limit the patent scope of the present application. Any equivalent implementation or change made without departing from the present application shall be included in the patent scope of the present application.

Claims

1. A multi-gene tandem recombinant protein, characterized in that, The multi-gene tandem recombinant protein is that the glycosyltransferase-encoding genes with nucleotide sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are inserted into the same expression vector in sequence and formed by simultaneously expressing the three glycosyltransferases in a host cell.

2. The multi-gene tandem recombination protein according to claim 1, characterized in that, The amino acid sequence of the glycosyltransferase encoded by the gene as shown in SEQ ID NO: 1 is as shown in SEQ ID NO: 4, the amino acid sequence of the glycosyltransferase encoded by the gene as shown in SEQ ID NO: 2 is as shown in SEQ ID NO: 5, and the amino acid sequence of the glycosyltransferase encoded by the gene as shown in SEQ ID NO: 3 is as shown in SEQ ID NO:

6.

3. A recombinant expression vector, characterized in that, The glycosyltransferase-encoding genes with nucleotide sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3.

4. An engineered bacterium, characterized in that, The product obtained after transforming the recombinant expression vector of claim 3.

5. The engineered bacterium of claim 4, wherein, The host bacteria of the engineered bacteria include yeasts.

6. Use of the multi-gene tandem recombinant protein of claim 1, the recombinant expression vector of claim 3 or the engineered bacteria of claim 4 in the preparation of an enzyme catalyst in one or more of the following reactions: (D1) catalyzing the generation of hydroxyl aescin monosaccharide from hydroxyl aescin; (D2) catalyzing the generation of hydroxyl aescin disaccharide from hydroxyl aescin monosaccharide; (D3) catalyzing the generation of hydroxyl aescin from hydroxyl aescin disaccharide.

7. Use of the multi-gene tandem recombinant protein of claim 1, the recombinant expression vector of claim 3 or the engineered bacteria of claim 4 in the preparation of hydroxyl aescin.

8. Use according to claim 7, characterized in that, The preparation of the hydroxyl aescin is based on hydroxyl aescin as a starting material.

9. Use of a composition comprising hydroxyphylllum root extract, chamomile extract, and spilanthes extract in the preparation of an antioxidant whitening product, characterized in that, The hydroxyl aescin is hydroxyl aescin with a purity of ≥ 95% produced by fermentation of the engineered bacteria of claim 4, and the mass ratio of the hydroxyl aescin, the golden chamomile extract and the purslane extract is 3:5:

2.

10. A process for the preparation of madecassoside, characterized in that, The engineered bacteria of claim 4 are cultured using YPD liquid medium to obtain seed liquid, and then the seed liquid is inoculated into YPG liquid medium containing hydroxyl aescin to culture and obtain.

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