A method for efficiently preparing hydroxyl asiaticoside based on yeast metabolic engineering and a compound cosmetic composition thereof

Abstract

The application discloses a kind of based on yeast metabolic engineering's hydroxyl asiaticoside high-efficiency preparation method and its compound cosmetic composition.The application is screened from asiaticoside transcriptome CaGlcT1, CaGlcT2 and CaRhaT three glycosyltransferases, after verifying its catalytic activity, multi-gene co-expression is carried out in saccharomyces cerevisiae, and hydroxyl asiaticoside can be catalyzed to synthesize hydroxyl asiaticoside.The conversion yield of hydroxyl asiaticoside reaches 582mg / L from 165mg / L after fermentation condition optimization and batch feeding fermentation, realizes 3.5 times yield leap, obtains the purity of hydroxyl asiaticoside reaches more than 95%, satisfies commercial application and research demand.Hydroxyl asiaticoside obtained is compounded with golden yellow chamomile extract and spilanthes extract, and plays the synergistic antioxidant whitening effect.The application provides a new scheme for green intelligent manufacturing of cosmetic raw materials.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and cosmetic raw material preparation technology, specifically to a highly efficient preparation method of hydroxyasiaticoside based on yeast metabolic engineering and its compound cosmetic composition. Background Technology

[0002] Madcassoside is one of the main active ingredients in Centella asiatica, possessing significant anti-inflammatory, antioxidant, and wound-healing pharmacological activities, and is widely used in the pharmaceutical and cosmetic fields. Currently, industrial production of madcassoside mainly relies on plant extraction methods (such as ethanol-ultrasound-assisted extraction and steam distillation), but faces 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 active ingredients is affected by environmental fluctuations, leading to unstable raw material supply and high costs; ② Low extraction efficiency: Ultrasound-assisted enzymatic hydrolysis (such as cellulase treatment) can increase the extraction rate to 1.92%, but multiple purification steps are still required, making the process complex and energy-intensive; ③ Difficult chemical synthesis: Madcassoside contains multiple glycosylation sites, resulting in complex chemical synthesis pathways, low purity, and complex processes.

[0003] The development of synthetic biology has provided innovative pathways for the heterologous synthesis of plant-derived natural products. Using microorganisms (such as Saccharomyces cerevisiae) as hosts, and through modular assembly and optimization of metabolic pathways, the efficient synthesis of natural products such as asiaticoside can be achieved. Compared with traditional extraction processes, asiaticoside prepared using synthetic biology techniques not only significantly reduces production costs but also strictly ensures product quality and safety. By optimizing metabolic pathways, high-purity asiaticoside can be produced on a large scale to meet the needs of commercial applications and scientific research experiments. This innovative synthetic strategy based on gene editing and metabolic engineering is reshaping the production model of plant-derived active ingredients, providing sustainable solutions for the modern biopharmaceutical industry.

[0004] In the fields of pharmacology and cosmetics, asiaticoside has been demonstrated to possess a variety of biological functions. In Centella asiatica, key genes involved in multiple steps of triterpenoid biosynthesis have been identified, including farnesyl pyrophosphate synthase (CaFPS), squalene synthase (CaSQS), β-amyrin synthase (CaβAS), and dammarane synthase (CaDDS). Modifications to the triterpenoid skeleton include oxidation, substitution, or glycosylation via P450 monooxygenases, UDP-Glc glycosyltransferases, and other enzymes. Glycosyltransferases are key enzymes in plant glycosylation processes and are crucial for the bioactivity of saponins. Existing studies have shown that glycosyltransferases such as UGT73AD1, UGT94M2, and CaUGT4 have individually demonstrated asiaticoside monosaccharide glycosylation activity (e.g., UGT73AD1 catalyzes the formation of asiaticoside monosaccharides), but the current technology has not reported on the tandem co-expression of multiple glycosyltransferases to achieve a multi-step glycosylation reaction from asiaticoside to asiaticoside.

[0005] Most whitening and antioxidant products on the market currently have a single mechanism of action, and single plant extracts often cannot comprehensively address the antioxidant problem. Therefore, it is necessary to develop a combination with multiple pathways that can effectively and instantly whiten and brighten skin with minimal side effects. Plant cultivation cycles are typically long, raw material supply is unstable, and costs are high. Furthermore, natural plant active ingredients generally suffer from poor solubility, low permeability, and low absorption rates. Therefore, applying synthetic biology to overcome the catalytic efficiency bottleneck of existing enzyme combinations, combining novel glycosyltransferase genes, producing and developing an active raw material, and applying it to an efficient, industrially scalable biomanufacturing system to achieve low-cost, high-yield conversion of inexpensive substrates into high-value hydroxyasiaticoside is of significant practical importance. Summary of the Invention

[0006] The first objective of this invention is to provide a multi-gene tandem recombinant protein, which is formed by sequentially inserting glycosyltransferase encoding genes, such as those shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, into the same expression vector, and by simultaneously expressing these three glycosyltransferases (UGTs) in host cells.

[0007] Transcriptome sequencing was used to analyze different tissues (roots, stems, and leaves) of Centella asiatica. Using bioinformatics methods such as local BLAST, sequence alignment, and phylogenetic analysis, three functional UGTs were identified and screened, named CaGlcT1, CaGlcT2, and CaRhaT, respectively. Their nucleotide sequences correspond to SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and their amino acid sequences correspond to SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0008] A second objective of this invention is to provide a recombinant expression vector containing a glycosyltransferase encoding gene with nucleotide sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0009] The vectors described in this invention are well known to those skilled in the art, including but not limited to: plasmids, bacteriophages, viral vectors, etc. In one embodiment of this invention, the vector is the pY26TEF-GPD vector.

[0010] A third objective of this invention is to provide an engineered bacterium obtained by transforming the aforementioned recombinant expression vector.

[0011] Preferably, the host bacteria of the engineered bacteria include, but are not limited to, yeast.

[0012] A fourth objective of this invention is to provide the application of the above-mentioned multi-gene tandem recombinant proteins, recombinant expression vectors, or engineered bacteria as enzyme catalysts in the preparation of one or more of the following reactions:

[0013] (D1) catalyzes the formation of hydroxyasiatic acid monosaccharide from hydroxyasiatic acid;

[0014] (D2) catalyzes the formation of hydroxyasiatic acid monosaccharide from hydroxyasiatic acid disaccharide;

[0015] (D3) catalyzes the formation of hydroxyascorbic acid disaccharide glycoside into hydroxyascorbic acid glycoside.

[0016] The fifth objective of this invention is to provide the application of the above-mentioned multi-gene tandem recombinant protein, recombinant expression vector or engineered bacteria in the preparation of asiaticoside.

[0017] Preferably, the preparation of the asiaticoside is based on asiatic acid as the starting material.

[0018] The sixth objective of this invention is to provide the application of a composition of asiaticoside, golden chamomile extract, and purslane extract in the preparation of an antioxidant whitening product, wherein the asiaticoside is asiaticoside with a purity ≥95% produced by fermentation with the aforementioned engineered bacteria, and the preferred mass ratio of asiaticoside, golden chamomile extract, and purslane extract is 3:5:2.

[0019] Preferably, the antioxidant whitening products include, but are not limited to, face masks, moisturizing creams, lotions, or facial cleansers.

[0020] The seventh objective of this invention is to provide a method for preparing asiaticoside, comprising the following steps: culturing the above-mentioned engineered bacteria in YPD liquid medium to obtain a seed culture; and then inoculating the seed culture in YPG liquid medium containing asiaticoside to obtain the final product.

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

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

[0023] This invention screened and identified three glycosyltransferases (CaGlcT1, CaGlcT2, and CaRhaT) involved in the biosynthesis of asiaticoside through a combination of genomic and transcriptomic methods. An expression vector was constructed using multi-gene tandem recombination to achieve heterologous expression in *Saccharomyces cerevisiae*. Optimized fermentation conditions significantly increased the yield of asiaticoside in *Saccharomyces cerevisiae*. Compared to the control experiment using the three-gene tandem expression of UGT73AD1, UGT94M2, and CaUGT4, the asiaticoside yield was only 118 mg / L, significantly lower than the gene combination of this invention (165 mg / L). The compound composition (asiaticoside: chamomile extract: purslane extract = 3:5:2) showed synergistic effects, demonstrating superior performance in antioxidant (DPPH scavenging rate 88.24%), inhibition of melanin production (relative content reduction 54.58%), and anti-inflammatory transepidermal water loss (TEWL value reduction 31.42%), significantly outperforming single components. Attached Figure Description

[0024] Figure 1 This describes the synthetic pathway from asiatic acid to asiaticoside.

[0025] Figure 2 This is an SDS-PAGE electrophoresis image of multi-gene tandem recombinant proteins. 1 represents the multi-gene tandem recombinant proteins CaGlcT1, CaGlcT2, and CaRhaT; 2 represents the multi-gene tandem recombinant proteins UGT73AD1, UGT94M2, and CaUGT4; M represents the marker.

[0026] Figure 3 The results are obtained by HPLC analysis of asiatic acid standards, asiaticoside standards, and asiatic acid standards plus multi-gene (CaGlcT1, CaGlcT2, and CaRhaT) tandem recombinant proteins.

[0027] Figure 4 MS / MS results for the synthesis of asiaticoside in Saccharomyces cerevisiae. Detailed Implementation

[0028] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0029] Unless otherwise specified, all experimental methods involved in this technical solution employ general techniques in the field of biochemistry. All materials, reagents, and equipment required for the experiments, unless otherwise indicated, can be obtained through conventional commercial channels.

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

[0031] (1) Using identified functional glycosyltransferase genes, 143 potential glycosylation modification-related genes were obtained from the measured Centella asiatica transcriptome via local Blast. Phylogenetic association analysis was performed between the screening results and known pentacyclic triterpenoid glycosyltransferases using phylogenetic tree construction technology.

[0032] (2) Transcriptome expression profile data were integrated to analyze the specific expression patterns of each candidate gene in root, stem, and leaf tissues. Functional verification experiments were conducted on high-expression targets in leaf tissues, and finally, glycosyltransferase genes CaGlcT1, CaGlcT2, and CaRhaT with catalytic functions were screened out.

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

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

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

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

[0037] Fresh Centella asiatica leaf tissue was collected, and total RNA was isolated using the EASYspin Plant RNA Rapid Extraction Kit (Aidlab). cDNA was then reverse transcribed using a reverse transcription kit (Novozymes). Specific amplification primers were designed.

[0038] Table 1 Primer sequences

[0039]

[0040] PCR amplification was performed using KOD plus high-fidelity DNA polymerase. The 50 μL reaction mixture contained: 1.0 μL each of forward and reverse primers (10 ng / μL), 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 a final volume of 50 μL. The thermal cycling parameters were: pre-denaturation at 98℃ for 2 min; cycling phase (30 cycles): 98℃ for 10 s, 60℃ for 30 s, and 68℃ for 2 min; final extension at 72℃ for 5 min. The amplified products were verified by 1% agarose gel electrophoresis and purified using an Axygen gel extraction kit.

[0041] Enzyme digestion-ligation reaction: The pY26TEF-GPD vector was double-digested to obtain a linearized pY26TEF-GPD fragment. The linearized vector was amplified using PrimeSTAR GXL high-fidelity DNA polymerase. After column purification, the digestion product was mixed with CaGlcT1 at a 1:3 molar ratio, and BsaI-HFv2 and T4 DNA ligase (NEB) were added. The mixture was ligated overnight at 16°C.

[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. The cells were incubated at 37°C for 16 h. After initial 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] The recombinant plasmid 1 was linearized as described above. The enzyme digestion product was purified by column chromatography and then mixed with CaGlcT2 at a molar ratio of 1:3. After ligation verification as described above, recombinant plasmid 2 was obtained.

[0044] The recombinant plasmid 2 was linearized as described above. The enzyme digestion product was purified by column chromatography and then mixed with CaRhaT at a molar ratio of 1:3. After ligation verification as described above, the multi-gene tandem recombinant protein was obtained.

[0045] Example 3: Transformation, Induced Expression and Product Synthesis of Saccharomyces cerevisiae

[0046] Electroporation was used to study the logarithmic growth phase of Saccharomyces cerevisiae (OD). 600Transformation was performed using a solution of 0.8 g / mL. After pretreatment with 100 mM LiAc / 10 mM DTT, competent cells were prepared by washing three times with electroporation buffer. 5-10 μL of recombinant plasmid was mixed with 80 μL of competent cells and transferred to a pre-chilled electroporation cuvette (0.2 cm) on ice for 5 min, followed by an application of a 1.5 kV, 25 μF, 200 Ω electrical pulse (5 ms). Immediately after transformation, 1 mL of pre-chilled 1 M sorbitol solution was added. The recovery culture was performed using a two-step method: after standing, 1 mL of YPD liquid medium (preparation method: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, dissolved in deionized water, sterilized at 115 °C for 20 min) was added, followed by recovery at 30 °C and 150 rpm for 2 h with shaking.

[0047] In the screening and validation stage, the revived bacterial culture was plated on YPD solid medium (preparation method: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, 20 g / L agar, dissolved in deionized water, sterilized at 115℃ for 20 min), and incubated at 30℃ for 72 h. Single colonies were then randomly selected for PCR validation. Confirmed positive clones were inoculated into 5 mL of YPD liquid medium and incubated at 30℃, 200 rpm for 24 h until OD500 reached. 600 =1.5 Preparation of seed culture. Precursor addition stage: The seed culture was mixed with YPG liquid medium containing 1mM asiaticoside at a volume ratio of 50:1 (preparation method: D-galactose 20g / L, peptone 20g / L, yeast extract 10g / L, dissolved in deionized water, sterilized at 115℃ for 20min), and cultured at 20℃ and 150rpm for 72h to promote the synthesis of asiaticoside.

[0048] Example 4: Optimization of fermentation conditions for engineered strains

[0049] To achieve efficient operation of the engineered strain fermentation system, this invention conducted a comprehensive and in-depth multi-dimensional optimization study, systematically fine-tuning the carbon-nitrogen source ratio and induction parameters. Under a 50 mL YPG liquid medium (preparation method: 20 g / L D-galactose, 20 g / L peptone, 10 g / L yeast extract dissolved in deionized water, sterilized at 115℃ for 20 min), the culture medium composition and culture conditions of the engineered strain were comprehensively optimized, specifically including D-galactose concentration, yeast extract concentration, peptone concentration, and OD during fermentation. 600 The optimal culture conditions were determined by factors such as D-galactose concentration 25 g / L, yeast extract concentration 15 g / L, peptone concentration 20 g / L, and OD200 concentration 25 g / L. 600=1.0, induction temperature 25℃. Before optimization (initial process without fermentation condition optimization), the conversion yield of asiaticoside was only 125 mg / L, while after optimization, the conversion yield of asiaticoside was 165 mg / L, which is 32% higher than before optimization, confirming that the metabolic regulation strategy of the present invention 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 engineered strains

[0054] Metabolic regulation fermentation of the engineered bacteria was carried out in a 5L fermenter containing 2L of culture medium. After obtaining a secondary seed culture through stepwise expansion in YPD liquid medium, the strain was transferred to the fermenter at a 10% inoculum rate, and 5mM asiatic acid precursor was added according to the method in Example 3. Based on previously optimized parameters, a two-stage dynamic feeding strategy was established:

[0055] Initial culture stage: The initial concentration of D-galactose was increased to 50 g / L to enhance induction efficiency. Dissolved oxygen was maintained at ≥30% and pH was stabilized at 5.5 using a stirring rate of 400 rpm and an aeration rate of 1.2 vvm. After 18 h of culture, when the carbon source was nearly depleted, fed medium I (formulation: 120 g / L D-galactose, 5 g / L yeast extract, 2 g / L KH2PO4, 1 g / L MgSO4·7H2O, solvent: water) was started at a constant feed rate (0.5 g / L / h) to precisely control the D-galactose concentration in the tank at 1.0 g / L. After 48 h, the biomass of the strain tended to stabilize, and OD... 600 The count reached 169.51, completing the bacterial cell proliferation stage.

[0056] Secondary metabolic stage: The strategy was switched to an ethanol + D-galactose complex carbon source. Initially, ethanol was added at a low rate of 0.1 g / L / h, while maintaining the D-galactose concentration at 1.0 g / L to ensure continuous activation of the GAL1 promoter. At 48 h, the ethanol concentration was high, so the ethanol was added at a lower rate. After 90 h, the ethanol concentration dropped to 18.2 g / L (below the preset maintenance limit of 20 g / L), and the ethanol addition rate was increased to stabilize the ethanol concentration within the range of 20-25 g / L. This range was determined in the previous optimization to be conducive to the efficient synthesis of the target product until the end of fermentation. Monitoring throughout the process showed that the product synthesis rate surged after the microbial community concentration increased, with a final yield of 582 mg / L, 3.5 times higher than the initial process in Example 4 (165 mg / L), meeting industrial production standards.

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

[0058] The fermentation broth in the 5L fermenter was centrifuged using a disc centrifuge (8000×g, 10min) to collect the cells. The cells were then resuspended in pre-cooled lysis buffer (50mM Tris-HCl pH 8.0, 1mM EDTA) and sonicated (300W, 5 seconds on / 5 seconds off, total duration 10min). After centrifugation again using a disc centrifuge, the supernatant was collected. The supernatant was used to pre-adsorb the concentrate onto HPD700 macroporous resin, and then further purified by passing it through a C18 reversed-phase column. The purified concentrate was filtered through a 0.22μm filter membrane and used for HPLC and LC-MS analysis.

[0059] Chromatographic separation of the purified concentrate was performed using HPLC with an Agilent Zorbax Extend C-18 column (250 × 4.6 mm, 5 μm). Mobile phase A consisted of acetonitrile, and mobile phase B consisted of a 2 mM aqueous solution of β-cyclodextrin (containing 0.1% phosphoric acid). The initial ratio (A:B) was 20:80, with gradient elution conditions: 0-8 min acetonitrile ratio 20%-30%; 8-15 min acetonitrile ratio 30%-40%; 15-25 min acetonitrile ratio 40%-60%; 25-30 min acetonitrile ratio 80%, flow rate 1 mL / min, and column temperature 30 °C. Mass spectrometry was performed using a Q-TOF 6545 system in ESI negative ion mode, with a scan range of m / z 100-1500, ion source temperature of 150 °C, and desolvation gas temperature of 350 °C.

[0060] The standard curve for the determination of hydroxyasiaticoside by HPLC showed good linearity (r = 0.9995), with a limit of quantitation of 0.2188 μg. The purity of the concentrated solution after HPLC analysis reached 95%. Mass spectrometry fragment ion comparison revealed characteristic spectra of the compound and the standard. Figure 1 To.

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

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

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

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

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

[0066] The specific steps are as follows:

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

[0068] Transformation, induction, and fermentation expression of Saccharomyces cerevisiae: Recombinant plasmid 3 was transformed into Saccharomyces cerevisiae for induction expression according to the conditions of Example 3. Subsequently, induction and fermentation expression were carried out under the same culture conditions (i.e., optimized fermentation conditions) according to Example 4. The yield of hydroxyascorbic acid was used as the control yield.

[0069] Product detection and yield analysis: The yield of asiaticoside was determined using the HPLC method described in Example 6. The results showed that the CaGlcT1, CaGlcT2, and CaRhaT gene combination used in this invention, through optimization of enzyme sequence and regulatory elements, achieved a significantly higher yield of asiaticoside (165 mg / L) than the control group combination of UGT73AD1, UGT94M2, and CaUGT4 (118 mg / L), achieving unexpected technical results.

[0070] Example 8: Combination of hydroxyascorbic acid with extracts of golden chamomile and purslane

[0071] The purified asiaticoside solution from Example 6 was combined with golden chamomile extract and purslane extract to prepare an antioxidant whitening product. The composition, by total mass percentage (100%), comprises the following components: asiaticoside, golden chamomile extract, purslane extract, and water as the balance.

[0072] The method for preparing the above composition includes the following steps:

[0073] 1) Mix the golden chamomile extract with water to prepare a golden chamomile extract solution;

[0074] 2) The purslane extract and the asiaticoside solution were mixed and homogenized by a high-pressure microfluidic homogenizer (using a Y-shaped interactive homogenizing chamber, operating pressure of 800-1000 bar, flow rate of 5 L / h, temperature of 25℃±2℃, and 3 cycles) to obtain a mixture of plant extracts.

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

[0076] Subsequently, hydroxyascorbic acid, golden chamomile extract, and purslane extract were combined in several groups with different mass percentages. The following is the mass combination of the raw materials (the total mass of the composition is 10g).

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

[0078]

[0079] To verify the optimal synergistic ratio of m-hydroxyasiaticoside, golden chamomile extract, and purslane extract in different compositions, and to verify the relationship between the components in the compositions, see Table 3 for details, and the following performance tests were conducted.

[0080] Example 9: Antioxidant Performance Test—DPPH Free Radical Scavenging

[0081] The principle behind the determination of DPPH free radical scavenging capacity is based on the characteristic colorimetric 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 scavenging efficiency can be quantitatively characterized by detecting the degree of absorbance decay using spectrophotometry.

[0082] Experimental Design: Dissolve DPPH powder in anhydrous ethanol to prepare a 20 mM stock solution (the working concentration needs to be adjusted based on the preliminary experimental results). Take 1 mL of the test sample and 1 mL of DPPH solution (final concentration 10 mM), mix thoroughly under light-protected conditions, let stand at room temperature for 30 min, and then measure the absorbance at 517 nm (denoted as A1). Set up two control groups: 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 has 3 replicates, and the data are averaged to eliminate systematic errors.

[0083] Calculate the DPPH radical scavenging rate of each test sample according to the following formula and record it in Table 4 below.

[0084] Clearance rate (%) = (1 - (A1 - A2) / A3) × 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: Efficacy Test for Inhibiting Melanin

[0089] Experimental groups: ① Blank control group: containing zebrafish embryos and standard dilution water, with one blank control group set up for each experiment; ② Composition test group: different compositions and zebrafish embryos.

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

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

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

[0093]

[0094] Example 11: Test on transepidermal water loss

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

[0096] (2) Test Method: 190 female volunteers were selected. Volunteers had no systemic diseases or other skin conditions, had not used other skincare products in the past 2-3 days, were not pregnant or breastfeeding, and volunteered to participate in the entire trial. The 190 volunteers were randomly divided into 19 groups of 10 people each. The product was used twice daily, once in the morning and once in the evening, for 28 consecutive days. The blank control group used only the base of the product—water. The TEWL values ​​of each group of volunteers were tested before use and on day 28 after use. The test results are shown in Table 6.

[0097] Table 6. Change rate of TEWL value of different compositions after epidermal water loss

[0098]

[0099] This invention screened three glycosyltransferase genes (CaGlcT1, CaGlcT2, and CaRhaT) from the Centella asiatica transcriptome, verified their catalytic activity, and then co-expressed them in Saccharomyces cerevisiae. Upon addition of the substrate asiatic acid, these genes heterologously catalyzed the production of asiaticoside. Furthermore, after optimization of fermentation conditions and fed-batch fermentation, the yield of asiaticoside reached 165 mg / L, significantly higher than the 118 mg / L yield achieved using the tandem expression of the UGT73AD1, UGT94M2, and CaUGT4 genes in the control experiment. The purity of the purified asiaticoside reached over 95%, meeting the needs of commercial applications and research. Furthermore, a synergistic effect was achieved when the purified asiaticoside was combined with chamomile extract and purslane extract at a mass ratio of 3:5:2. This combination demonstrated superior performance in DPPH scavenging, melanin inhibition, and anti-inflammatory transepidermal water loss tests compared to the single-component formulation.

[0100] Since asiaticoside is currently unavailable and difficult to extract or synthesize, we constructed an expression vector using a multi-gene tandem recombination approach by screening three glycosyltransferase genes (CaGlcT1, CaGlcT2, and CaRhaT) from the Centella asiatica transcriptome. This vector was then transformed into Saccharomyces cerevisiae, and asiaticoside was synthesized in Saccharomyces cerevisiae by feeding it to the yeast as a substrate, thus achieving asiaticoside synthesis. The catalytic steps of the multi-gene tandem recombination protein are as follows: Figure 1 As shown, the SDS-PAGE detection results of protein expression are as follows: Figure 2 As shown, the liquid phase detection results are as follows: Figure 3 As shown, CaGlcT1 catalyzes the synthesis of asiatic acid monosaccharide from asiatic acid, CaGlcT2 catalyzes the synthesis of asiatic acid disaccharide from asiatic acid monosaccharide, and CaRhaT is responsible for catalyzing the formation of asiaticoside from asiatic acid disaccharide. The MS / MS detection results of asiaticoside synthesis in Saccharomyces cerevisiae are as follows: Figure 4 As shown.

[0101] >SEQ ID NO:1

[0102]

[0103] >SEQ ID NO:2

[0104]

[0105] >SEQ ID NO:3

[0106]

[0107] >SEQ ID NO:4

[0108] MASNIQQLHFVLVPLMSQSHIIPLTDFGKLLAQRGVVVTMIKTPLNAVRCKPIIDGAQNANLNIQLASLHFPTREVGLPEGVENLDELSMKSLKTLGINFFQANEMLREPLEKLLAEMVPRPSCIISTDALPWTNEVAWKFKIPRYSFNTISCFSLVLFHKLKISRVHESVTSDSESFVVPGLPDKIELKRSQLPEWVKRTSNDNNSHVMDKLKATEHLPRGVLVNTFEEMEPRYVEEFKKQEKLVFCVGSVSLCNTDQSDMSSRGNKASVEEDSCLKWLDSMKPCSVIYVCFGSLSNIPYPQLIELGLGLEASNRPFIWIIRKGDYSPRLEKWLEEYRFEEKVKGRGLIIRGWAPLVLILSHPSVGGFFTHCGWNSILEAVCEGMPMITWPMFAEQFYNERFVVHVVKIGVPVGVEEALTSVEDEEKFELLVTSSQVKEAIDELMYGEDAGERRKRARELGEKAKKAIEDGGSSYLYITNLIEDVRQQNSSRF*

[0109] >SEQ ID NO:5MVTNSKDNNALRILMFPWLGHGHISPYLELAKKLSRKDSKIYFRSTPINLKPIKNKILDYNSIELIEFPLPSSQELPPHYHKTTGLPPHLLPALKDAFEMASPQLSNILDTITPDLLIYDIYQPWVPKLASSYKIPAVHFQTTGATAISYFYRLSMNLKTAFPSTIIHLKDIELVRMLETGGGDESDPENEQDRDRVFDSILGSVEILLIKSSREIEGKYIDCLSDFIKKKIVSVGPLVQEFVGNDQENDDVITWLSKKEPFSTVYVSFGTESFLSKKDLEVLAHGLELSGVNFIWALKFPEAEKITKVEEALPQGFLERVGEKGLVLGGWVPQAKILNHSSIGGFVSHCGWSSVIESLSFGVPIIAMPLQNDQPLNARLVVEVGVGLEVEKDDKLEFGREEVARVVKEVVVEEKSRETFGKRVKELSEVLKVKADEDVDNAIKELKRLCENNLRKVE*

[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] MASNIQQLHFVLVPLMSQSHIIPLTDFGKLLAQRGVVVTMITTPLNAVRCKPIIDGAQNANLNIQLASLHFPTREVGLPEGVENLDELSMKSLKTLGINFFQANEMLREPLEKLLAEMVPRPSCIISTDALPWTNEVAWKFKIPRYSFNTISCFSLVLFHKLKISRVHESVTSDSESFVVPGLPDKIELKRSQLPEWVKRTSNDNNSHVMDKLKATEHLPRGVLVNTFEEMEPRYVEEFKKQEKLVFCVGSVSLCNTDQSDMSSRGNKASVEEDSCLKWLDSMKPCSVIYVCFGSLSNIPYPQLIELGLGLEASNRPFIWIIRKGDYSPRLEKWLEEYRFEEKVKGRGLIIRGWAPQVLILSHPSVGGFFTHCGWNSILEAVCEGMPMITWPMFAEQFYNERFVVDVVKIGVPVGVEEALTSVEDEEKFELLVTSSQVKEAIDELMDGEDAGERRKRARELGEKAKKAIEDGGSSYLYITNLIEDVRQQNSSRF*

[0132] >SEQ ID NO:17

[0133] MVTNSKDNNALRILMFPWLGHGHISPYLELAKKLSRKDSKIYFCSTPINLKPIKNKILDYNSIELIEFPLPSSQELPPHYHTTTGLPPHLLPALKDAFEMASPQLSNILDTITPDLLIYDIYQPWVPKLASSYKIPAVHFQTTGATAISYFYRLSMNLKTAFPSTIIHLKDIELVRMLETGGGDESDPENEQDRDRVFDSILGSVEILLIKSSREIEGKYIDCLSDFIKKKIVSVGPLVQEFVGNDQENDDVITWLSKKEPFSTVYVSFGTESFLSKKDLEELAHGLELSGVNFIWALKFPEAEKITKVEEALPQGFLERVGEKGLVLGGWVPQAKILNHSSIGGFVSHCGWSSVIESLSFGVPIIAMPLQNDQPLNARLVVEVGVGLEVEKDDKLEFGREEVARVVKEVVVEEKSRETFGKRVKELSEVLKVKADEDVDNAIKELKRLCENNLRKVE*

[0134] >SEQ ID NO:18

[0135] MAIKEHSNLRVLMFPWLAYGHISPYLELAKKLSKRNFHIYFCSTPVNLNSIKTRLTPSNDDFNAKSLELVELHLPTLPNLPPHLHTTNGLPPELDSTLVKAADMARPNFEDIMKDLKPDLIIYDVMQSWVSDLASWYAIPAVHLQILPATALSYIRCMFGGKQFPSPAIFIRPAEIKIMTKGAPKYEEGEDPMTASFKRSCEIVLIQSSKNFENEYIDFLSDLVDKKVVPVGPLIQAAAPPKEEDDKIMEWLNRKEADSTVLVSFGSQYYLSKEELEELAYGLELSQVNFVWVIRFPKGEEVKAVEVLPQGFMDRVGDRGLLVEGWAPQTKILEHSSIGGFVSHCGWNSATESMSLGVPIIAIPMVIEQPLNCRRVVELGVGMEVMKDENSEFDREEVARVIREVVVEKSGEEIRRKAKELSENVRHTSDEEVNNVAQELRKLCNKKLDAHPF*

[0136] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. A multi-gene tandem recombinant protein, characterized in that, The multi-gene tandem recombinant protein is formed by sequentially inserting the glycosyltransferase encoding genes, such as those shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, into the same expression vector, thereby simultaneously expressing these three glycosyltransferases in the host cell.

2. A recombinant expression vector, characterized in that, The recombinant expression vector contains glycosyltransferase encoding genes shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in sequence.

3. An engineered bacterium, characterized in that, The recombinant expression vector of claim 2 is transformed into a host bacterium, wherein the host bacterium is yeast.

4. The use of the multi-gene tandem recombinant protein of claim 1, the recombinant expression vector of claim 2, or the engineered bacteria of claim 3 as enzyme catalysts in the preparation of one or more of the following reactions: (D1) Catalyzes the formation of hydroxyasiatic acid from hydroxyasiatic acid monosaccharide; (D2) Catalyzes the formation of asiatic acid monosaccharide from asiatic acid disaccharide; (D3) catalyzes the formation of asiaticoside from hydroxyasiatic acid disaccharide.

5. The use of the multi-gene tandem recombinant protein of claim 1, the recombinant expression vector of claim 2, or the engineered bacteria of claim 3 in the preparation of asiaticoside.

6. The application according to claim 5, characterized in that, The preparation of asiaticoside uses asiatic acid as the starting material.

7. A method for preparing hydroxyasiaticoside, characterized in that, The engineered bacteria described in claim 3 were cultured in YPD liquid medium to obtain a seed culture; then the seed culture was inoculated into YPG liquid medium containing asiatic acid and cultured to obtain the final product.

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