A recombinant Escherichia coli for producing mevalonic acid and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a recombinant Escherichia coli for producing mevalonic acid and its application. The present invention uses Escherichia coli MG1655 as the host cell, and transfers the HMG-CoA synthase MvaS gene and HMG-CoA reductase MvaE gene derived from Lactobacillus casei, the acetyl-CoA acetyltransferase atoB gene derived from Escherichia coli, the phosphoketolase fxpk gene derived from Bifidobacterium adolescentis, and the fructose-1,6-bisphosphatase fbp gene derived from Escherichia coli into the Escherichia coli host cell. The recombinant Escherichia coli constructed by the present invention can be used for the synthesis of mevalonic acid. Using glucose as the substrate, after fermentation in a 5 L fermenter at 37 °C for 48 hours, the yield of mevalonic acid can reach 87.86 g / L. It has the advantages of short fermentation time and high yield, meeting the requirements of industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant Escherichia coli for producing mevalonic acid and its application. Background Art

[0002] Mevalonic acid widely exists in various organisms. It is a key intermediate in cell biology and an important precursor for the synthesis of terpenoids, participating in the synthesis of terpenoids (such as cholesterol, steroid hormones, bile acids, ubiquinone, and dolichol), defense agents, growth hormones, etc., and plays an extremely important regulatory role in organisms. Initially, mevalonic acid was used as an acetic acid substitute growth factor for lactic acid bacteria in alcohol fermentation and was called divalonic acid. Later, Gakuzao Tamura in Japan also discovered this substance in the growth factor of the koji mold (Aspergillus oryzae) used for brewing sake, and named it hiotic acid in Japanese. Later, it was uniformly called mevalonic acid.

[0003] Mevalonic acid is highly soluble in water and polar organic solvents, and its aqueous solution is weakly acidic. Under acidic conditions, it is extremely prone to lactonization reaction to produce mevalonlactone C6H 10 O3 (mevalonlactone), and the two can be converted into each other under corresponding conditions, such as: When mevalonlactone is added to water, it immediately undergoes ring-opening to form mevalonic acid. Mevalonlactone has a small molecular weight and is easy to penetrate. Mevalonic acid has a hydrophilic structure and can form a network crosslink on the skin surface, locking in moisture, maintaining a smooth skin feel, and having a significant moisturizing effect. It is a very important cosmetic raw material.

[0004] Currently, the synthesis of mevalonic acid mainly relies on chemical methods. Using 4-chloro-2-butanone as the raw material, through steps such as esterification, Reformatsky reaction, hydrolysis, and cyclization, a mevalonlactone product is finally obtained (Hu Xiao, Zhai Jianfeng, Wang Lixiang, et al. Study on the synthesis of mevalonlactone [J]. Chemical Industry Times, 2009, 23(07): 46 - 47). However, the product finally obtained by the chemical synthesis method is a racemate, that is, a mixture of equal proportions of R configuration and S configuration. If a pure R configuration product is to be obtained, a complex separation process is still required.

[0005] Biosynthesis methods can solve the problem of racemates and only synthesize R-configured mevalonic acid with physiological functions

Scalable production of mechanically tunable block polymers from sugar, PNAS, 2014

[0006] Patent document CN115820527A discloses a recombinant Halomonas for producing mevalonic acid, a construction method and applications thereof. The construction method is as follows: 1) Knock out genes phaB and phaC in Halomonas sp. TD01; 2) Targetedly integrate the gene mvaElac and the gene mvaSlac expression units into the genomic G7 locus of the strain obtained in step 1) to obtain a recombinant strain with the mvaElac and mvaSlac expression units integrated at the G7 locus; 3) Targetedly integrate the gene mvaEef and mvaSef expression units into the genomic G4 locus of the strain obtained in step 2) to obtain a recombinant Halomonas for producing mevalonic acid.

[0007] However, the existing technologies for biosynthesizing mevalonic acid have a relatively long fermentation time, generally requiring more than 80 - 120 h, resulting in relatively high costs. Therefore, researching and developing a biosynthetic method for mevalonic acid with a short fermentation time and high yield is a current research hotspot. Summary of the Invention

[0008] To overcome the deficiencies in the prior art, the present invention provides an Escherichia coli engineering bacterium for efficiently synthesizing mevalonic acid. The present invention uses Escherichia coli MG1655 as the host cell, heterologously expresses the HMG-CoA synthase MvaS gene and the HMG-CoA reductase MvaE gene derived from Lactobacillus casei , overexpresses the acetyl-CoA acetyltransferase atoB gene derived from Escherichia coli, heterologously expresses the phosphoketolase fxpk gene derived from Bifidobacterium adolescentis , and overexpresses the fructose-1,6-bisphosphatase fbp gene derived from Escherichia coli. The constructed engineering strain can achieve the microbial synthesis of mevalonic acid.

[0009] The specific technical solution of the present invention is as follows:

[0010] The present invention provides a recombinant Escherichia coli for producing mevalonic acid. The recombinant Escherichia coli MG1655 LS01 ( Escherichia coli MG1655 LS01) was deposited at the China Center for Type Culture Collection on November 11, 2024, with the deposit number: CCTCC NO: M 20242500.

[0011] Furthermore, the recombinant Escherichia coli MG1655 LS01 is obtained by transferring the HMG-CoA synthase MvaS gene derived from Lactobacillus casei ( Lactobacillus casei ), the HMG-CoA reductase MvaE gene derived from Lactobacillus casei ( Lactobacillus casei ), the acetyl-CoA acetyltransferase atoB gene derived from Escherichia coli, the phosphoketolase fxpk gene derived from Bifidobacterium adolescentis ( Bifidobacterium adolescentis ), and the fructose-1,6-bisphosphatase fbp gene derived from Escherichia coli into an Escherichia coli host cell.

[0012] Furthermore, the amino acid sequence of the protein encoded by the HMG-CoA synthase MvaS gene is as shown in SEQ ID NO.6; the amino acid sequence of the protein encoded by the HMG-CoA reductase MvaE gene is as shown in SEQ ID NO.7; the amino acid sequence of the protein encoded by the acetyl-CoA acetyltransferase atoB gene is as shown in SEQ ID NO.8; the amino acid sequence of the protein encoded by the phosphoketolase fxpk gene is as shown in SEQ ID NO.9; the amino acid sequence of the protein encoded by the fructose-1,6-bisphosphatase fbp gene is as shown in SEQ ID NO.10.

[0013] Furthermore, the recombinant Escherichia coli MG1655 LS01 contains at least two recombinant vectors, and the recombinant vectors are selected from at least one of pGEX, pR6K, pMAL, pTrcHis, pBAD, and pQE.

[0014] Furthermore, the recombinant Escherichia coli MG1655 LS01 contains two recombinant vectors, one of which contains the fructose-1,6-bisphosphatase fbp gene and the phosphoketolase fxpk gene, and the other contains the acetyl-CoA acetyltransferase atoB gene, the HMG-CoA synthase MvaS gene, and the HMG-CoA reductase MvaE gene.

[0015] Furthermore, the present invention also provides a method for constructing the recombinant Escherichia coli for producing mevalonic acid, comprising the following steps:

[0016] Step S1, constructing a recombinant vector containing the fructose-1,6-bisphosphatase fbp gene and the phosphoketolase fxpk gene;

[0017] Step S2, constructing a recombinant vector containing the acetyl-CoA acetyltransferase atoB gene, the HMG-CoA synthase MvaS gene, and the HMG-CoA reductase MvaE gene;

[0018] Step S3: Co-transform the recombinant vector obtained in Step S1 and the recombinant vector obtained in Step S2 into Escherichia coli, and that's it.

[0019] In addition, the present invention also provides a method for fermentatively producing mevalonic acid. Using the recombinant Escherichia coli MG1655 LS01 for producing mevalonic acid as the fermentation strain, with glucose as the substrate, cultivate at a temperature of 20 - 37 °C until OD600 = 10 - 15, then add IPTG (isopropyl-β-D-thiogalactoside) inducer to induce the synthesis of mevalonic acid, and that's it.

[0020] Furthermore, the fermentation conditions are as follows: the fermentation temperature is 20 - 37 °C, the rotation speed is 100 - 600 rpm, the dissolved oxygen is controlled between 30 - 40%, the pH is controlled between 5.0 - 6.5, and continue to ferment for 48 - 60 h after adding the inducer.

[0021] Furthermore, the concentration of the IPTG inducer is 0.5 mmol / L.

[0022] In addition, the present invention also claims the application of the R-configured mevalonic acid obtained by the method for fermentatively producing mevalonic acid in the preparation of cosmetics.

[0023] Furthermore, the cosmetics are products with the function of enhancing skin barrier, products with the function of enhancing mitochondria and regulating lipid metabolism, products with anti-wrinkle and firming effects, or beauty and skincare products.

[0024] Compared with the prior art, the recombinant Escherichia coli for producing mevalonic acid provided by the present invention has the following advantages:

[0025] (1) For the recombinant Escherichia coli for producing mevalonic acid provided by the present invention, using glucose as the substrate, after fermenting in a 5 L fermenter at 37 °C for 48 hours, the yield of mevalonic acid can reach 87.86 g / L, which has the advantages of short fermentation time and high yield, meeting the requirements of industrial production.

[0026] (2) The R-configured mevalonic acid fermentatively produced by using the recombinant Escherichia coli for producing mevalonic acid provided by the present invention has the function of enhancing skin barrier, enhancing mitochondria, regulating lipid metabolism, anti-wrinkle and firming effects. It is an ideal cosmetic raw material and has broad application prospects in the fields of cosmetics, etc.

[0027] (3) Compared with the traditional chemical synthesis method, the present invention directly synthesizes the physiologically functional R-configured mevalonic acid by recombinant Escherichia coli fermentation method, without complex separation steps, improving the purity and consistency of the product.

[0028] Microbial information of recombinant Escherichia coli MG1655 LS01 for producing mevalonic acid provided by the present invention: The recombinant Escherichia coli MG1655 LS01 ( Escherichia coli MG1655 LS01) was deposited at the China Center for Type Culture Collection on November 11, 2024, with the deposit number: CCTCC NO: M 20242500, and the deposit address: Wuhan University, Wuhan, China, Zip code: 430072. Description of the Drawings

[0029] Figure 1 It is a metabolic schematic diagram of heterologous synthesis of mevalonic acid in Escherichia coli.

[0030] Figure 2 It is a map of the recombinant plasmid pGEX-mvaE-mvaS-atoB constructed in Example 4.

[0031] Figure 3 It is a map of the recombinant plasmid pR6K-fxpk-fbp constructed in Example 4.

[0032] Figure 4 It is a yield chart of mevalonic acid fermentation synthesized by different recombinant strains in each group.

[0033] Figure 5 It is a bar chart of the relative expression level of the FLG gene.

[0034] Figure 6 It is a bar chart of the relative expression level of the IVL gene.

[0035] Figure 7 It is a bar chart of the relative expression level of the LOR gene.

[0036] Figure 8 It is a bar chart of the relative expression level of the TGM1 gene.

[0037] Figure 9 It is a detection result chart of the fluorescence intensity of zebrafish body muscle mitochondria.

[0038] Figure 10 It is a detection result chart of cholesterol content.

[0039] Figure 11 It is a bar chart of the relative expression level of the zebrafish elastin gene.

[0040] Figure 12 It is a bar chart of the relative expression level of the zebrafish type I collagen gene. Detailed Embodiments

[0041] The present invention will be further described below through specific embodiments. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of the present invention. As long as they do not depart from the basic idea of the present invention, they are within the scope of the present invention.

[0042] I. Strains and primers involved in the present invention

[0043] The strains and primers involved in Examples 1 to 4 of the present invention are shown in Tables 1 and 2.

[0044] Table 1 Strains involved in Examples 1 to 4

[0045] Strain Name Genotype MG1655 K-12, λ,,, MVA-1 MG1655 pGEX-mvaE-mvaS MVA-2 MG1655 pGEX-mvaE-mvaS-atoB MVA-3 MG1655 pGEX-mvaE-mvaS-atoB&pR6K-fxpk MVA-4 MG1655 pGEX-mvaE-mvaS-atoB&pR6K-fxpk-fbp

[0046] Table 2 Primer sequences used in Examples 1 to 4

[0047] Primer Name Sequence (5′-3′) pR6K-f CCGCTGGCGATAACGAGTGAtgatcggcacgtaagaggttcc pR6K-r AATTCACCTAACGTTTTCATTATATCTCCTTATTgctagtattatacct pR6K-r1 CCAATAACAGGACTCGTCATTATATCTCCTTATTgctagtattatacct fbp-f actagcAATAAGGAGATATAATGAAAACGTTAGGTGAATTTATTG fbp-r GCCAATAACAGGACTCGTCATTTACGCGTCCGGGAACT fxpk-f TGAGTTCCCGGACGCGTAAATGACGAGTCCTGTTATTGGC fxpk-f1 actagcAATAAGGAGATATAATGACGAGTCCTGTTATTGGC fxpk-r aacctcttacgtgccgatcaTCACTCGTTATCGCCAGCGgtt pGEX-f GAGAACCGTGACctcgagTAAggatccccggaattcccg pGEX-r CTGACGATGACACAATTTTTcatgaatactgtttcctgtgtgaa pGEX-r1 ATCTAGTATTTCTCCTCTTTAATgaatactgtttcctgtgtgaa atoB-f tcacacaggaaacagtattcatgAAAAATTGTGTCATCGTCagt atoB-r ATACCGATCTTCATCTAGTATTTCTCCTCTTTAATTTAATTCAACCGTTCAATC mvaS-f ACGGTTGAATtaaATTAAAGAGGAGAAATACTAGATGAAGATCGGTATCGAC mvaS-f1 aggaaacagtattcATTAAAGAGGAGAAATACTAGATGAAGATCGGTATCGAC mvaS-r CGTAGAATTTCATCTAGTATTTCTCCTCTTTAATTTAGTGAGTGGAGGTAGAG mvaE-f TCCACTCACTAAATTAAAGAGGAGAAATACTAGATGAAATTCTACGAAAAAAC mvaE-r gtcgacccgggaattccggggatccttactcgagGTCACGGTT

[0048] II. The culture media involved in Examples 1 to 4 of the present invention are as follows:

[0049] (1) LB medium formula: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride. For solid medium, add 1.5 - 2.0% agar powder.

[0050] (2) Fermentation medium:

[0051] Fermentation medium formula: 10 g / L glucose, 7.5 g / L K2HPO4, 2.0 g / L citric acid monohydrate, 0.5 g / L yeast extract, 2.0 g / L MgSO4·7H2O, 0.3 g / L ferric ammonium citrate, 0.008 g / L thiamine hydrochloride, 0.008 g / L D-(+)-biotin, 0.008 g / L nicotinic acid, 0.032 g / L vitamin B6, 1 mL / L trace metal solution.

[0052] The formula of the trace metal solution: 10 g / L sodium chloride, 40 g / L citric acid, 1.0 g / L ZnSO4·7H2O, 30 g / L MnSO4·H2O, 0.1 g / L CuSO4·5H2O, 0.1 g / L H3BO3, 0.1 g / L Na2MoO4·2H2O, 1.0 g / L FeSO4·7H2O, 1.0 g / L CoCl2·6H2O.

[0053] III. The nucleotide sequences and amino acid sequences involved in Examples 1 to 4 of the present invention are as follows:

[0054] (1) Derived from Lactobacillus casei (Lactobacillus casei The nucleotide sequence of the HMG-CoA synthase MvaS gene of () is (SEQ ID NO.1):

[0055]

[0056] (2) The nucleotide sequence of the HMG-CoA reductase MvaE gene derived from Lactobacillus casei ( Lactobacillus casei ) is (SEQ ID NO.2):

[0057]

[0058] (3)The nucleotide sequence of the acetyl-CoA acetyltransferase atoB gene derived from Escherichia coli is (SEQ ID NO.3):

[0059]

[0060] (4) The nucleotide sequence of the phosphoketolase fxpk gene derived from Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) is (SEQ ID NO.4):

[0061]

[0062] (5) The nucleotide sequence of the fructose-1,6-bisphosphatase fbp gene derived from Escherichia coli is (SEQ ID NO.4):

[0063] ATGAAAACGTTAGGTGAATTTATTGTCGAAAAGCAGCACGAGTTTTCTCATGCTACCGGTGAGCTCACTGCTTTGCTGTCGGCAATAAAACTGGGCGCCAAGATTATCCATCGCGATATCAACAAAGCAGGACTGGTTGATATCCTGGGTGCCAGCGGTGCTGAGAACGTGCAGGGCGAGGTTCAGCAGAAACTCGACTTGTTCGCTAATGAAAAACTGAAAGCCGCACTGAAAGCACGCGATATCGTTGCGGGCATTGCCTCTGAAGAAGAAGATGAGATTGTCGTCTTTGAAGGCTGTGAACACGCAAAATACGTGGTGCTGATGGACCCCCTGGATGGCTCGTCCAACATCGATGTTAACGTCTCTGTCGGTACCATTTTCTCCATCTACCGCCGCGTTACGCCTGTTGGCACGCCGGTAACGGAAGAAGATTTCCTCCAGCCTGGTAACAAACAGGTTGCGGCAGGTTACGTGGTATACGGCTCCTCTACCATGCTGGTTTACACCACCGGATGCGGTGTTCACGCCTTTACTTACGATCCTTCGCTCGGCGTTTTCTGCCTGTGCCAGGAACGGATGCGCTTCCCGGAGAAAGGCAAAACCTACTCCATCAACGAAGGAAACTACATTAAGTTTCCGAACGGGGTGAAGAAGTACATTAAATTCTGCCAGGAAGAAGATAAATCCACCAACCGCCCTTATACCTCACGTTATATCGGTTCACTGGTCGCGGATTTCCACCGTAACCTGCTGAAAGGCGGTATTTATCTCTACCCAAGCACCGCCAGCCACCCGGACGGCAAACTGCGTTTGCTGTATGAGTGCAACCCGATGGCATTCCTGGCGGAACAAGCGGGCGGTAAAGCGAGCGATGGCAAAGAGCGTATTCTGGATATCATCCCGGAAACCCTGCACCAGCGCCGTTCATTCTTTGTCGGCAACGACCATATGGTTGAAGATGTCGAACGCTTTATCCGTGAGTTCCCGGACGCGTAA

[0064] (6) The amino acid sequence of the protein encoded by the HMG-CoA synthase MvaS gene is (SEQ ID NO.6):

[0065] MKIGIDAIAMDTPDFYVDLVELAKVRGDEPDKYTIGIGQDEQAVPPSSQDIVTMGANAAAPLMTPAVRDSLGMVLVGTESGVDASKSAALFIHQLLDLPEWVRAAELKEACYGGTAALMMARDYVAGHPDKSVLVIAADIARYGLATAGEVTQGAGAVAMIVKADPRLITIESDSVYRSASINDFWRPVYQDTAIAQGKYSTEQYLAFFQAVWDRYQAENHLTAADFAGMTFHLPYTKMGKKALDLVLPEADDQTVKRLKERFDASTRYCRRIGNIYTGSLYLGLLSLLDHDATLQAGDRIGLFSYGSGAVAEFFSGILQPGFKDQLQSAAHARQLDDRQQLTAPEYEALFSDKVPYDPEDYRPDPAYFRGQFVLTGVVGQERLYEKRPANSSTSTH*

[0066] (7)The amino acid sequence of the protein encoded by the HMG-CoA reductase MvaE gene is (SEQ ID NO.7):

[0067] MKFYEKTPEERRAQLIREGSLTQADASLFAATLSLPAATEAKLIENSIGEFSLPLGIARNLLVNGQLHQVPLANEEPSVVAAASNGARIAAENGGVVARVDSHQVVAEVVLTDLADLEAAKQTVLAHQADIKAIVAAAHPSMIQRGGGLVGVAVSVLANRFLKIRLTLNPKEAMGANYANTVAEAVAAVVKTWLNGTILVSILTNTPAELVTATVQLDPPTLATETASGEVIAKKIVQLSELAFVDPERAVTHNKGILNGVIGAVLATGNDTRAIAASVGAFAAASGQYQPLSRWVMHAGKLEGTLQIPLPLGAVGGAIGALPLAQAARRLGGYRDLATMQQVIAALGLVQNLAALRALAGPGIQAGHMKLQANALAIAAGADERELPALVAALREGQMDLAHAKTYLANIRSNQKVGQSKNENRDLE*

[0068] (8) The amino acid sequence of the protein encoded by the acetyl-CoA acetyltransferase atoB gene is (SEQ ID NO.8):

[0069] MKNCVIVSAVRTAIGSFNGSLASTSAIDLGATVIKAAIERAKIDSQHVDEVIMGNVLQAGLGQNPARQALLKSGLAETVCGFTVNKVCGSGLKSVALAAQAIQAGQAQSIVAGGMENMSLAPYLLDAKARSGYRLGDGQVYDVILRDGLMCATHGYHMGITAENVAKEYGITREMQDELALHSQRKAAAAIESGAFTAEIVPVNVVTRKKTFVFSQDEFPKANSTAEALGALRPAFDKAGTVTAGNASGINDGAAALVIMEESAALAAGLTPLARIKSYASGGVPPALMGMGPVPATQKALQLAGLQLADIDLIEANEAFAAQFLAVGKNLGFDSEKVNVNGGAIALGHPIGASGARILVTLLHAMQARDKTLGLATLCIGGGQGIAMVIERLN*

[0070] (9) The amino acid sequence of the protein encoded by the phosphoketolase fxpk gene is (SEQ ID NO.9):

[0071] MTSPVIGTPWKKLNAPVSEEAIEGVDKYWRAANYLSIGQIYLRSNPLMKEPFTREDVKHRLVGHWGTTPGLNFLIGHINRLIADHQQNTVIIMGPGHGGPAGTAQSYLDGTYTEYFPNITKDEAGLQKFFRQFSYPGGIPSHYAPETPGSIHEGGELGYALSHAYGAVMNNPSLFVPAIVGDGEAETGPLATGWQSNKLINPRTDGIVLPILHLNGYKIANPTILSRISDEELHEFFHGMGYEPYEFVAGFDNEDHLSIHRRFAELFETVFDEICDIKAAAQTDDMTRPFYPMIIFRTPKGWTCPKFIDGKKTEGSWRSHQVPLASARDTEAHFEVLKNWLESYKPEELFDENGAVKPEVTAFMPTGELRIGENPNANGGRIREELKLPKLEDYEVKEVAEYGHGWGQLEATRRLGVYTRDIIKNNPDSFRIFGPDETASNRLQAAYDVTNKQWDAGYLSAQVDEHMAVTGQVTEQLSEHQMEGFLEGYLLTGRHGIWSSYESFVHVIDSMLNQHAKWLEATVREIPWRKPISSMNLLVSSHVWRQDHNGFSHQDPGVTSVLLNKCFNNDHVIGIYFPVDSNMLLAVAEKCYKSTNKINAIIAGKQPAATWLTLDEARAELEKGAAEWKWASNVKSNDEAQIVLAATGDVPTQEIMAAADKLDAMGIKFKVVNVVDLVKLQSAKENNEALSDEEFAELFTEDKPVLFAYHSYARDVRGLIYDRPNHDNFNVHGYEEQGSTTTPYDMVRVNNIDRYELQAEALRMIDADKYADKINELEAFRQEAFQFAVDNGYDHPDYTDWVYSGVNTNKQGAISATAATAGDNE*

[0072] (10) The amino acid sequence of the protein encoded by the fructose-1,6-bisphosphatase fbp gene is (SEQ ID NO.10):

[0073] MKTLGEFIVEKQHEFSHATGELTALLSAIKLGAKIIHRDINKAGLVDILGASGAENVQGEVQQKLDLFANEKLKAALKARDIVAGIASEEEDEIVVFEGCEHAKYVVLMDPLDGSSNIDVNVSVGTIFSIYRRVTPVGTPVTEEDFLQPGNKQVAAGYVVYGSSTMLVYTTGCGVHAFTYDPSLGVFCLCQERMRFPEKGKTYSINEGNYIKFPNGVKKYIKFCQEEDKSTNRPYTSRYIGSLVADFHRNLLKGGIYLYPSTASHPDGKLRLLYECNPMAFLAEQAGGKASDGKERILDIIPETLHQRRSFFVGNDHMVEDVERFIREFPDA*

[0074] Example 1. Construction of recombinant Escherichia coli MVA-1

[0075] Step S1. Construction of recombinant plasmid pGEX-mvaE-mvaS:

[0076] The HMG-CoA synthase MvaS gene and HMG-CoA reductase MvaE gene derived from Lactobacillus casei were both synthesized by Tsingke Company. Fragments mvaE, mvaS and linearized plasmid pGEX were amplified using primers mvaE-f / r, mvaS-f1 / r, pGEX-f / r1 respectively. Recombination was carried out using Novoprotein ClonExpress technology to obtain recombinant plasmid pGEX-mvaE-mvaS.

[0077] The recombinant plasmid pGEX-mvaE-mvaS was transferred into Escherichia coli DH5α by chemical transformation method, and spread onto LB solid medium plates containing 100 mg / L ampicillin resistance, and cultured overnight in a 37°C incubator. The grown monoclonal colonies were picked to extract plasmids and verified by sequencing to ensure that the inserted gene sequences were correct.

[0078] Step S2. Construction of MVA-1 engineering strain:

[0079] The recombinant plasmid pGEX-mvaE-mvaS prepared in Step S1 was transformed into competent cells of Escherichia coli MG 1655, spread onto LB solid medium containing 100 mg / L ampicillin resistance, and cultured overnight in a 37°C incubator. Monoclonal colonies were picked for LB liquid culture and stored in glycerol at -80°C to obtain recombinant strains E. coliMG1655 pGEX-mvaE-mvaS, namely strain MVA-1.

[0080] Example 2. Construction of recombinant Escherichia coli MVA-2

[0081] Step S1. Construction of recombinant plasmid pGEX-mvaE-mvaS-atoB:

[0082] Derived from Lactobacillus casei The HMG-CoA synthase MvaS gene and HMG-CoA reductase MvaE gene were both synthesized by Tsingke. Fragments mvaE, mvaS, and linearized plasmid pGEX were amplified using primers mvaE-f / r, mvaS-f1 / r, and pGEX-f / r1, respectively. Recombination was performed using Novoprotein ClonExpress technology to obtain recombinant plasmid pGEX-mvaE-mvaS.

[0083] Using the Escherichia coli genome as a template, the atoB fragment was amplified with primers atoB-f / r. The recombinant plasmid pGEX-mvaE-mvaS was linearized with primers amvaS-f / pGEX-r, and recombination was performed by Novoprotein ClonExpress technology to obtain recombinant plasmid pGEX-mvaE-mvaS-atoB.

[0084] The recombinant plasmid pGEX-mvaE-mvaS-atoB was transformed into Escherichia coli DH5α by chemical transformation method, and spread onto LB solid medium plates containing 100 mg / L ampicillin resistance, and cultured overnight in a 37°C incubator. The grown monoclonal colonies were used to extract plasmids and verified by sequencing to ensure that the inserted gene sequences were correct.

[0085] Step S2. Construction of MVA-2 engineering strain:

[0086] The recombinant plasmid pGEX-mvaE-mvaS-atoB prepared in Step S1 was transformed into the competent cells of Escherichia coli MG 1655, spread onto LB solid medium containing 100 mg / L ampicillin resistance, and cultured overnight in a 37°C incubator. Monoclonal colonies were picked for LB liquid culture and stored in glycerol at -80°C to obtain recombinant strain E. coli MG1655pGEX-mvaE-mvaS-atoB, namely strain MVA-2.

[0087] Example 3. Construction of recombinant Escherichia coli MVA-3

[0088] Step S1. Construction of recombinant plasmid pGEX-mvaE-mvaS-atoB:

[0089] Derived from Lactobacillus casei Both the HMG-CoA synthase MvaS gene and the HMG-CoA reductase MvaE gene were synthesized by Tsingke Biotechnology Company. Fragments mvaE, mvaS, and the linearized plasmid pGEX were obtained by amplification using primers mvaE-f / r, mvaS-f1 / r, and pGEX-f / r1, respectively. Recombinant plasmid pGEX-mvaE-mvaS was obtained by recombination using Novoprotein ClonExpress technology.

[0090] Using the Escherichia coli genome as a template, the atoB fragment was obtained by amplification with primers atoB-f / r. The recombinant plasmid pGEX-mvaE-mvaS was linearized using primers amvaS-f / pGEX-r, and recombinant plasmid pGEX-mvaE-mvaS-atoB was obtained by recombination using Novoprotein ClonExpress technology.

[0091] Step S2: Construction of recombinant plasmid pR6K-fxpk:

[0092] Derived from Bifidobacterium adolescentis The phosphoketolase fxpk gene was synthesized by Tsingke Biotechnology Company. The fxpk fragment and the linearized plasmid pR6K were obtained by amplification using primers fxpk-f1 / r and pR6K-f / r1, respectively. Recombinant plasmid pR6K-fxpk was obtained by recombination using Novoprotein ClonExpress technology.

[0093] The recombinant plasmids pGEX-mvaE-mvaS-atoB and pR6K-fxpk were transferred into Escherichia coli DH5α by chemical transformation method, and were respectively spread onto LB solid medium plates containing 30 mg / L chloramphenicol and 100 mg / L ampicillin resistance, and cultured overnight in a 37°C incubator. The plasmids of the grown monoclonal colonies were extracted and sequenced to verify that the inserted gene sequences were correct.

[0094] Step S3: Construction of MVA-3 engineering strain:

[0095] The recombinant plasmid pGEX-mvaE-mvaS-atoB prepared in step S1 and the recombinant plasmid pR6K-fxpk prepared in step S2 were simultaneously transformed into the competent cells of Escherichia coli MG 1655, spread onto LB solid medium containing 30 mg / L chloramphenicol and 100 mg / L ampicillin resistance, and cultured overnight in a 37°C incubator. Single colonies were picked for LB liquid culture and stored in glycerol at -80°C to obtain the recombinant strain E. coli MG1655 pGEX-mvaE-mvaS-atoB&pR6K-fxpk, namely MVA-3 strain.

[0096] Example 4. Construction of Recombinant Escherichia coli MVA-4

[0097] Step S1. Construction of Recombinant Plasmid pGEX-mvaE-mvaS-atoB:

[0098] The HMG-CoA synthase MvaS gene and HMG-CoA reductase MvaE gene derived from Lactobacillus casei were both synthesized by Tsingke. Fragments mvaE, mvaS and linearized plasmid pGEX were obtained by amplification using primers mvaE-f / r, mvaS-f1 / r, pGEX-f / r1 respectively. Recombination was carried out using Novoprotein ClonExpress technology to obtain recombinant plasmid pGEX-mvaE-mvaS.

[0099] Using the Escherichia coli genome as a template, the atoB fragment was obtained by amplification with primers atoB-f / r. The recombinant plasmid pGEX-mvaE-mvaS was linearized with primers amvaS-f / pGEX-r, and recombination was carried out by Novoprotein ClonExpress technology to obtain recombinant plasmid pGEX-mvaE-mvaS-atoB (the recombinant plasmid pGEX-mvaE-mvaS-atoB is as shown in Figure 2 ).

[0100] Step S2. Construction of Recombinant Plasmid pR6K-fxpk-fbp:

[0101] The phosphoketolase fxpk gene derived from Bifidobacterium adolescentis was synthesized by Tsingke. Fragments fxpk and linearized plasmid pR6K were obtained by amplification using primers fxpk-f1 / r, pR6K-f / r1 respectively. Recombination was carried out using Novoprotein ClonExpress technology to obtain recombinant plasmid pR6K-fxpk. The fructose-1,6-bisphosphatase fbp gene derived from Escherichia coli was synthesized by Tsingke. Fragments fbp and linearized vector pR6K-fxpk were obtained by amplification using primers fbp-f / r, fxpk-f / pR6K-r respectively. Recombination was carried out using Novoprotein ClonExpress technology to obtain recombinant plasmid pR6K-fxpk-fbp (the recombinant plasmid pR6K-fxpk-fbp is as shown in Figure 3 ).

[0102] The recombinant plasmid pGEX-mvaE-mvaS-atoB and the recombinant plasmid pR6K-fxpk-fbp were transformed into Escherichia coli DH5α by chemical transformation method, and were respectively spread onto the LB solid medium plates containing 30 mg / L chloramphenicol and 100 mg / L ampicillin resistance, and cultured overnight in a 37 °C incubator. The plasmids of the grown monoclonal colonies were extracted and sequenced to verify that the inserted gene sequences were correct.

[0103] Step S3, Construction of MVA-4 engineering strain:

[0104] The recombinant plasmid pGEX-mvaE-mvaS-atoB prepared in Step S1 and the recombinant plasmid pR6K-fxpk-fbp prepared in Step S2 were simultaneously transformed into the competent cells of Escherichia coli MG1655, spread onto the LB solid medium containing 30 mg / L chloramphenicol and 100 mg / L ampicillin resistance, and cultured overnight in a 37 °C incubator. Single colonies were picked for LB liquid culture, stored in glycerol, and stored at -80 °C to obtain the recombinant strain E. coli MG1655 pGEX-mvaE-mvaS-atoB&pR6K-fxpk-fbp, namely MVA-4 strain (recombinant Escherichia coli MG1655 LS01).

[0105] Example 5, Fermentation process of recombinant Escherichia coli

[0106] (1) Seed liquid preparation:

[0107] The prepared recombinant Escherichia coli engineering strain was streaked on the LB solid medium to obtain single colonies. Single colonies were picked and inoculated into a 150 mL conical flask containing 30 mL of LB liquid medium, and cultured at 37 °C and 200 rpm for 12 h to prepare the seed liquid.

[0108] (2) Fermentation culture:

[0109] The prepared seed liquid was inoculated into a 5 L fermenter containing 3 L of fermentation medium at an inoculation amount of 1% (v / v). It was cultured at a rotation speed of 100 - 600 rpm, an aeration rate of 1 VVM, the dissolved oxygen controlled at about 40%, the glucose controlled below 5 g / L, the pH value controlled at 6.0, and the temperature at 30 °C until the OD600 value reached 12, and then 0.5 mmol / L IPTG was added for gene expression induction to initiate the expression and activity of related enzymes in the mevalonic acid synthesis pathway, and continued to ferment for 48 h to obtain the product.

[0110] Experimental Example 1, Yield detection experiment of mevalonic acid

[0111] 1. Experimental method:

[0112] The fermentation process of the recombinant Escherichia coli in Example 5 was adopted, and the recombinant Escherichia coli prepared in Examples 1 to 4 was used as the fermentation strain to ferment and produce mevalonic acid, which were respectively denoted as MVA-1, MVA-2, MVA-3, and MVA-4. During the fermentation process, after culturing for 12 hours, samples were taken every 2 h to detect the yield of mevalonic acid. Mevalonic acid was detected by high performance liquid chromatography (HPLC). The specific chromatographic detection conditions were as follows: the chromatographic column was AminexR HPX-87H ion exchange column, the detector was RID-10A differential refractive index detector, the column temperature was 65 °C, the detection time for each sample was 30 min, the mobile phase was 5 mmol / L sulfuric acid, and the flow rate was 0.6 mL / min.

[0113] 2. Experimental results:

[0114] The experimental results are as Figure 4 shown in Table 3.

[0115] 2.1. The detection results of the mevalonic acid yield of each group of recombinant Escherichia coli are as Figure 4 shown.

[0116] 2.2. The mevalonic acid yields of each group of recombinant Escherichia coli after 48 h of fermentation are shown in Table 3:

[0117] Table 3 Detection results of mevalonic acid yields of each group of recombinant Escherichia coli after 48 h of fermentation

[0118] Group Mevalonic acid production (g / L) Example 1 - MVA-1 strain 18.87 Example 2 - MVA-2 strain 43.41 Example 3 - MVA-3 strain 71.51 Example 4 - MVA-4 strain 87.86

[0119] As can be seen from Table 3, the recombinant Escherichia coli MG1655 LS01 ( Escherichia coli MG1655 LS01)) prepared in Example 4 of the present invention can produce a high amount of R-configured mevalonic acid in a short time, meeting the requirements of industrial production.

[0120] Example 2. Efficacy experiment on the effect of mevalonic acid in enhancing skin barrier

[0121] 1. Experimental materials:

[0122] Mevalonic acid fermented and produced by using the fermentation process of the recombinant Escherichia coli in Example 5 and using the recombinant Escherichia coli MG1655LS01 prepared in Example 4 as the fermentation strain.

[0123] 2. Experimental method:

[0124] The recombinant Escherichia coli MG1655 LS01 prepared in Example 4 was used as the fermentation strain to ferment and produce mevalonic acid, and its effect on enhancing the skin barrier was detected. The test sample was denoted as LightSyn / mevalonolactone. The test method referred to the Standard Operating Procedure for Testing the Skin Barrier Enhancement Effect of Cosmetics, which was as follows:

[0125] 2.1 System and sample volume:

[0126] Cell line: Human immortalized keratinocytes (HaCaT).

[0127] Cell passage number: At least passaged 2 times.

[0128] Cell amount per group in the experiment: 6×10 5 cells / well.

[0129] 2.2 Specific steps:

[0130] (1) Inoculate cells into a 6-well plate (6×10 5 cells / well), and culture at 37 °C and 5% CO2 for 24 h.

[0131] After incubation, gently rinse the cells with D-Hanks once or twice. Add fresh medium to the normal control group, and add fresh medium containing the sample at the corresponding concentration to the sample group. The detected concentrations (mass concentration, wt%) are: 0.156%, 0.312%, 0.625%. Culture at 37 °C and 5% CO2 for 24 h.

[0132] (3) Extract total RNA from each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of β-actin and the target gene.

[0133] (4) Use β-actin as the internal reference for gene expression, and calculate the relative RNA expression level of the target gene.

[0134] ;

[0135] 3. Experimental results:

[0136] The experimental results are shown in Table 4 and Figures 5 - 8 as follows.

[0137] 3.1 The relative expression levels of FLG, IVL, LOR, and TGM1 genes are shown in Table 4.

[0138] Table 4 Results of relative expression levels of FLG, IVL, LOR, and TGM1 genes

[0139] Detection concentration (%) Relative expression level of FLG gene Relative expression level of IVL gene Relative expression level of LOR gene Relative expression level of TGM1 gene 0.156 1.98 1.39 1.62 1.49 0.312 2.26 1.82 2.57 1.70 0.625 3.25 3.36 3.03 2.38

[0140] 3.2. Detection results of relative expression levels of FLG, IVL, LOR, and TGM1 genes are as follows Figures 5 - 8 as shown

[0141] Figure 5 This is the bar graph of the relative expression level of the FLG gene. Compared with the normal control group, ***p < 0.001. Figure 6 This is the bar graph of the relative expression level of the IVL gene. Compared with the normal control group, *p < 0.05, ***p < 0.001. Figure 7 This is the bar graph of the relative expression level of the LOR gene. Compared with the normal control group, *p < 0.05, ***p < 0.001. Figure 8 This is the bar graph of the relative expression level of the TGM1 gene. Compared with the normal control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0142] From Table 4 and Figures 5 - 8 it can be seen that the relative expression levels of FLG, IVL, LOR, and TGM1 genes in the sample LightSyn / mevalonolactone group are significantly increased compared with the normal control group, revealing that this sample has the effect of enhancing the skin barrier.

[0143] Experimental Example 3. Efficacy Experiment on the Effect of Mevalonic Acid in Enhancing Mitochondria

[0144] 1. Experimental materials:

[0145] Using the fermentation process of the recombinant Escherichia coli in Example 5, mevalonic acid was fermented and produced using the recombinant Escherichia coli MG1655LS01 prepared in Example 4 as the fermentation strain.

[0146] 2. Experimental methods:

[0147] To detect the effect of mevalonic acid fermented and produced using the recombinant Escherichia coli MG1655 LS01 prepared in Example 4 as the fermentation strain in enhancing mitochondria, the test sample was designated as LightSyn / mevalonolactone. The detection method referred to the Standard Operating Procedure for the Evaluation Experiment of Enhancing Mitochondria in Zebrafish, as follows:

[0148] 2.1. System and sample size:

[0149] Experimental system: Transgenic mitochondrial green fluorescent zebrafish (CZ222).

[0150] Zebrafish age: 4 days post-fertilization (4 dpf).

[0151] Sample size for each group of experiments: 15 tails (N = 10).

[0152] Adult fish rearing and breeding method: It meets the requirements of international AAALAC accreditation (accreditation number: 001458).

[0153] 2.2. Specific steps:

[0154] (1) Randomly select zebrafish and place them in a 6-well plate, with 15 fish in each well.

[0155] (2) Administer the sample in aqueous solution. The detected concentration of the sample (mass concentration, wt%) is 0.1%. At the same time, set up a normal control group and a model control group, with a volume of 3 mL in each well.

[0156] (3) Incubate in the dark at 28 °C for 22 h.

[0157] (4) Except for the normal control group, establish a zebrafish mitochondrial damage model by administering cobalt chloride hexahydrate in aqueous solution to the remaining experimental groups.

[0158] (5) Incubate in the dark at 28 °C for 4 h.

[0159] After the incubation, randomly select 10 zebrafish from each experimental group and place them under a fluorescence microscope for photography. Use advanced image processing software to analyze and collect data, analyze the mitochondrial fluorescence intensity (S) of the body muscles of zebrafish, calculate the mitochondrial enhancement effect of the sample according to the formula, and determine whether it has a mitochondrial enhancement effect.

[0160] ;

[0161] 3. Experimental results:

[0162] The experimental results are shown in Table 4 and Figure 9 as follows.

[0163] 3.1. The detection results of the mitochondrial enhancement effect of the sample LightSyn / mevalonolactone are shown in Table 5:

[0164] Table 5 Detection results of the mitochondrial enhancement effect of the sample LightSyn / mevalonolactone

[0165] Detection concentration (%) Effect (%) p value Detection result 0.1 97 < 0.001 Significant

[0166] 3.2. The detection results of the mitochondrial fluorescence intensity of the body muscles of zebrafish in the sample LightSyn / mevalonolactone group are as Figure 9 follows.

[0167] As can be seen from Table 5 and Figure 9 it can be known that the mitochondrial fluorescence intensity of the body muscles of zebrafish in the sample LightSyn / mevalonolactone group is significantly increased compared with the model control group, revealing that this sample has a mitochondrial enhancement effect.

[0168] Experimental Example 4: Efficacy Experiment on the Role of Mevalonic Acid in Regulating Lipid Metabolism

[0169] 1. Experimental Materials:

[0170] The mevalonic acid fermented and produced using the fermentation process of the recombinant Escherichia coli in Example 5, with the recombinant Escherichia coli MG1655LS01 prepared in Example 4 as the fermentation strain.

[0171] 2. Experimental Method:

[0172] To detect the role of mevalonic acid fermented and produced using the recombinant Escherichia coli MG1655 LS01 prepared in Example 4 as the fermentation strain in regulating lipid metabolism, the test sample is denoted as LightSyn / mevalonolactone, and the detection method refers to the Standard Operating Procedure for the Evaluation Experiment on the Role of Zebrafish in Regulating Lipid Metabolism, specifically as follows:

[0173] 2.1 System and Sample Size:

[0174] Fish strain used: Wild-type AB strain zebrafish.

[0175] Zebrafish age: 6 hours post-fertilization (6 hpf).

[0176] Sample size for each group of experiments: 30 tails (three biological replicates, N = 3).

[0177] Adult fish rearing and breeding method: Meeting the requirements of international AAALAC certification (certification number: 001458).

[0178] 2.2 Specific Steps:

[0179] (1) Randomly select zebrafish and place them in a 6-well plate, with 30 tails in each well.

[0180] (2) Administer the sample in aqueous solution. The detected concentration of the sample (mass concentration, wt%) is 0.1%. At the same time, set up a normal control group, with a volume of 3 mL in each well. Three biological replicates are conducted.

[0181] (3) Incubate in the dark at 28 °C for 120 h.

[0182] (4) Collect the zebrafish samples from each experimental group, grind and homogenize them using a grinder, centrifuge to obtain the supernatant, and use a BCA protein concentration assay kit to measure the protein concentration of each experimental group.

[0183] (5) Use a cholesterol detection kit for measurement, collect data using an enzyme-linked immunosorbent assay reader, analyze the OD500 values of each experimental group, calculate the cholesterol content (C) based on the OD500 values, and calculate and determine whether the sample has the effect of regulating lipid metabolism according to the formula.

[0184] ;

[0185] 3. Experimental results:

[0186] The experimental results are shown in Table 6 and Figure 10 as follows.

[0187] 3.1. The detection results of the lipid metabolism regulation effect of the sample LightSyn / mevalonolactone are shown in Table 6:

[0188] Table 6 Detection results of the lipid metabolism regulation effect of the sample LightSyn / mevalonolactone

[0189] Detection concentration (%) Effect (%) p value Detection result 0.1 8 < 0.001 Significant

[0190] 3.2. The cholesterol content results of the sample LightSyn / mevalonolactone group are as Figure 10 follows.

[0191] Figure 10 is the cholesterol content result chart of the sample LightSyn / mevalonolactone group, where: compared with the normal control group, ***p < 0.001.

[0192] From Table 6 and Figure 10 it can be seen that the cholesterol content of the sample LightSyn / mevalonolactone group is significantly increased compared with the normal control group, revealing that this sample has the effect of regulating lipid metabolism.

[0193] Experimental Example Five Anti-wrinkle and Firming Efficacy Experiment of Mevalonic Acid

[0194] 1. Experimental materials:

[0195] Using the fermentation process of the recombinant Escherichia coli in Example 5, mevalonic acid fermented and produced with the recombinant Escherichia coli MG1655LS01 prepared in Example 4 as the fermentation strain.

[0196] 2. Experimental method:

[0197] To detect the anti-wrinkle and firming efficacy of mevalonic acid fermented and produced with the recombinant Escherichia coli MG1655 LS01 prepared in Example 4 as the fermentation strain, the detection method refers to "Anti-wrinkle and Firming Efficacy of Cosmetics - Zebrafish Elastin Gene Expression Promotion Test Method", specifically as follows:

[0198] Thirty-six 6-day-old zebrafish were divided into three groups and exposed to solutions of 0.025 g / L, 0.0025 g / L, and 0.0005 g / L of the small fish 15 sample (LightSyn / mevalonic acid lactone) respectively. At the same time, a blank control group was set up. After 24 h of exposure, RNA was extracted from the zebrafish, cDNA was synthesized, and real-time PCR amplification was carried out. Using β-actin as the housekeeping gene and Ct as the amplification result, the relative expression level of Elna was calculated and statistical analysis was performed.

[0199] 3. Experimental results:

[0200] The experimental results are shown in Table 7 and Figure 11 as follows.

[0201] 3.1. The detection results of the promotion rate of the expression of the zebrafish elastin gene Elna are shown in Table 7.

[0202] Table 7 Detection results of the promotion rate of the expression of the zebrafish elastin gene Elna

[0203] Test concentration Result Evaluation of anti-wrinkle and firming efficacy 0.025 g / L 24%(p=0.017) Significant 0.0025 g / L 107%(p=0.000080) Significant 0.0005 g / L 67% (p=0.0033) Significant

[0204] 3.2. The detection results of the relative expression level of the zebrafish elastin gene are as Figure 11 follows.

[0205] Figure 11 is the bar chart of the relative expression level of the zebrafish elastin gene, where: *p < 0.05.

[0206] From Table 7 and Figure 11 it can be seen that the promotion rates of the expression of the zebrafish Elna gene by the sample at the test concentrations of 0.025 g / L, 0.0025 g / L, and 0.0005 g / L are 24% (p = 0.017), 107% (p = 0.000080), and 67% (p = 0.0033) respectively. The sample can significantly promote the expression of the zebrafish Elna gene, has the effect of promoting elastin regeneration, and supports the claims of anti-wrinkle and firming effects.

[0207] Experimental Example VI. Anti-wrinkle and firming effect experiment of mevalonic acid

[0208] 1. Experimental materials:

[0209] Mevalonic acid produced by fermenting the recombinant Escherichia coli MG1655LS01 prepared in Example 4 using the fermentation process of the recombinant Escherichia coli in Example 5.

[0210] 2. Experimental method:

[0211] Detect the anti-wrinkle and firming effects of mevalonic acid fermented and produced using the recombinant Escherichia coli MG1655 LS01 prepared in Example 4 as the fermentation strain. The detection method refers to "Anti-wrinkle and Firming Effects of Cosmetics - Promoting Test Method for Zebrafish Type I Collagen Gene Expression", specifically as follows:

[0212] Thirty-six 6-day-old zebrafish were divided into 3 groups and exposed to solutions of 0.025 g / L, 0.0025 g / L, and 0.0005 g / L of the sample for small fish 15 (LightSyn / mevalonolactone) respectively. Meanwhile, a blank control group was set up. After 24 h of exposure, RNA extraction, cDNA synthesis, and real-time PCR amplification were performed on the zebrafish. Using β-actin as the housekeeping gene and Ct as the amplification result, calculate the relative expression levels of col1a1a, col1a1b, and col1a2 and conduct statistical analysis.

[0213] 3. Experimental results:

[0214] The experimental results are shown in Table 8 and Figure 12 as follows.

[0215] 3.1. The detection results of the relative expression promotion rate of zebrafish type I collagen gene are shown in Table 8.

[0216] Table 8 Detection results of the relative expression promotion rate of zebrafish type I collagen gene

[0217] Test concentration Promotion rate of zebrafish type I collagen gene col1a1a expression Promotion rate of zebrafish type I collagen gene col1a1b expression Promotion rate of zebrafish type I collagen gene col1a2 expression Evaluation of anti-wrinkle and firming efficacy 0.025 g / L 41% (p=0.0014) 101% (p=0.000034) 48% (p=0.0010) Significant 0.0025 g / L 41% (p=0.011) 49% (p=0.0088) 75% (p=0.0019) Significant 0.0005 g / L 55% (p=0.000033) 122% (p=0.00039) 68% (p=0.015) Significant

[0218] 3.2. The detection results of the relative expression levels of zebrafish type I collagen gene are as Figure 12 shown.

[0219] Figure 12 is a bar chart of the relative expression levels of zebrafish type I collagen gene, where: *p < 0.05.

[0220] From Table 8 and Figure 12 it can be seen that the sample can significantly promote the expression of zebrafish type I collagen gene, has the effect of promoting the regeneration of type I collagen, and supports the claims of anti-wrinkle and firming effects.

[0221] The above embodiments merely illustrate the principles and effects of the present invention, rather than limiting the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A recombinant Escherichia coli for producing mevalonic acid, characterized in that, The recombinant Escherichia coli is obtained by transferring the HMG-CoA synthase mvaS gene derived from Lactobacillus casei ( Lactobacillus casei ), the HMG-CoA reductase mvaE gene derived from Lactobacillus casei ( Lactobacillus casei ), the acetyl-CoA acetyltransferase atoB gene derived from Escherichia coli shown in SEQ ID NO. 3, the phosphoketolase fxpk gene derived from Bifidobacterium adolescentis ( Bifidobacterium adolescentis ), and the fructose-1,6-bisphosphatase fbp gene derived from Escherichia coli shown in SEQ ID NO. 5 into Escherichia coli DH5α; and the recombinant Escherichia coli contains two recombinant plasmids, one of which is pGEX-mvaE-mvaS-atoB and the other is pR6K-fxpk-fbp.

2. A recombinant Escherichia coli for producing mevalonic acid, characterized in that, The recombinant Escherichia coli ( Escherichia coli ) MG1655 LS01 was deposited at the China Center for Type Culture Collection on November 11, 2024, with the deposit number: CCTCC NO: M 20242500.

3. A method for constructing the recombinant Escherichia coli as described in claim 1, characterized in that, comprising the following steps: Step S1, constructing a recombinant vector containing the fructose-1,6-bisphosphatase fbp gene and the phosphoketolase fxpk gene; Step S2, constructing a recombinant vector containing the acetyl-CoA acetyltransferase atoB gene, the HMG-CoA synthase mvaS gene and the HMG-CoA reductase mvaE gene; Step S3, simultaneously transforming the recombinant vector obtained in Step S1 and the recombinant vector obtained in Step S2 into Escherichia coli, thus obtaining.

4. A method for fermentatively producing mevalonic acid, characterized in that, Using the recombinant Escherichia coli for producing mevalonic acid as described in claim 1 or 2 as the fermentation strain, using glucose as the substrate, culturing at a temperature of 20-37 °C until the OD 600 reaches 10-15, adding an IPTG inducer to induce the synthesis of mevalonic acid, thus obtaining the product.

5. The method for fermentatively producing mevalonic acid according to claim 4, wherein, The fermentation conditions are as follows: the fermentation temperature is 37 °C, the rotation speed is 200 rpm, the dissolved oxygen is controlled between 30% and 40%, the pH is controlled between 5.0 and 6.5, and after adding an inducer, continue to ferment for 48 - 60 h; the concentration of the IPTG inducer is 0.5 mmol / L.

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