Recombinant escherichia coli for producing melonic acid and application of recombinant escherichia coli

Abstract

The invention belongs to the technical field of biology, and particularly relates to recombinant escherichia coli for producing melonic acid and application of the recombinant escherichia coli. Escherichia coli MG1655 is used as a host cell, and HMG-CoA synthetase MvaS gene and HMG-CoA reductase MvaE gene derived from lactobacillus casei, acetyl-coenzyme A acetyltransferase atoB gene derived from escherichia coli, phosphoketoenzyme fxpk gene derived from bifidobacterium adolescentis and fructose-1, 3-D-glucanase derived from escherichia coli are synthesized into an HMG-CoA synthetase MvaS gene and HMG-CoA reductase MvaE gene derived from lactobacillus casei. And transferring the 2, 6-diphosphatase fbp gene into Escherichia coli host cells to obtain the gene. The recombinant Escherichia coli constructed by the invention can be used for synthesis of the melonic acid, the yield of the melonic acid can reach 87.86 g / L after the recombinant Escherichia coli takes glucose as a substrate and is fermented for 48 hours in a 5L fermentation tank at 37 DEG C, and the recombinant Escherichia coli has the advantages of short fermentation time and high yield and meets the requirements of industrial production.

Description

Technical Field

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

[0002] Mevalonic acid is widely present in many organisms. It is a key intermediate in cellular organisms and an important precursor for the synthesis of terpenes. It participates in the synthesis of terpenes (cholesterol, steroid hormones, bile acid, ubiquinone and galactol, etc.), defense agents, growth hormones, etc., and plays an extremely important regulatory role in organisms. Initially, mevalonic acid was used as a growth factor for lactic acid bacteria instead of acetic acid for alcohol fermentation and was called divalonic acid. Later, Japanese Gakuzo Tamura also discovered this substance in the growth factor of Aspergillus oryzae used in brewing Japanese sake. The Japanese name was hiotic acid, and later it was uniformly called mevalonic acid.

[0003] Mevalonate is very soluble in water and polar organic solvents, and its aqueous solution is weakly acidic. Under acidic conditions, it is very easy to undergo lactonization reaction to produce mevalonate lactone C6H 10 O3 (mevalonlactone), the two can be converted into each other under corresponding conditions, such as: When mevalonate lactone is added to water, it immediately opens its ring to form mevalonate. Mevalonate lactone has a small molecular weight and is easy to penetrate. Mevalonate has a hydrophilic structure and can form a network cross-linking on the skin surface to lock in moisture, keep the skin smooth, and has a significant moisturizing effect. It is a very important cosmetic raw material.

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

[0005] Biosynthesis can solve the problem of racemates and only synthesize R-configuration mevalonic acid with physiological functions [Scalable production of mechanically tunable block polymers from sugar, PNAS, 2014]. For example, patent document CN104762242A discloses a bacterium for producing mevalonic acid using methanol as a raw material, wherein the bacterium belongs to the genus Methylobacterium (Methylobacterium ), and the bacterium is modified to express HMG-CoA synthetase (HMGS) and HMG-CoA reductase (HMGR). The Methylobacterium genus ( Methylobacterium ) can be used to produce mevalonate.

[0006] Patent document CN115820527A discloses a recombinant Halomonas sp. producing mevalonic acid, a construction method and an application thereof, wherein the construction method comprises: 1) knocking out the phaB and phaC genes in Halomonas sp. TD01; 2) integrating the mvaElac and mvaSlac expression units into the G7 site of the genome of the strain obtained in step 1); obtaining a recombinant strain having the mvaElac and mvaSlac expression units integrated into the G7 site; 3) integrating the mvaEef and mvaSef expression units into the G4 site of the genome of the strain obtained in step 2), obtaining a recombinant Halomonas sp. producing mevalonic acid.

[0007] However, the existing biosynthetic technology of mevalonic acid has a long fermentation time, generally requiring more than 80 to 120 hours, resulting in high costs. Therefore, research and development of a biosynthetic method of mevalonic acid with short fermentation time and high yield is currently a hot topic. Summary of the invention

[0008] In order to overcome the shortcomings of the prior art, the present invention provides an Escherichia coli engineered bacterium that efficiently synthesizes mevalonate. The present invention uses Escherichia coli MG1655 as a host cell, and heterologously expresses Lactobacillus casei The HMG-CoA synthase MvaS gene and HMG-CoA reductase MvaE gene from Escherichia coli were overexpressed, and the acetyl-CoA acetyltransferase atoB gene from Bifidobacterium adolescentis The engineered strain constructed by overexpressing the phosphoketolase fxpk gene from Escherichia coli and the fructose-1,6-bisphosphatase fbp gene can realize the microbial synthesis of mevalonic acid.

[0009] The specific technical solutions of the present invention are as follows: The present invention provides a recombinant Escherichia coli for producing mevalonic acid, wherein the recombinant Escherichia coli MG1655 LS01 ( Escherichia coli MG1655 LS01) was deposited in the China Center for Type Culture Collection on November 11, 2024, with the deposit number: CCTCC NO: M 20242500.

[0010] Furthermore, the recombinant Escherichia coli MG1655 LS01 is derived from Lactobacillus casei ( Lactobacillus casei ) of the HMG-CoA synthetase MvaS gene, the HMG-CoA synthetase MvaS gene from Lactobacillus casei ( Lactobacillus casei ) of HMG-CoA reductase MvaE gene, acetyl-CoA acetyltransferase atoB gene from Escherichia coli as shown in SEQ ID NO.3, and Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) and the fructose-1,6-bisphosphatase fbp gene from Escherichia coli were transferred into Escherichia coli host cells.

[0011] Furthermore, the amino acid sequence of the protein encoded by the HMG-CoA synthetase MvaS gene is shown in SEQ ID NO.6; the amino acid sequence of the protein encoded by the HMG-CoA reductase MvaE gene is shown in SEQ ID NO.7; the amino acid sequence of the protein encoded by the acetyl-CoA acetyltransferase atoB gene is shown in SEQ ID NO.8; the amino acid sequence of the protein encoded by the phosphoketolase fxpk gene is shown in SEQ ID NO.9; and the amino acid sequence of the protein encoded by the fructose-1,6-bisphosphatase fbp gene is shown in SEQ ID NO.10.

[0012] 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.

[0013] 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 recombinant vector contains the acetyl-CoA acetyltransferase atoB gene, the HMG-CoA synthetase MvaS gene and the HMG-CoA reductase MvaE gene.

[0014] Furthermore, the present invention also provides a method for constructing the recombinant Escherichia coli for producing mevalonic acid, 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 comprising an acetyl-CoA acetyltransferase atoB gene, an HMG-CoA synthetase MvaS gene, and an HMG-CoA reductase MvaE gene; Step S3, transforming the recombinant vector prepared in step S1 and the recombinant vector prepared in step S2 into Escherichia coli at the same time, to obtain.

[0015] In addition, the present invention also provides a method for producing mevalonic acid by fermentation, wherein the recombinant Escherichia coli MG1655 LS01 for producing mevalonic acid is used as a fermentation strain, glucose is used as a substrate, and the culture is carried out at a temperature of 20-37°C until OD600 is 10-15, and then IPTG (isopropyl-β-D-thiogalactoside) inducer is added to induce the synthesis of mevalonic acid to obtain the product.

[0016] Furthermore, the fermentation conditions are as follows: fermentation temperature is 20-37°C, rotation speed is 100-600 rpm, dissolved oxygen is controlled between 30-40%, pH is controlled between 5.0-6.5, and fermentation is continued for 48-60 h after adding the inducer.

[0017] Furthermore, the concentration of the IPTG inducer is 0.5 mmol / L.

[0018] In addition, the present invention also seeks to protect the use of R-configuration mevalonic acid obtained by the method for producing mevalonic acid by fermentation in the preparation of cosmetics.

[0019] Furthermore, the cosmetics are products that have the function of enhancing the skin barrier, products that have the function of enhancing mitochondria and regulating lipid metabolism, products that have anti-wrinkle and firming effects, or beauty and skin care products.

[0020] Compared with the prior art, the recombinant Escherichia coli for producing mevalonic acid provided by the present invention has the following advantages: (1) The recombinant Escherichia coli for producing mevalonic acid provided by the present invention uses glucose as a substrate. After fermentation at 37°C in a 5 L fermenter 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 needs of industrial production.

[0021] (2) The R-configuration mevalonic acid produced by fermentation with the recombinant Escherichia coli for producing mevalonic acid provided by the present invention has the effects of enhancing the skin barrier function, strengthening mitochondria, regulating lipid metabolism, and anti-wrinkle and firming effects. It is an ideal cosmetic raw material and has broad application prospects in the fields of cosmetics.

[0022] (3) Compared with the traditional chemical synthesis method, the present invention uses the recombinant E. coli fermentation method to directly synthesize R-configuration mevalonic acid with physiological function, without the need for complex separation steps, thereby improving the purity and consistency of the product.

[0023] The microbial information of the recombinant Escherichia coli MG1655 LS01 for producing mevalonic acid provided by the present invention is as follows: the recombinant Escherichia coli MG1655 LS01 ( Escherichia coli MG1655 LS01) was deposited in 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, Postal Code: 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the metabolism of heterologous synthesis of mevalonic acid in Escherichia coli.

[0025] Figure 2 This is a map of the recombinant plasmid pGEX-mvaE-mvaS-atoB constructed in Example 4.

[0026] Figure 3 This is a map of the recombinant plasmid pR6K-fxpk-fbp constructed in Example 4.

[0027] Figure 4 This is a graph showing the production of mevalonic acid produced by fermentation of different recombinant strains in each group.

[0028] Figure 5 It is a bar graph of the relative expression of FLG gene.

[0029] Figure 6 It is a bar graph of relative expression of IVL genes.

[0030] Figure 7 It is a bar graph of the relative expression of LOR genes.

[0031] Figure 8 It is a bar graph of the relative expression of TGM1 gene.

[0032] Figure 9 This is the result of detecting the fluorescence intensity of mitochondria in zebrafish body muscles.

[0033] Figure 10 This is a graph showing cholesterol content test results.

[0034] Figure 11 This is a bar graph of the relative expression of zebrafish elastin gene.

[0035] Figure 12 This is a bar graph of the relative expression of type I collagen gene in zebrafish. DETAILED DESCRIPTION

[0036] The present invention is further illustrated below through the description of specific implementation modes, but 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, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0037] 1. Strains and primers involved in the present invention The strains and primers involved in Examples 1 to 4 of the present invention are shown in Tables 1 and 2.

[0038] Table 1 Strains involved in Examples 1 to 4 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 Table 2 Primer sequences used in Examples 1 to 4 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 ATACCGATCTTCATCTAGTATTTCTCCTCTTAATTTAATTCAACCGTTCAATC mvaS-f ACGGTTGAATtaaATTAAAGAGGAGAAATACTAGATGAAGATCGGTATCGAC mvaS-f1 aggaaacagtattcATTAAAGAGGAGAAATACTAGATGAAGATCGGTATCGAC mvaS-r CGTAGAATTTCATCTAGTATTTCTCCTTCTTTAATTTAGTGAGTGGAGGTAGAG mvaE-f TCCACTCACTAAATTAAAGAGGAGAAATACTAGATGAAATTCTACGAAAAAAC mvaE-r gtcgacccgggaattccggggatccttactcgagGTCACGGTT 2. The culture medium involved in Examples 1 to 4 of the present invention is as follows: (1) LB medium formula: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 1.5-2.0% agar powder for solid culture medium.

[0039] (2) Fermentation medium: 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 niacin, 0.032 g / L vitamin B6, 1 mL / L trace metal solution.

[0040] The formula of the trace metal solution is: 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, and 1.0 g / L CoCl2·6H2O.

[0041] 3. The nucleotide sequences and amino acid sequences involved in Examples 1 to 4 of the present invention are as follows: (1) Derived from Lactobacillus casei ( Lactobacillus casei ) is (SEQ ID NO.1): (2) Derived from Lactobacillus casei ( Lactobacillus casei ) is (SEQ ID NO.2): (3) The nucleotide sequence of the acetyl-CoA acetyltransferase atoB gene from Escherichia coli is (SEQ ID NO.3): (4) Derived from Bifidobacterium adolescentis ( Bifidobacterium adolescentis The nucleotide sequence of the phosphoketolase fxpk gene of ) is (SEQ ID NO.4): (5) The nucleotide sequence of the fructose-1,6-bisphosphatase fbp gene from Escherichia coli is (SEQ ID NO.4): ATGAAAACGTTAGGTGAATTTATTGTCGAAAAGCAGCACGAGTTTTCTCATGCTACCGGTGAGCTCACTGCTTTGCTGTCGGCAATAAAACTGGGCGCCAAGATTATCCATCGCGATATCAACAAAGCAGGACTGGTTGATATCCTGGGTGCCAGCGGTGCTGAGAACGTGCAGGGCGAGGTTCAGCAGAAACTCGACTTGTTCGCTAATGAAAAACTGAAAGCCGCACTGAAAGCACGCGATATCGTTGCGGGCATTGCCTCTGAAGAAGAAGATGAGATTGTCGTCTTTGAAGGCTGTGAACACGCAAAATACGTGGTGCTGATGGACCCCCTGGATGGCTCGTCCAACATCGATGTTAACGTCTCTGTCGGTACCATTTTCTCCATCTACCGCCGCGTTACGCCTGTTGGCACGCCGGTAACGGAAGAAGATTTCCTCCAGCCTGGTAACAAACAGGTTGCGGCAGGTTACGTGGTATACGGCTCCTCTACCATGCTGGTTTACACCACCGGATGCGGTGTTCACGCCTTTACTTACGATCCTTCGCTCGGCGTTTTCTGCCTGTGCCAGGAACGGATGCGCTTCCCGGAGAAAGGCAAAACCTACTCCATCAACGAAGGAAACTACATTAAGTTTCCGAACGGGGTGAAGAAGTACATTAAATTCTGCCAGGAAGAAGATAAATCCACCAACCGCCCTTATACCTCACGTTATATCGGTTCACTGGTCGCGGATTTCCACCGTAACCTGCTGAAAGGCGGTATTTATCTCTACCCAAGCACCGCCAGCCACCCGGACGGCAAACTGCGTTTGCTGTATGAGTGCAACCCGATGGCATTCCTGGCGGAACAAGCGGGCGGTAAAGCGAGCGATGGCAAAGAGCGTATTCTGGATATCATCCCGGAAACCCTGCACCAGCGCCGTTCATTCTTTGTCGGCAACGACCATATGGTTGAAGATGTCGAACGCTTTATCCGTGAGTTCCCGGACGCGTAA (6)The amino acid sequence of the protein encoded by the HMG-CoA synthase MvaS gene is (SEQ ID NO.6): MKIGIDAIAMDTPDFYVDLVELAKVRGDEPDKYTIGIGQDEQAVPPSSQDIVTMGANAAAPLMTPAVRDSLGMVLVGTESGVDASKSAALFIHQLLDLPEWVRAAELKEACYGGTAALMMARDYVAGHPDKSVLVIAADIARYGLATAGEVTQGAGAVAMIVKADPRLITIESDSVYRSASINDFWRPVYQDTAIAQGKYSTEQYLAFFQAVWDRYQAENHLTAADFAGMTFHLPYTKMGKKALDLVLPEADDQTVKRLKERFDASTRYCRRIGNIYTGSLYLGLLSLLDHDATLQAGDRIGLFSYGSGAVAEFFSGILQPGFKDQLQSAAHARQLDDRQQLTAPEYEALFSDKVPYDPEDYRPDPAYFRGQFVLTGVVGQERLYEKRPANSSTSTH* (7)The amino acid sequence of the protein encoded by the HMG-CoA reductase MvaE gene is (SEQ ID NO.7): MKFYEKTPEERRAQLIREGSLTQADASLFAATLSLPAATEAKLIENSIGEFSLPLGIARNLLVNGQLHQVPLANEEPSVVAAASNGARIAAENGGVVARVDSHQVVAEVVLTDLADLEAAKQTVLAHQADIKAIVAAAHPSMIQRGGGLVGVAVSVLANRFLKIRLTLNPKEAMGANYANTVAEAVAAVVKTWLNGTILVSILTNTPAELVTATVQLDPPTLATETASGEVIAKKIVQLSELAFVDPERAVTHNKGILNGVIGAVLATGNDTRAIAASVGAFAAASGQYQPLSRWVMHAGKLEGTLQIPLPLGAVGGAIGALPLAQAARRLGGYRDLATMQQVIAALGLVQNLAALRALAGPGIQAGHMKLQANALAIAAGADERELPALVAALREGQMDLAHAKTYLANIRSNQKVGQSKNENRDLE* (8) The amino acid sequence of the protein encoded by the acetyl-CoA acetyltransferase atoB gene is (SEQ ID NO.8): MKNCVIVSAVRTAIGSFNGSLASTSAIDLGATVIKAAIERAKIDSQHVDEVIMGNVLQAGLGQNPARQALLKSGLAETVCGFTVNKVCGSGLKSVALAAQAIQAGQAQSIVAGGMENMSLAPYLLDAKARSGYRLGDGQVYDVILRDGLMCATHGYHMGITAENVAKEYGITREMQDELALHSQRKAAAAIESGAFT AEIVPVNVVTRKKTFVFSQDEFPKANSTAEALGALRPAFDKAGTVTAGNASGINDGAAALVIMEESAALAAGLTPLARIKSYASGGVPPALMGMGPVPATQKALQLAGLQLADIDLIEANEAFAAQFLAVGKNLGFDSEKVNVNGGAIALGHPIGASGARILVTLLHAMQARDKTLGLATLCIGGGQGIAMVIERLN* (9) The amino acid sequence of the protein encoded by the phosphoketolase fxpk gene is (SEQ ID NO.9): MTSPVIGTPWKKLNAPVSEEAIEGVDKYWRAANYLSIGQIYLRSNPLMKEPFTREDVKHRLVGHWGTTPGLNFLIGHINRLIADHQQNTVIIMGPGHGGPAGTAQSYLDGTYTEYFPNITKDEAGLQKFFRQFSYPGGIPSHYAPETPGSIHEGGELGYALSHAYGAVMNNPSLFVPAIVGDGEAETGPLATGWQSNKLINPRTDGIVLPILHLNGYKIANPTILSRISDEELHEFFHGMGYEPYEFVAGFDNEDHLSIHRRFAELFETVFDEICDIKAAAQTDDMTRPFYPMIIFRTPKGWTCPKFIDGKKTEGSWRSHQVPLASARDTEAHFEVLKNWLESYKPEELFDENGAVKPEVTAFMPTGELRIGENPNANGGRIREELKLPKLEDYEVKEVAEYGHGWGQLEATRRLGVYTRDIIKNNPDSFRIFGPDETASNRLQAAYDVTNKQWDAGYLSAQVDEHMAVTGQVTEQLSEHQMEGFLEGYLLTGRHGIWSSYESFVHVIDSMLNQHAKWLEATVREIPWRKPISSMNLLVSSHVWRQDHNGFSHQDPGVTSVLLNKCFNNDHVIGIYFPVDSNMLLAVAEKCYKSTNKINAIIAGKQPAATWLTLDEARAELEKGAAEWKWASNVKSNDEAQIVLAATGDVPTQEIMAAADKLDAMGIKFKVVNVVDLVKLQSAKENNEALSDEEFAELFTEDKPVLFAYHSYARDVRGLIYDRPNHDNFNVHGYEEQGSTTTPYDMVRVNNIDRYELQAEALRMIDADKYADKINELEAFRQEAFQFAVDNGYDHPDYTDWVYSGVNTNKQGAISATAATAGDNE* (10)The amino acid sequence of the protein encoded by the fructose-1,6-bisphosphatase fbp gene is (SEQ ID NO.10): MKTLGEFIVEKQHEFSHATGELTALLSAIKLGAKIIHRDINKAGLVDILGASGAENVQGEVQQKLDLFANEKLKAALKARDIVAGIASEEEDEIVVFEGCEHAKYVVLMDPLDGSSNIDVNVSVGTIFSIYRRVTPVGTPVTEEDFLQPGNKQVAAGYVVYGSSTM LVYTTGCGVHAFTYDPSLGVFCLCQERMRFPEKGKTYSINEGNYIKFPNGVKKYIKFCQEEDKSTNRPYTSRYIGSLVADFHRNLLKGGIYLYPSTASHPDGKLRLLYECNPMAFLAEQAGGKASDGKERILDIIPETLHQRRSFFVGNDHMVEDVERFIREFPDA* Example 1. Construction of MVA-1 recombinant Escherichia coli Step S1, construction of recombinant plasmid pGEX-mvaE-mvaS: From Lactobacillus casei The HMG-CoA synthetase MvaS gene and HMG-CoA reductase MvaE gene were synthesized by Qingke Company. The primers mvaE-f / r, mvaS-f1 / r, and pGEX-f / r1 were used to amplify the fragments mvaE, mvaS, and the linearized plasmid pGEX. The recombinant plasmid pGEX-mvaE-mvaS was obtained by recombination using the ClonExpress technology of Novozymes.

[0042] The recombinant plasmid pGEX-mvaE-mvaS was transferred into Escherichia coli DH5α by chemical transformation, spread on LB solid culture medium plates containing 100 mg / L ampicillin resistance, and cultured in a 37°C incubator overnight. The plasmid of the grown single clone was extracted and sequenced to ensure that the inserted gene sequence was correct.

[0043] Step S2: Construction of MVA-1 engineered strain: The recombinant plasmid pGEX-mvaE-mvaS prepared in step S1 was transformed into competent cells of Escherichia coli MG 1655, spread on LB solid medium containing 100 mg / L ampicillin resistance, and cultured in a 37°C incubator overnight. A single clone was picked for LB liquid culture and stored in glycerol at -80°C to obtain a recombinant strain. E. coli MG1655 pGEX-mvaE-mvaS, i.e., MVA-1 strain.

[0044] Example 2: Construction of MVA-2 recombinant Escherichia coli Step S1, construction of recombinant plasmid pGEX-mvaE-mvaS-atoB: From Lactobacillus casei The HMG-CoA synthetase MvaS gene and HMG-CoA reductase MvaE gene were synthesized by Qingke Company. The primers mvaE-f / r, mvaS-f1 / r, and pGEX-f / r1 were used to amplify the fragments mvaE, mvaS, and the linearized plasmid pGEX. The recombinant plasmid pGEX-mvaE-mvaS was obtained by recombination using the ClonExpress technology of Novozymes.

[0045] Using the Escherichia coli genome as a template, the atoB fragment was amplified using primers atoB-f / r, and the recombinant plasmid pGEX-mvaE-mvaS was linearized using primers amvaS-f / pGEX-r. The recombinant plasmid pGEX-mvaE-mvaS-atoB was obtained by recombination using the ClonExpress technology of Novazonics.

[0046] The recombinant plasmid pGEX-mvaE-mvaS-atoB was transferred into Escherichia coli DH5α by chemical transformation, spread on LB solid culture medium plates containing 100 mg / L ampicillin resistance, and cultured in a 37°C incubator overnight. The plasmid was extracted from the grown single clones and sequenced to ensure that the inserted gene sequence was correct.

[0047] Step S2: Construction of MVA-2 engineering strain: The recombinant plasmid pGEX-mvaE-mvaS-atoB prepared in step S1 was transformed into competent cells of Escherichia coli MG 1655, spread on LB solid medium containing 100 mg / L ampicillin resistance, and cultured in a 37°C incubator overnight. A single clone was selected for LB liquid culture and stored in glycerol at -80°C to obtain a recombinant strain. E. coli MG1655pGEX-mvaE-mvaS-atoB, i.e., MVA-2 strain.

[0048] Example 3: Construction of MVA-3 recombinant Escherichia coli Step S1, construction of recombinant plasmid pGEX-mvaE-mvaS-atoB: From Lactobacillus caseiThe HMG-CoA synthetase MvaS gene and HMG-CoA reductase MvaE gene were synthesized by Qingke Company. The primers mvaE-f / r, mvaS-f1 / r, and pGEX-f / r1 were used to amplify the fragments mvaE, mvaS, and the linearized plasmid pGEX. The recombinant plasmid pGEX-mvaE-mvaS was obtained by recombination using the ClonExpress technology of Novozymes.

[0049] Using the Escherichia coli genome as a template, the atoB fragment was amplified using primers atoB-f / r, and the recombinant plasmid pGEX-mvaE-mvaS was linearized using primers amvaS-f / pGEX-r. The recombinant plasmid pGEX-mvaE-mvaS-atoB was obtained by recombination using the ClonExpress technology of Novazonics.

[0050] Step S2, construction of recombinant plasmid pR6K-fxpk: From Bifidobacterium adolescentis The phosphoketolase fxpk gene was synthesized by Qingke Co., Ltd. The fxpk fragment and the linearized plasmid pR6K were amplified using primers fxpk-f1 / r and pR6K-f / r1, respectively, and recombined using the ClonExpress technology of Novozymes to obtain the recombinant plasmid pR6K-fxpk.

[0051] The recombinant plasmids pGEX-mvaE-mvaS-atoB and pR6K-fxpk were transformed into Escherichia coli DH5α by chemical transformation, respectively spread on LB solid culture medium plates containing 30 mg / L chloramphenicol and 100 mg / L ampicillin resistance, and cultured in a 37°C incubator overnight. The plasmids of the grown single clones were extracted and sequenced to ensure that the inserted gene sequence was correct.

[0052] Step S3: Construction of MVA-3 engineering strain: 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 competent cells of Escherichia coli MG 1655, spread on LB solid medium containing 30 mg / L chloramphenicol and 100 mg / L ampicillin resistance, and cultured in a 37°C incubator overnight. Pick a single clone for LB liquid culture, store it in glycerol, and store it at -80°C to obtain a recombinant strain E. coli MG1655 pGEX-mvaE-mvaS-atoB&pR6K-fxpk, i.e., MVA-3 strain.

[0053] Example 4: Construction of MVA-4 recombinant Escherichia coli Step S1, construction of recombinant plasmid pGEX-mvaE-mvaS-atoB: From Lactobacillus casei The HMG-CoA synthetase MvaS gene and HMG-CoA reductase MvaE gene were synthesized by Qingke Company. The primers mvaE-f / r, mvaS-f1 / r, and pGEX-f / r1 were used to amplify the fragments mvaE, mvaS, and the linearized plasmid pGEX. The recombinant plasmid pGEX-mvaE-mvaS was obtained by recombination using the ClonExpress technology of Novozymes.

[0054] Using the Escherichia coli genome as a template, the atoB fragment was amplified with primers atoB-f / r, and the recombinant plasmid pGEX-mvaE-mvaS was linearized using primers amvaS-f / pGEX-r. The recombinant plasmid pGEX-mvaE-mvaS-atoB was obtained by recombination using the ClonExpress technology of Norwegian. Figure 2 as shown).

[0055] Step S2, construction of recombinant plasmid pR6K-fxpk-fbp: From Bifidobacterium adolescentis The phosphoketolase fxpk gene was synthesized by Qingke Company. The primers fxpk-f1 / r and pR6K-f / r1 were used to amplify the fxpk fragment and the linearized plasmid pR6K, respectively, and the recombinant plasmid pR6K-fxpk was obtained by recombination using the ClonExpress technology of Novazines. The fructose-1,6-bisphosphatase fbp gene derived from Escherichia coli was synthesized by Qingke Company. The primers fbp-f / r and fxpk-f / pR6K-r were used to amplify the fragment fbp and the linearized vector pR6K-fxpk, respectively, and the recombinant plasmid pR6K-fxpk-fbp was obtained by recombination using the ClonExpress technology of Novazines (the recombinant plasmid pR6K-fxpk-fbp is as shown in Figure 3 as shown).

[0056] The recombinant plasmid pGEX-mvaE-mvaS-atoB and the recombinant plasmid pR6K-fxpk-fbp were transferred into Escherichia coli DH5α by chemical transformation, and spread on LB solid culture medium plates containing 30 mg / L chloramphenicol and 100 mg / L ampicillin resistance, respectively, and cultured in a 37°C incubator overnight. The plasmids of the grown single clones were extracted and sequenced to ensure that the inserted gene sequence was correct.

[0057] Step S3: Construction of MVA-4 engineering strain: 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 competent cells of Escherichia coli MG 1655, spread on LB solid medium containing 30 mg / L chloramphenicol and 100 mg / L ampicillin resistance, and cultured in a 37°C incubator overnight. A single clone was picked for LB liquid culture and stored in glycerol at -80°C to obtain a recombinant strain. E. coli MG1655 pGEX-mvaE-mvaS-atoB&pR6K-fxpk-fbp, i.e., MVA-4 strain (recombinant Escherichia coli MG1655 LS01).

[0058] Example 5: Fermentation process of recombinant Escherichia coli (1) Seed solution preparation: The prepared recombinant Escherichia coli engineered strain was streaked on LB solid culture medium to obtain a single colony, and a single colony was picked and inoculated into a 150 mL conical flask containing 30 mL LB liquid culture medium, and cultured at 37°C and 200 rpm for 12 h to prepare a seed solution.

[0059] (2) Fermentation culture: The prepared seed liquid was inoculated at a 1% (v / v) inoculation rate into a 5 L fermentation tank containing 3 L fermentation medium, and cultured at a rotation speed of 100-600 rpm, aeration volume of 1 VVM, dissolved oxygen controlled at about 40%, glucose controlled at less than 5 g / L, pH value controlled at 6.0, and temperature of 30°C until the OD600 value reached 12. 0.5 mmol / L IPTG was added for gene expression induction to start the expression and activity of related enzymes in the mevalonate synthesis pathway, and the fermentation culture was continued for 48 h to obtain the product.

[0060] Experimental Example 1: Production Detection Experiment of Mevalonic Acid 1. Experimental methods: The fermentation process of the recombinant Escherichia coli of Example 5 was adopted, and the recombinant Escherichia coli prepared in Examples 1 to 4 were used as fermentation strains to ferment and produce mevalonic acid, which were recorded as MVA-1, MVA-2, MVA-3 and MVA-4, respectively. During the fermentation process, after culturing for 12 hours, samples were taken every 2 hours to detect the yield of mevalonic acid. Mevalonic acid was detected by high performance liquid chromatography HPLC. The chromatographic detection conditions were as follows: the chromatographic column was an AminexR HPX-87H ion exchange column, the detector was a RID-10A differential 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.

[0061] 2. Experimental results: The experimental results are as follows Figure 4 And as shown in Table 3.

[0062] 2.1. The results of the detection of mevalonic acid production of each group of recombinant Escherichia coli are as follows Figure 4 shown.

[0063] 2.2. The yield of mevalonic acid after 48 h of fermentation by recombinant E. coli in each group is shown in Table 3: Table 3 Results of the production of mevalonic acid by recombinant E. coli in each group after 48 h of fermentation 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 As shown in Table 3, the recombinant Escherichia coli MG1655 LS01 ( Escherichia coli MG1655 LS01) can produce a high amount of R-configuration methylvalonic acid in a short time, meeting the needs of industrial production.

[0064] Example 2: Experimental study on the efficacy of mevalonic acid in enhancing the skin barrier function 1. Experimental materials: The fermentation process of the recombinant Escherichia coli of Example 5 was adopted, and the recombinant Escherichia coli MG1655LS01 prepared in Example 4 was used as the fermentation strain to produce mevalonic acid.

[0065] 2. Experimental methods: The skin barrier enhancement effect of mevalonate produced by fermentation of the recombinant Escherichia coli MG1655 LS01 prepared in Example 4 was detected. The test sample was recorded as LightSyn / mevalonate lactone. The detection method was referred to the "Standard Operating Procedure for Cosmetic Skin Barrier Enhancement Test", which is as follows: 2.1. System and sample size: Cell line: human immortalized keratinocytes (HaCaT).

[0066] Cell generation number: at least 2 generations.

[0067] The number of cells per group: 6×10 5 Pieces / hole.

[0068] 2.2 Specific steps: (1) Inoculate cells in a 6-well plate (6×10 5 / well) and cultured at 37 ℃, 5% CO2 for 24 h.

[0069] (2) After the incubation, the cells were gently rinsed once or twice with D-Hanks. Fresh culture medium was added to the normal control group, and fresh culture medium containing samples of corresponding concentrations was added to the sample group. The detection concentrations (mass concentration, wt%) were: 0.156%, 0.312%, and 0.625%. The cells were cultured at 37°C and 5% CO2 for 24 h.

[0070] (3) Total RNA was extracted from each experimental group, cDNA was synthesized, and the gene expression of β-actin and target genes was detected by q-PCR.

[0071] (4) Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene.

[0072] ; 3. Experimental results: The experimental results are shown in Table 4 and Figure 5~Figure 8 shown.

[0073] 3.1. The relative expression levels of FLG, IVL, LOR and TGM1 genes are shown in Table 4.

[0074] Table 4 Relative expression results of FLG, IVL, LOR and TGM1 genes Detection concentration (%) Relative expression of FLG gene IVL gene relative expression Relative expression of LOR gene Relative expression 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 3.2. The results of relative expression of FLG, IVL, LOR and TGM1 genes are shown in Figure 5~Figure 8 shown.

[0075] Figure 5 The bar graph shows the relative expression of FLG gene. Compared with the normal control group, ***p < 0.001. Figure 6 The bar graph shows the relative expression of IVL genes. Compared with the normal control group, *p < 0.05, ***p < 0.001. Figure 7 The bar graph shows the relative expression of LOR genes. Compared with the normal control group, *p < 0.05, ***p < 0.001. Figure 8 The bar graph shows the relative expression of TGM1 gene. Compared with the normal control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0076] From Table 4 and Figure 5~Figure 8 It can be seen that the relative expression levels of FLG, IVL, LOR and TGM1 genes in the LightSyn / methylvalonolactone group were significantly increased compared with the normal control group, revealing that the sample has the effect of enhancing the skin barrier.

[0077] Experimental Example 3: Experiment on the efficacy of methylvalonate in enhancing mitochondrial function 1. Experimental materials: The fermentation process of the recombinant Escherichia coli of Example 5 was adopted, and the recombinant Escherichia coli MG1655LS01 prepared in Example 4 was used as the fermentation strain to produce mevalonic acid.

[0078] 2. Experimental methods: The mitochondrial enhancement effect of mevalonate produced by fermentation of the recombinant Escherichia coli MG1655 LS01 prepared in Example 4 was detected. The test sample was recorded as LightSyn / mevalonate lactone. The detection method was referred to the "Standard Operating Procedure for Evaluation of Mitochondrial Enhancement in Zebrafish" and was as follows: 2.1. System and sample size: Experimental system: Transgenic mitochondrial green fluorescent zebrafish (CZ222).

[0079] Zebrafish age: 4 days post fertilization (4 dpf).

[0080] The sample size of each experimental group was 15 (N=10).

[0081] Adult fish rearing and breeding methods: in line with the requirements of the international AAALAC certification (certification number: 001458).

[0082] 2.2 Specific steps: (1) Randomly select zebrafish and place them in a 6-well plate, with 15 zebrafish in each well.

[0083] (2) For water-soluble samples, the sample detection concentration (mass concentration, wt%) was 0.1%. A normal control group and a model control group were set up at the same time. The volume of each well was 3 mL.

[0084] (3) Incubate at 28°C in the dark for 22 h.

[0085] (4) Except for the normal control group, all other experimental groups were given cobalt chloride hexahydrate to establish the zebrafish mitochondrial damage model.

[0086] (5) Incubate at 28°C in the dark for 4 h.

[0087] After the incubation, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Advanced image processing software was used to analyze and collect data, and the fluorescence intensity (S) of mitochondria in the zebrafish body muscles was analyzed. The enhanced mitochondrial effect of the sample was calculated according to the formula to determine whether it had the enhanced mitochondrial effect.

[0088] ; 3. Experimental results: The experimental results are shown in Table 4 and Fig. 9 shown.

[0089] 3.1. The test results of sample LightSyn / Mevalonolactone for enhancing mitochondrial effects are shown in Table 5: Table 5 Test results of sample LightSyn / Mevalonolactone enhancing mitochondrial effect Detection concentration (%) effect(%) p-value Test results 0.1 97 < 0.001 Significant 3.2. The results of the fluorescence intensity test of zebrafish body muscle mitochondria in the LightSyn / Mevalonolactone group are as follows Fig. 9 shown.

[0090] From Table 5 and Fig. 9 It can be seen that the fluorescence intensity of mitochondria in the zebrafish body muscle of the LightSyn / methylvalonolactone group was significantly increased compared with the model control group, revealing that the sample has an enhancing mitochondrial effect.

[0091] Experimental Example 4: Efficacy experiment of mevalonic acid in regulating lipid metabolism 1. Experimental materials: The fermentation process of the recombinant Escherichia coli of Example 5 was adopted, and the recombinant Escherichia coli MG1655LS01 prepared in Example 4 was used as the fermentation strain to produce mevalonic acid.

[0092] 2. Experimental methods: The lipid metabolism regulating effect of mevalonate produced by fermentation of the recombinant Escherichia coli MG1655 LS01 prepared in Example 4 was detected. The detected sample was recorded as LightSyn / mevalonate lactone. The detection method was referred to the "Standard Operating Procedure for Evaluation of Lipid Metabolism Regulation in Zebrafish", which is as follows: 2.1. System and sample size: Fish strain used: Wild-type AB strain zebrafish.

[0093] Zebrafish age: 6 hours post fertilization (6 hpf).

[0094] The number of experimental samples in each group was 30 (three biological replicates, N=3).

[0095] Adult fish rearing and breeding methods: in line with the requirements of the international AAALAC certification (certification number: 001458).

[0096] 2.2 Specific steps: (1) Randomly select zebrafish and place them in a 6-well plate, with 30 zebrafish in each well.

[0097] (2) Water-soluble administration samples, the sample detection concentration (mass concentration, wt%) was 0.1%, and a normal control group was set up at the same time, with a volume of 3 mL per well. Three biological replicates were performed.

[0098] (3) Incubate at 28°C in the dark for 120 h.

[0099] (4) Collect zebrafish samples from each experimental group, grind them into homogenate using a grinder, centrifuge and obtain the supernatant, and use the BCA protein concentration assay kit to determine the protein concentration of each experimental group.

[0100] (5) Use a cholesterol detection kit to measure, use an ELISA instrument to collect data, analyze the OD500 value of each experimental group, calculate the cholesterol content (C) based on the OD500 value, and calculate and determine whether the sample has the effect of regulating lipid metabolism based on the formula.

[0101] ; 3. Experimental results: The experimental results are shown in Table 6 and Fig.10 shown.

[0102] 3.1. The test results of the sample LightSyn / Mevalonolactone regulating lipid metabolism are shown in Table 6: Table 6 Test results of the effect of sample LightSyn / Mevalonolactone on regulating lipid metabolism Detection concentration (%) effect(%) p-value Test results 0.1 8 < 0.001 Significant 3.2. The cholesterol content results of the LightSyn / Mevalonate group are as follows: Fig.10 shown.

[0103] Fig.10 The cholesterol content results of the LightSyn / Mevalonolactone group, where: compared with the normal control group, ***p < 0.001.

[0104] From Table 6 and Fig.10 It can be seen that the cholesterol content of the LightSyn / methylvalonate group was significantly increased compared with the normal control group, revealing that the sample has the effect of regulating lipid metabolism.

[0105] Experimental Example 5: Anti-wrinkle and firming effect experiment of methylvalonate 1. Experimental materials: The fermentation process of the recombinant Escherichia coli of Example 5 was adopted, and the recombinant Escherichia coli MG1655LS01 prepared in Example 4 was used as the fermentation strain to produce mevalonic acid.

[0106] 2. Experimental methods: The anti-wrinkle and firming effects of mevalonate produced by fermentation using the recombinant Escherichia coli MG1655 LS01 prepared in Example 4 as the fermentation strain were tested. The testing method was referred to the "Anti-wrinkle and Firming Effects of Cosmetics - Test Method for Promoting Expression of Zebrafish Elastin Gene", which is as follows: Thirty-six six-day-old zebrafish were divided into three groups and exposed to 0.025 g / L, 0.0025 g / L and 0.0005 g / L Xiaoyu 15 sample (LightSyn / methylvalonolactone) solutions, respectively. A blank control group was also set up. After 24 h of exposure, RNA was extracted from the zebrafish, cDNA was synthesized, and real-time PCR amplification was performed. β-actin was used as the housekeeping gene and Ct was used as the amplification result. The relative expression level of Elna was calculated and statistical analysis was performed.

[0107] 3. Experimental results: The experimental results are shown in Table 7 and Fig.11 shown.

[0108] 3.1. The results of the zebrafish elastin gene Elna expression promotion rate test are shown in Table 7.

[0109] Table 7 Results of the expression promotion rate test of zebrafish elastin gene Elna Test concentration result Anti-wrinkle and firming efficacy evaluation 0.025g / L 24%(p=0.017) Significant 0.0025g / L 107%(p=0.000080) Significant 0.0005g / L 67% (p=0.0033) Significant 3.2. Results of relative expression of zebrafish elastin gene Fig.11 shown.

[0110] Fig.11 It is a bar graph of the relative expression of zebrafish elastin gene, where: *p<0.05.

[0111] From Table 7 and Fig.11 It can be seen that the samples at the test concentrations of 0.025g / L, 0.0025g / L and 0.0005g / L have promotion rates of 24% (p=0.017), 107% (p=0.000080) and 67% (p=0.0033) on zebrafish Elna gene expression respectively. The samples can significantly promote zebrafish Elna gene expression and have the effect of promoting elastin regeneration, supporting the anti-wrinkle and firming efficacy claims.

[0112] Experimental Example 6: Experiment on the Anti-wrinkle and Firming Effects of Mevalonate 1. Experimental materials: The fermentation process of the recombinant Escherichia coli of Example 5 was adopted, and the recombinant Escherichia coli MG1655LS01 prepared in Example 4 was used as the fermentation strain to produce mevalonic acid.

[0113] 2. Experimental methods: The anti-wrinkle and firming effects of mevalonate produced by fermentation of the recombinant Escherichia coli MG1655 LS01 prepared in Example 4 were detected. The detection method was referred to the "Anti-wrinkle and Firming Effect of Cosmetics - Test Method for Promoting Expression of Type I Collagen Gene in Zebrafish", which is as follows: Thirty-six six-day-old zebrafish were divided into three groups and exposed to 0.025 g / L, 0.0025 g / L and 0.0005 g / L LightSyn / methylvalonolactone solutions, respectively. A blank control group was also set up. After 24 h of exposure, RNA was extracted from the zebrafish, cDNA was synthesized, and real-time PCR amplification was performed. β-actin was used as the housekeeping gene and Ct was used as the amplification result. The relative expression levels of col1a1a, col1a1b and col1a2 were calculated and statistically analyzed.

[0114] 3. Experimental results: The experimental results are shown in Table 8 and Fig.12 shown.

[0115] 3.1. The results of the relative expression promotion rate test of zebrafish type I collagen gene are shown in Table 8.

[0116] Table 8 Results of relative expression promotion rate of zebrafish type I collagen gene Test concentration Expression 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 Anti-wrinkle and firming efficacy evaluation 0.025g / L 41% (p=0.0014) 101% (p=0.000034) 48% (p=0.0010) Significant 0.0025g / L 41% (p=0.011) 49% (p=0.0088) 75% (p=0.0019) Significant 0.0005g / L 55% (p=0.000033) 122% (p=0.00039) 68% (p=0.015) Significant 3.2. The relative expression results of zebrafish type I collagen gene are as follows Fig.12 shown.

[0117] Fig.12 This is a bar graph of the relative expression of type I collagen gene in zebrafish, where: *p<0.05.

[0118] From Table 8 and Fig.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 anti-wrinkle and firming effect claims.

[0119] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall 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 MG1655 LS01 ( Escherichia coli MG1655 LS01) was deposited in the China Center for Type Culture Collection on November 11, 2024, with the deposit number: CCTCC NO: M 20242500.

2. The recombinant Escherichia coli for producing mevalonic acid according to claim 1, characterized in that The recombinant Escherichia coli MG1655 LS01 is derived from Lactobacillus casei ( Lactobacillus casei ) of the HMG-CoA synthetase MvaS gene, the HMG-CoA synthetase MvaS gene from Lactobacillus casei ( Lactobacillus casei ) of HMG-CoA reductase MvaE gene, acetyl-CoA acetyltransferase atoB gene from Escherichia coli as shown in SEQ ID NO.3, and Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) and the fructose-1,6-bisphosphatase fbp gene from Escherichia coli were transferred into Escherichia coli host cells.

3. The recombinant Escherichia coli for producing mevalonic acid according to claim 2, characterized in that: The amino acid sequence of the protein encoded by the HMG-CoA synthetase MvaS gene is shown in SEQ ID NO.6; the amino acid sequence of the protein encoded by the HMG-CoA reductase MvaE gene is shown in SEQ ID NO.7; the amino acid sequence of the protein encoded by the acetyl-CoA acetyltransferase atoB gene is shown in SEQ ID NO.8; The amino acid sequence of the protein encoded by the phosphoketolase fxpk gene is shown in SEQ ID NO.9; the amino acid sequence of the protein encoded by the fructose-1,6-bisphosphatase fbp gene is shown in SEQ ID NO.

10.

4. The recombinant Escherichia coli for producing mevalonic acid according to claim 1, characterized in that The recombinant Escherichia coli MG1655 LS01 comprises at least two recombinant vectors, and the recombinant vectors are selected from at least one of pGEX, pR6K, pMAL, pTrcHis, pBAD, and pQE.

5. The recombinant Escherichia coli for producing mevalonic acid according to claim 4, characterized in that The recombinant Escherichia coli MG1655 LS01 comprises two recombinant vectors, one of which comprises the fructose-1,6-bisphosphatase fbp gene and the phosphoketolase fxpk gene, and the other comprises the acetyl-CoA acetyltransferase atoB gene, the HMG-CoA synthetase MvaS gene and the HMG-CoA reductase MvaE gene.

6. A method for constructing a recombinant Escherichia coli for producing mevalonic acid according to any one of claims 1 to 5, characterized in that: The following steps are involved: 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 comprising acetyl-CoA acetyltransferase atoB gene, HMG-CoA synthetase MvaS gene and HMG-CoA reductase MvaE gene; Step S3, transforming the recombinant vector prepared in step S1 and the recombinant vector prepared in step S2 into Escherichia coli at the same time, to obtain.

7. A method for producing mevalonic acid by fermentation, characterized in that: The recombinant Escherichia coli MG1655 LS01 for producing mevalonic acid according to any one of claims 1 to 5 is used as a fermentation strain, glucose is used as a substrate, and the culture is carried out at a temperature of 20 to 37° C. until OD600 is 10 to 15, and then an IPTG inducer is added to induce mevalonic acid synthesis to obtain the product.

8. The method for producing mevalonic acid by fermentation according to claim 7, characterized in that: The fermentation conditions are as follows: fermentation temperature is 20-37°C, rotation speed is 100-600 rpm, dissolved oxygen is controlled between 30-40%, pH is controlled between 5.0-6.5, and fermentation is continued for 48-60 h after adding the inducer; the concentration of the IPTG inducer is 0.5 mmol / L.

9. Use of mevalonic acid obtained by the method for producing mevalonic acid by fermentation as claimed in claim 7 or 8 in the preparation of cosmetics.

10. The use according to claim 9, characterized in that The cosmetics are products that have the function of enhancing the skin barrier, products that have the function of enhancing mitochondria and regulating lipid metabolism, products that have anti-wrinkle and firming effects, or beauty and skin care products.

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