Construction method of nicotinamide engineering bacteria for cosmetics

Abstract

The invention discloses a construction method of nicotinamide engineering bacteria for cosmetics, which comprises the following steps: amplifying target DNA, recovering the target DNA, carrying out seamless cloning or CPEC connection on the recovered DNA fragment and a vector, carrying out seamless cloning by using a seamless cloning kit of an all-type gold company, carrying out connection by using a sterile ultrapure water complementing system, and carrying out plasmid construction. The method comprises the following steps: inoculating a liquid culture medium with a strain which is verified to be correct, carrying out plasmid extraction on the obtained bacterial liquid by using an AxyPrep plasmid small extraction kit, carrying out shake flask small test on enzyme activity, thallus propagation and protein expression after chassis strain gene editing, adding a substrate 3-cyanopyridine solution into the crushed thallus for reaction, and carrying out HPLC (High Performance Liquid Chromatography) detection on nicotinamide. According to the method, the nitrile hydratase is expressed through metabolism of microorganisms, conversion from 3-cyanopyridine to nicotinamide is achieved through one-step reaction after cell disruption, the enzyme activity of the nitrile hydratase is improved, and meanwhile the generation amount of the by-product nicotinic acid is reduced.

Description

Technical Field

[0001] This invention relates to the field of whole-cell microbial catalysis technology, and in particular to a method for constructing engineered bacteria for cosmetic use containing nicotinamide. Background Technology

[0002] Niacinamide is a water-soluble vitamin, also known as vitamin B3 or nicotinamide, and is the amide form of nicotinic acid. It has important physiological functions and a wide range of applications. Physiologically, nicotinamide is a core component of cellular energy metabolism, participating in intracellular redox reactions to help convert nutrients into energy. It can also activate poly(ADP-ribose) polymerase (PARP) to participate in DNA damage repair, maintain cellular genome stability, and regulate cell signaling, influencing processes such as cell proliferation, differentiation, and apoptosis.

[0003] In the field of skin care, niacinamide has garnered significant attention due to its multiple benefits. It can inhibit melanin synthesis and transport, reducing melanin deposition in the epidermis and thus achieving a whitening effect; simultaneously, it promotes collagen synthesis, increasing skin elasticity and firmness, and delaying aging; it can also promote the synthesis of lipids in the stratum corneum, particularly the production of ceramides, enhancing the skin barrier function, reducing moisture loss, and improving moisturizing ability; furthermore, niacinamide has anti-inflammatory effects, inhibiting the release of inflammatory factors and alleviating symptoms such as redness and itching, making it suitable for treating skin problems such as acne and dermatitis. In the medical field, niacinamide also has wide applications, regulating lipid metabolism, lowering LDL cholesterol levels, and raising HDL cholesterol levels, thus assisting in the treatment of cardiovascular diseases; it also participates in energy metabolism and DNA repair, protecting nerve cells and slowing the progression of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases.

[0004] In China, research on nicotinamide and its derivatives mainly focuses on anti-aging and disease treatment. For example, studies have shown that nicotinamide adenine dinucleotide (NAD⁺) and its precursors play an important role in preventing and treating age-related diseases, effectively delaying the aging process by regulating cell metabolism and gene expression. Internationally, research on nicotinamide in skin care and disease treatment is more extensive. Studies have shown that nicotinamide can effectively inhibit melanin synthesis and transport, reduce skin pigmentation, and promote collagen synthesis, thus delaying skin aging. Furthermore, nicotinamide has also shown good effects in treating skin diseases such as acne and psoriasis. In the medical field, nicotinamide is used to regulate lipid metabolism, assist in the treatment of cardiovascular diseases, and play an adjunctive role in the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0005] Currently, the main method for producing nicotinamide is chemical synthesis. This method primarily involves the hydrolysis of 3-methylpyridine (3-cyanopyridine) in water and a catalyst (such as manganese dioxide) to produce nicotinamide. However, the product purity is low, requiring further purification; the catalyst activity and selectivity also need improvement. Alternatively, it can be synthesized via photonitrosation-isomerization and Beckmann rearrangement, or potassium permanganate oxidation and amidation. However, this method has drawbacks such as the need for toxic and harmful gases like nitrosyl chloride, high requirements for the operating environment and safety, relatively complex photochemical reaction equipment, numerous process steps requiring various reagents and catalysts, and the need for high-pressure reaction equipment and relatively harsh reaction conditions.

[0006] In contrast, microbial enzymatic processes, as an environmentally friendly production method, cleverly utilize the natural biosynthetic capabilities of microbial strains to manufacture nicotinamide, offering a safer and greener option. This method not only reduces the introduction of harmful substances but also demonstrates the enormous potential of biotechnology in modern production.

[0007] However, since niacinamide is the amide form of nicotinic acid, the biosynthesis of niacinamide easily produces the byproduct nicotinic acid, which is highly irritating to the skin. This greatly limits the amount of niacinamide that can be added to cosmetics. Summary of the Invention

[0008] The purpose of this invention is to provide a method for constructing engineered bacteria for cosmetic use with nicotinamide, in order to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution: The method for constructing engineered bacteria for cosmetic nicotinamide includes the following steps: S1: Use FastPfu Fly high-fidelity enzyme is used as an amplification enzyme kit, which uses DNA fragments, plasmids, or genomes containing the target fragment as templates and correspondingly designed primers for PCR amplification. S2: Separate DNA fragments by agarose gel electrophoresis, centrifuge, melt, and vortex. Add the melted gel to the adsorption column, centrifuge a second time, add SPW Buffer to the adsorption column, centrifuge a third time, place the adsorption column in a sterile dry centrifuge tube and place it in an oven, and add sterile ultrapure water to elute the DNA. S3: Perform seamless cloning or CPEC ligation of the recovered DNA fragments with the vector (homologous sequences are required). For seamless cloning, use the Seamless Cloning Kit from TransGen Corporation. Add 6 μl of Mix enzyme, add the fragments and vectors to the system at a molar ratio of 1:3, and make up the system to 10 μl with sterile ultrapure water. Ligate at 50°C for 20 min. S4: Inoculate the verified strain into liquid culture medium and incubate at 37 °C and 250 rpm on a constant temperature shaking incubator for 8-10 hours until the bacterial culture OD reaches its limit. 600 The concentration was 0.8-1.2, and the obtained bacterial culture was used for plasmid extraction using the AxyPrep plasmid mini-prep kit; S5: Two-step method for E. coli gene knockout based on Cas9 recombination system; S6: Dilute the bacterial culture stored in the glycerol tube by 10. -4 The culture was then spread onto Kan-resistant LB agar plates and incubated at 37°C for 16 h. Single colonies were picked and inoculated into Kan-resistant LB shake flasks at a final Kan concentration of 50 mg / L. The flasks were incubated at 37°C and 250 rpm for 7 h. TB medium was then transferred at a 2% transfer rate, and the culture was incubated at 37°C and 250 rpm for 1 h. IPTG at a final concentration of 0.05 mM was added to induce nitrile hydratase expression, and the culture conditions were adjusted to 25°C and 220 rpm. Enzyme activity was measured after 16 h of incubation. S7: Dilute the bacterial culture stored in the glycerol tube by 10. -4 The culture was then spread onto Kan-resistant LB agar plates and incubated at 37°C for 16 hours. A single colony was picked and inoculated into a Kan-resistant LB flask with a final Kan concentration of 50 mg / L. The flask was then incubated overnight at 37°C and 200 rpm for 13 hours, and the OD was measured. 600 Approximately 3-4 hours later, using this as seed culture, inoculate 5 L or 10 L fermenters to culture the cells. The temperature is controlled at 30℃ to avoid the formation of inclusion bodies. At the same time, add IPTG to the tank at a final concentration of 0.05 mM to induce the expression of nitrile hydratase, and add Kan antibiotic at a final concentration of 50 mg / L. After fermentation culture for 32 h, collect the cells from the tank, wash the cells with 1×PBS buffer, and then lyse the cells. S8: The nicotinamide was detected by HPLC after the lysed bacterial cells were reacted with a 3-cyanopyridine substrate solution.

[0010] Further, in S1, after PCR amplification, 3-5 mL of PCR product is aspirated for nucleic acid gel electrophoresis to check if the size of the target gene band is correct. If the electrophoresis result is consistent with expectations, the DNA fragment is recovered. If a plasmid is used as a template, 1 mL of Fast Digest gel electrophoresis solution needs to be added to the PCR product. FrontThe mixture is prepared in a 1 mL enzyme / 50 mL system and a 1 mL buffer / 10 mL system, and incubated overnight at 37 °C for digestion before DNA recovery and purification to remove template plasmids and avoid false positives. When amplifying the target fragment or vector, it is crucial to use enzymes with high fidelity to prevent mutations in the amplified DNA fragment. Primers should be checked for hairpin formation. The number of amplification cycles should not be excessive, as too many cycles may lead to enzyme inactivation in the later stages of amplification, resulting in DNA mutations.

[0011] Furthermore, S2 specifically includes the following steps: S21: DNA fragments were separated by agarose gel electrophoresis, using freshly prepared 1×TAE Buffer as the electrophoresis buffer; S22: When the desired DNA fragment is completely separated on the agarose gel, use a sterile scalpel to cut off the desired DNA fragment, removing as much excess gel as possible; S23: Transfer the gel block containing the target fragment to a 1.5 ml centrifuge tube and weigh it to obtain the weight of the gel block; S24: Add an equal volume of XP2 Binding Buffer to the centrifuge tube; calculate the required volume of XP2 Binding Buffer to be added based on 1 g / ml. S25: Incubate at 50-60℃ for 7 min or until the gel is completely melted, shaking the mixture every 2-3 min; if the gel block is large or the gel concentration is high, the incubation time can be extended appropriately until the colloid is completely melted; S26: Add the melted gel to the adsorption column, centrifuge at 10,000 rpm for 1 min at room temperature, and discard the filtrate; S27: Add 700 μl SPW Buffer to the adsorption column, centrifuge at 10000 rpm for 1 min at room temperature, discard the filtrate, and repeat S27 once; S28: Place the adsorption column in a new, sterile, dry 1.5 ml centrifuge tube and place it in an oven for 2 min; S29: Add 15-30 μl of sterile ultrapure water to elute DNA.

[0012] Furthermore, in S3, the CPEC method for ligating the vector and fragment can be performed using a high-fidelity enzyme via PCR amplification. The molar ratio of fragment to vector is also added at 1:3. Amplification is carried out according to the PCR program using the enzyme, with 5-10 cycles. Before transformation, the PCR product needs to be thoroughly mixed by pipetting to prevent DNA from clumping together and forming large molecules. The ligated DNA is then transferred into competent cells via electroporation or chemical transfer. After incubation, the cells are plated onto corresponding antibiotic plates and cultured until single colonies appear.

[0013] Furthermore, S4 includes the following operational steps: S41: Centrifuge the bacterial solution to collect the bacterial cells; S42: Add 250 μl of S1 resuspended bacterial cells to the bacterial cells; S43: Add 250 μl of S2 and let stand until the bacterial solution becomes clear; S44: Add 350 μl of S3, and invert the centrifuge tube to mix thoroughly; S45: After centrifugation for 10 min, transfer the supernatant DNA solution to the adsorption column and centrifuge for 1 min to remove the waste liquid; S46: Add 500 μl W1 to the adsorption column, centrifuge for 1 min, and remove the waste liquid; S47: Add 700 μl W2 centrifuge for 1 min, remove waste liquid, and repeat S47 once; S48: Centrifuge again for 2 min to completely remove the liquid from the adsorption column, and place the adsorption column in an oven; S49: Add 60 μl of sterile ultrapure water to the adsorption column to elute the plasmid.

[0014] Furthermore, S5 specifically includes the following steps: S51: Design primers to perform point mutations on the pECgRNA vector (replace the sequence after J23119 promoter in the pECgRNA vector with the N20 sequence to the region before the gRNA scaffold), using the Takara kit.

[0015]

[0016] Take 19 μL of the PCR product, add 1 μL of dpn1 enzyme digestion (to remove non-mutant plasmid template), and incubate at 37°C for 2 h.

[0017] Transform 10 μL of the above enzyme digestion product into E. coli DH5α. After confirming the spectinomycin resistance sequencing result is correct, extract the plasmid for later use.

[0018] S52: Primers were designed using overlap extension PCR to amplify homologous arm fragments; S53: Prepare competent states by converting pEcCas9 to BL21(DE3); S54: Extract genomic DNA from culture and send it for sequencing to check the knockout status; S55: Eliminate plasmids in the bacterial culture.

[0019] Furthermore, in S6, HPLC was used to detect the amount of nicotinamide generated in the reaction solution. The concentration of nicotinamide in the reaction solution was calculated based on the standard curve, and the enzyme activity was calculated using the following formula: Enzyme activity calculation formula: Enzyme activity (U / mL) = Δ[nicotinamide] * Vreaction / Δt / Venzyme Wherein, Δ[nicotinamide] is the amount of nicotinamide generated in the reaction solution, in mol / L; Δt is the reaction time, in s; Vreaction is the total volume of the reaction system, in L; and Venzyme is the volume of the enzyme solution, in L.

[0020] Furthermore, the electrotransferring of the pECgRNA-N20:Δ2=1:9 mixture in S53 into the electrocompetent cells specifically includes the following steps: Take 50 μL of pEcCAS9 BL21(DE3) bacteria from 5 mL LB of the previous day and transfer it to 50 mL LB. Add 10 mM arabinose and Kana antibiotic at a 2% transfer rate. Shake at 37°C and 200 rpm for 1 hour. When the OD600 reaches about 0.2, add 10 mM arabinose and shake at 37°C and 200 rpm for 1 hour. When the OD600 is about 0.5-0.6, collect the bacteria and let it stand on ice for 5-10 minutes. Preparation of competent cells: Centrifuge all the above bacteria with OD600≈0.4 at 4℃ for 3500rpm for 10min and remove the supernatant. Resuspend in 30mL of pre-chilled 10% glycerol and centrifuge at 3500rpm for 10min to remove the supernatant. Repeat twice. Finally, resuspend in 2mL of 10% glycerol and aliquot into pre-chilled 1.5mL centrifuge tubes, 80µl each. Electroporation: The amplified and digested ushA fragment (concentration above 200) and pEcgRNA-N2O (concentration above 60) were added to 80 μL of competent cells. The addition amount was 1500-2000 ng of ushA fragment and 500-600 ng of pEcgRNA-N2O. After mixing, the mixture was placed on ice for 20 min. The electrode cup was placed on ice for 10 min in advance. The mixture was added to the electroporation cup. Electroporation was performed according to the instrument's preset program, selecting the electroporation cup program as 1 mm. After electroporation, 800-1000 μL of antibiotic-free LB was added and mixed. The mixture was then incubated at 37℃ and 200 rpm for 1 h before plating (with streptomycin and KAN resistance).

[0021] Furthermore, S55 specifically includes the following steps: pECgRNA plasmid was removed with rhamnose (10 mM); pEcCas9 plasmid was removed using glucose (5 g / L) + sucrose (10 g / L); The successfully knocked-out bacterial culture was inoculated into 2 ml of LB medium containing rhamnose (10 mM) + kan (50 ug / ml) and incubated overnight at 37°C and 220 rpm with shaking. Then, dilute and plate the culture onto 50 μg / ml LB agar plates and incubate overnight at 37°C. Pick a single colony, shake to remove it, dilute again, and plate onto LB agar plates containing spectinomycin. If no single colony grows, it indicates that the pECgRNA plasmid has been eliminated. This bacterial culture A can be used for the next experiment. Inoculate bacterial culture A into liquid LB agar containing glucose (5 g / L), incubate overnight at 37°C and 200 rpm, then dilute and plate onto LB agar plates containing glucose (5 g / L) + sucrose (10 g / L). After single colonies without KAN resistance are picked and incubated, they are diluted and plated onto plates containing KAN resistance and plates without KAN resistance for screening. If the colony cannot grow on plates containing KAN resistance but can grow on plates without KAN resistance, it indicates that the pEcCas9 plasmid has been eliminated.

[0022] Set HPLC chromatographic conditions: Chromatographic column: Usually an octadecylsilane bonded silica (C18) column is used, with a size of 4.6 mm × 150 mm.

[0023] Mobile phase A: 0.1% - 0.2% sodium heptanesulfonate aqueous solution, pH adjusted to 1.9 - 2.5.

[0024] Mobile phase B: Methanol.

[0025] Mobile phase C: Isopropanol.

[0026] Mixing ratio: Mobile phases A, B, and C are mixed in a ratio of 91:7:2.

[0027] Flow rate: 0.8 - 1.2 mL / min, preferably 1.0 mL / min.

[0028] Detection wavelength: 250 - 280 nm, preferably 267 nm.

[0029] Column temperature: 25 - 35℃, preferably 30℃.

[0030] Injection volume: 20 μL.

[0031] Preparation of extract: Add 50 mL of acetonitrile and 10 mL of glacial acetic acid to 800 mL of water, mix well, and then dilute to 1000 mL with water.

[0032] Test solution: Accurately weigh approximately 0.1 g of the test sample and place it in a 100 mL volumetric flask. Dissolve and dilute to volume with the extraction buffer. Then, accurately measure 5 mL into a 100 mL volumetric flask, dilute and dilute to volume with the extraction buffer to prepare a solution with a final concentration of 5 μg / mL.

[0033] Reference solution: Take nicotinamide standard, dissolve and dilute it with extract to prepare a solution containing 5.0 μg nicotinamide per 1 mL.

[0034] Furthermore, in S8, the HPLC detection of nicotinamide specifically includes the following steps: Set HPLC chromatographic conditions: Set the instrument parameters according to the chromatographic conditions described above; Accurately measure 20 μL of the test solution and inject it into the liquid chromatograph, then record the chromatogram. Take another 20 μL of nicotinamide reference solution, inject it into the liquid chromatograph, and record the chromatogram; Content calculation: The content of nicotinamide in the test sample is calculated by peak area using the external standard method.

[0035] Compared with the prior art, the beneficial effects of the present invention are: This invention constructs an engineered strain for synthesizing cosmetic-grade nicotinamide, reducing the synthesis of the byproduct nicotinic acid, breaking through existing technological bottlenecks, and enabling the production of nicotinamide with low nicotinic acid content to develop in a more efficient, economical, and environmentally friendly direction. This invention uses microorganisms to express nitrile hydratase through their own metabolism, achieving the conversion of 3-cyanopyridine to nicotinamide in a one-step reaction after cell lysis. By gene editing of the chassis strain's genome and random mutation of the nitrile hydratase, the enzyme activity of the nitrile hydratase is increased, while the amount of byproduct nicotinic acid generated is reduced. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The method for constructing engineered bacteria for cosmetic nicotinamide includes the following steps: Step 1: Constructing the strain The gene sequence encoding nitrile hydratase (SEQ ID NO. 1) was amplified using primers. Simultaneously, primers were designed to amplify the pET42a vector. Using a seamless cloning method, the nitrile hydratase was ligated to the multiple cloning site of the pET42a vector, named pET42a-NH. After seamless cloning, the vector was transformed into BL21(DE3) chemocompetent cells, and after successful sequencing, the cells were preserved.

[0038] The base sequence of the nitrile hydratase in the engineered bacteria is shown in SEQ ID NO.1.

[0039] The amino acid sequence of the nitrile hydratase in the engineered bacteria is shown in SEQ ID NO.2. .

[0040] Step 2: Random mutation to enhance nitrile hydratase activity Using the pET-NH plasmid containing the gene encoding nitrile hydratase (nucleotide sequence SEQ ID NO. 1, amino acid sequence SEQ ID NO. 2) as a template, PCR amplification was performed using primers, and mutations were randomly introduced.

[0041] PCR reaction system (50 μL): template pET-RZ 0.5–20 ng, 1× Taq Buffer (without Mg2+), 0.2 mM dNTP, 0.3 mM MnCl2, 2 mM MgCl2, forward and reverse primers T7 F and T7 R 0.2 μM each, Taq DNA polymerase 5 U.

[0042] PCR conditions: (1) 95℃ pre-denaturation for 5 min; (2) 95℃ denaturation for 15 s; (3) 60℃ annealing for 5 s; (4) 72℃ extension for 30 s, repeating steps 2-4 for a total of 30 cycles; (5) final extension at 72℃ for 3 min, then storage at 4℃. The PCR product was analyzed by agarose gel electrophoresis and then recovered by gel extraction. The recovered product was then used as primers to amplify the complete plasmid.

[0043] PCR system (50 μL): 2× Phanta Max buffer, 0.2 mM dNTPs, 2.5 U Phanta Max high-fidelity polymerase, 50 ng gel-recovered product, 20 ng pET-NH plasmid.

[0044] PCR conditions: (1) Pre-denaturation at 95℃ for 5 min; (2) Denaturation at 95℃ for 15 s, annealing at 60℃ for 5 s, extension at 72℃ for 3.5 min, 35 cycles in total for step 2; (3) Final extension at 72℃ for 5 min, storage at 4℃.

[0045] The amplified PCR product was digested with restriction enzyme DpnI at 37℃ for 3 h, inactivated at 65℃ for 10 min, transformed into E. coli BL21(DE3), plated on LB plates containing kanamycin (50 μg / mL), and incubated overnight at 37℃.

[0046] Step 3: Mutant Screening 3-Cyanopyridine exhibits a strong growth inhibitory effect on microbial cells, while nitrile hydratase can catalyze the synthesis of nicotinamide from 3-cyanopyridine. To screen for nitrile hydratases with high activity and strong catalytic performance, a high-concentration 3-cyanopyridine culture medium environment was created in the laboratory. After inoculating mutants, IPTG was added to induce nitrile hydratase expression. Through growth pressure screening, strains capable of expressing nitrile hydratase at high concentrations of 3-cyanopyridine were successfully screened. After amplifying the gene encoding the nitrile hydratase, DNA sequencing was performed. The results showed mutations in L198I, K271T, and S366T (nucleotide sequence SEQ ID NO. 3, amino acid sequence SEQ ID NO. 4). The nucleotide sequence is shown in SEQ ID NO. 3. The amino acid sequence is shown in SEQ ID NO. 4. MTGSHGRDGDHHGHHHDRDHDNHLDPMTARVMALETILTEKGMVDPDALDAIIDTYETKVGPRNGASVVAKAWSDPDYADWLARDATAAIASLGFTGRQGEHMQAVFNTPERHNLVVCTLCSCYPWSVLGLPPVWYKSPPYR SRAVSDPRGVLREFGVALPDGVSVRVWDSTAELRYLVVPERPAGTEGLSEAALAAIVTRKSMIGTERDLSPHAAPETAARTAPTISAVGTASGRSRRRQTSRCSMRPGSAAPSPGRSPPVRWAIGRSTTAAPPVRIATRPTI TVRPITRSGPRALRRCSCATASSAIANCAPGGPSTRPCRRTASRRPMPSRRPLPRAVRPTAIPKAARPFSRRATGSARGTCTRAITSACPPMPARRPAPSKPFRVSMSSRMPAPRATTMSRTGSTRWSSTHSRCGAATLRPT TPSPSMPGSPILRTPETGIAASPGLPRDAAGEPVFFAPWQAKAFAMTVALNERGILAWTDWAAALGRACASLPAAGPSPEATADAYFTAWLVALEEILTARALVSANAVDAAQAVWHRAAEATPHGTPIRFEAGLPNPHDRKL Step 4: Gene Editing of Chassis Strains Gene editing of strain BL21(DE3) was performed using a two-step Cas9 recombination system to knock out the gene encoding Escherichia coli autoamide hydrolase. pncA It blocks the breakdown and metabolism of nicotinamide into nicotinic acid.

[0047] Step 5: Activation of microbial strains Dilute the bacterial culture preserved in the glycerol tube by 10%. -4 The culture was then spread onto Kan-resistant LB agar plates and incubated at 37°C for 16 hours. A single colony was picked and inoculated into a Kan-resistant LB flask with a final Kan concentration of 50 mg / L. The flask was then incubated overnight at 37°C and 200 rpm for 13 hours, and the OD was measured. 600Approximately 3-4 hours later, this was used as seed culture to inoculate 5 L or 10 L fermenters to culture the cells. The temperature was controlled at 30℃ to avoid the formation of inclusion bodies. At the same time, IPTG at a final concentration of 0.05 mM was added to the tank to induce the expression of nitrile hydratase, and Kan antibiotic at a final concentration of 50 mg / L was added. After fermentation for 32 h, the cells were collected from the tank and washed with 1×PBS buffer before cell disruption.

[0048] Step 6: Enzyme activity assay The amount of nicotinamide formed in the reaction solution was detected using HPLC. The concentration of nicotinamide in the reaction solution was calculated based on the standard curve, and then the enzyme activity was calculated.

[0049] Data Processing: Enzyme Activity Calculation Formula: Enzyme Activity (U / mL) = Δ[Nicotinamide] * V_reaction / Δt / V_enzyme Wherein, Δ[nicotinamide] is the amount of nicotinamide generated in the reaction solution (mol / L), Δt is the reaction time (s), Vreaction is the total volume of the reaction system (L), and Venzyme is the volume of the enzyme solution (L).

[0050] Tests showed that the highest enzyme activity in shake flasks was 600 u / mL, and the highest enzyme activity in a 50L fermenter was 9895 u / mL.

[0051] Step 7: Small-scale catalytic testing in a 1L system, reaction conditions are shown in Table 1 below:

[0052] First catalytic small-scale test: Using unmutated wild-type nitrile hydratase, the nicotinamide yield reached about 190 g / L, and the nicotinic acid accumulation was about 500 ppm.

[0053]

[0054] Second catalytic trial: Using a mutant but unedited engineered strain for catalysis, the nicotinamide yield reached approximately 450 g / L, and the nicotinic acid accumulation was approximately 500 ppm.

[0055]

[0056] The third catalytic trial: using a mutated engineered strain with gene editing of the chassis strain for catalysis, the nicotinamide yield reached about 450 g / L, and the nicotinic acid accumulation was below 10 ppm.

[0057] The primers for this embodiment are listed below:

[0058]

[0059] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing engineered bacteria for cosmetic use containing nicotinamide, characterized in that, Includes the following steps: S1: Use FastPfu Fly high-fidelity enzyme is used as an amplification enzyme kit, which uses DNA fragments, plasmids, or genomes containing the target fragment as templates and correspondingly designed primers for PCR amplification. S2: Separate DNA fragments by agarose gel electrophoresis, centrifuge, melt, and vortex. Add the melted gel to the adsorption column, centrifuge a second time, add SPW Buffer to the adsorption column, centrifuge a third time, place the adsorption column in a sterile dry centrifuge tube and place it in an oven, and add sterile ultrapure water to elute the DNA. S3: Perform seamless cloning or CPEC ligation of the recovered DNA fragments with the vector. Use a seamless cloning kit, add 6 μl of Mix enzyme, and make up the system with sterile ultrapure water for ligation. S4: Inoculate the verified strain into liquid culture medium and incubate at 37 °C and 250 rpm on a constant temperature shaking incubator for 8-10 hours until the bacterial culture OD reaches its limit. 600 The concentration was 0.8-1.2, and the obtained bacterial culture was used for plasmid extraction using the AxyPrep plasmid mini-prep kit; S5: Two-step method for E. coli gene knockout based on Cas9 recombination system; S6: Dilute the bacterial culture stored in the glycerol tube by 10. -4 After spreading the culture onto Kan-resistant LB plates, a single colony was picked and inoculated into a Kan-resistant LB shake flask. TB medium was then transferred at a transfer rate of 2%. After culturing for 1 h, IPTG at a final concentration of 0.05 mM was added to induce the expression of nitrile hydratase. After culturing for 16 h, samples were taken to determine the enzyme activity. S7: Dilute the bacterial culture stored in the glycerol tube by 10. -4 After spreading the bacteria onto Kan-resistant LB agar plates, a single colony was picked and inoculated into a Kan-resistant LB shake flask. The plates were incubated overnight at 37°C and 200 rpm for 13 hours. OD values ​​were then measured. 600 3-4. Using this as seed culture, inoculate 5 L or 10 L fermenters to culture the cells. Add IPTG to the tank at a final concentration of 0.05 mM to induce the expression of nitrile hydratase. Add Kan antibiotic at a final concentration of 50 mg / L. After fermentation for 32 h, collect the cells from the tank and wash them with 1×PBS buffer before cell disruption. S8: The nicotinamide was detected by HPLC after the lysed bacterial cells were reacted with a 3-cyanopyridine substrate solution.

2. The method for constructing engineered nicotinamide bacteria for cosmetic use according to claim 1, characterized in that, In S1, after PCR amplification, 3-5 mL of PCR product is aspirated for nucleic acid gel electrophoresis to check if the band size of the target gene is correct. If the electrophoresis result is consistent with expectations, the DNA fragment is recovered. If plasmid is used as a template, 1 mL of Fast Digest is added to the PCR product. Dpn The enzyme was used in a 50 mL system, and the buffer was used in a 1 mL system. The mixture was incubated overnight at 37 °C for digestion before DNA recovery and purification.

3. The method for constructing engineered nicotinamide bacteria for cosmetic use according to claim 1, characterized in that, S2 specifically includes the following steps: S21: DNA fragments were separated by agarose gel electrophoresis, using freshly prepared 1×TAE Buffer as the electrophoresis buffer; S22: Once the desired DNA fragment is completely separated on the agarose gel, cut the desired DNA fragment using a sterile scalpel; S23: Transfer the gel block containing the target fragment to a 1.5 ml centrifuge tube and weigh it to obtain the weight of the gel block; S24: Add an equal volume of XP2 Binding Buffer to the centrifuge tube; calculate the required volume of XP2 Binding Buffer to be added based on 1 g / ml. S25: Incubate at 50-60℃ for 7 min or until the gel is completely melted, shaking the mixture every 2-3 min; S26: Add the melted gel to the adsorption column, centrifuge at 10,000 rpm for 1 min at room temperature, and discard the filtrate; S27: Add 700 μl SPW Buffer to the adsorption column, centrifuge at 10000 rpm for 1 min at room temperature, discard the filtrate, and repeat S27 once; S28: Place the adsorption column in a new, sterile, dry 1.5 ml centrifuge tube and place it in an oven for 2 min; S29: Add 15-30 μl of sterile ultrapure water to elute DNA.

4. The method for constructing engineered nicotinamide bacteria for cosmetic use according to claim 1, characterized in that, In S3, the fragment and vector are added to the system at a molar ratio of 1:

3. Amplification is performed according to the PCR program of the enzyme used, and the number of cycles can be set to 5-10. The ligated DNA is transferred into competent cells by electroporation or chemical transfer. After incubation, it is plated onto the corresponding resistant plates for culture until a single colony appears.

5. The method for constructing engineered nicotinamide bacteria for cosmetic use according to claim 1, characterized in that, S4 includes the following operation steps: S41: Centrifuge the bacterial solution to collect the bacterial cells; S42: Add 250 μl of S1 resuspended bacterial cells to the bacterial cells; S43: Add 250 μl of S2 and let stand until the bacterial solution becomes clear; S44: Add 350 μl of S3, and invert the centrifuge tube to mix thoroughly; S45: After centrifugation for 10 min, transfer the supernatant DNA solution to the adsorption column and centrifuge for 1 min to remove the waste liquid; S46: Add 500 μl W1 to the adsorption column, centrifuge for 1 min, and remove the waste liquid; S47: Add 700 μl W2 centrifuge for 1 min, remove waste liquid, and repeat S47 once; S48: Centrifuge again for 2 min to completely remove the liquid from the adsorption column, and place the adsorption column in an oven; S49: Add 60 μl of sterile ultrapure water to the adsorption column to elute the plasmid.

6. The method for constructing engineered nicotinamide bacteria for cosmetic use according to claim 1, characterized in that, S5 specifically includes the following steps: S51: Design primers to perform point mutations on the pECgRNA vector; S52: Primers were designed using overlap extension PCR to amplify homologous arm fragments; S53: Prepare competent states by converting pEcCas9 to BL21(DE3); S54: Extract genomic DNA from culture and send it for sequencing to check the knockout status; S55: Eliminate plasmids in the bacterial culture.

7. The method for constructing engineered nicotinamide bacteria for cosmetic use according to claim 1, characterized in that, In S6, HPLC was used to detect the amount of nicotinamide formed in the reaction solution. The concentration of nicotinamide in the reaction solution was calculated based on the standard curve, and the enzyme activity was calculated using the following formula: Enzyme activity calculation formula: Enzyme activity (U / mL) = Δ[nicotinamide] * Vreaction / Δt / Venzyme Wherein, Δ[nicotinamide] is the amount of nicotinamide generated in the reaction solution, in mol / L; Δt is the reaction time, in s; Vreaction is the total volume of the reaction system, in L; and Venzyme is the volume of the enzyme solution, in L.

8. The method for constructing engineered bacteria for cosmetic nicotinamide according to claim 6, characterized in that, The mixture of pECgRNA-N20:Δ2=1:9 in S53 is electrotransferred into 3 electrocompetent cells, specifically including the following steps: Take 50 μL of pEcCAS9 BL21(DE3) bacteria from 5 mL LB of the previous day and transfer it to 50 mL LB. Add 10 mM arabinose and Kana antibiotic at a 2% transfer rate. Shake at 37°C and 200 rpm for 1 hour. When the OD600 reaches about 0.2, add 10 mM arabinose and shake at 37°C and 200 rpm for 1 hour. When the OD600 is about 0.5-0.6, collect the bacteria and let it stand on ice for 5-10 minutes. Preparation of competent cells: Centrifuge all the above bacteria with OD600≈0.4 at 4℃ for 3500rpm for 10min and remove the supernatant. Resuspend in 30mL of pre-chilled 10% glycerol and centrifuge at 3500rpm for 10min to remove the supernatant. Repeat twice. Finally, resuspend in 2mL of 10% glycerol and aliquot into pre-chilled 1.5mL centrifuge tubes, 80µl each. Electroporation: The amplified and digested ushA fragment and pEcgRNA-N2O were added to 80 μL of competent cells. The addition amount was 1500-2000 ng of ushA fragment and 500-600 ng of pEcgRNA-N2O. After mixing, the mixture was placed on ice for 20 min. The electrode cup was placed on ice for 10 min in advance. The mixture was then added to the electroporation cup. Electroporation was performed according to the instrument's preset program, selecting the electroporation cup program as 1 mm. After electroporation, 800-1000 μL of antibiotic-free LB was added and mixed. The mixture was then incubated at 37℃ and 200 rpm for 1 h before plating.

9. The method for constructing engineered nicotinamide bacteria for cosmetic use according to claim 6, characterized in that, S55 specifically includes the following steps: Remove pECgRNA plasmids with rhamnose; Remove pEcCas9 plasmid using glucose and sucrose; The successfully knocked-out bacterial culture was inoculated into 2 ml of rhamnose + Kan LB medium and incubated overnight at 37°C and 220 rpm with shaking. Then dilute and plate on Kan LB solid medium, incubate overnight at 37°C. Pick a single colony, shake the culture, dilute and plate on LB plates containing spectinomycin, and incubate. If no single colony grows, it means that the pECgRNA plasmid has been cleared. This bacterial culture A can be used for the next experiment. Inoculate bacterial culture A into liquid LB agar containing glucose, incubate overnight at 37°C and 200 rpm, then dilute and plate onto LB agar plates containing glucose and sucrose. Pick single colonies that do not respond to the culture and dilute them separately onto plates containing KAN resistance and plates without KAN resistance for screening. If the colony cannot grow on plates containing KAN resistance but can grow on plates without KAN resistance, it indicates that the pEcCas9 plasmid has been eliminated.

10. The method for constructing engineered nicotinamide bacteria for cosmetic use according to claim 1, characterized in that, In S8, the HPLC detection of nicotinamide specifically includes the following steps: Set HPLC chromatographic conditions; Accurately measure 20 μL of the test solution and inject it into the liquid chromatograph, then record the chromatogram. Take another 20 μL of nicotinamide reference solution, inject it into the liquid chromatograph, and record the chromatogram; Content calculation: The content of nicotinamide in the test sample is calculated by peak area using the external standard method.

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