Recombinant corynebacterium glutamicum for producing beta- arbutin and construction method and application thereof
By constructing recombinant Corynebacterium glutamicum and optimizing the fermentation medium, the problem of low production efficiency of β-arbutin was solved, achieving efficient and green biomanufacturing, and significantly increasing the number and yield of cells on the carrier.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING TECH UNIV
- Filing Date
- 2025-11-14
- Publication Date
- 2026-07-21
Smart Images
Figure CN121109272B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, specifically to a recombinant glutamate-producing Corynebacterium praecox, its construction method, and its application. Background Technology
[0002] β-Arbutin is a hydroquinone glucoside compound naturally found in various plants. Due to its significant inhibitory effect on tyrosinase activity, it has long been widely used in the cosmetics industry as a highly effective whitening ingredient. Furthermore, it exhibits various biological activities in the pharmaceutical industry, including anti-inflammatory and antibacterial properties, demonstrating promising application potential. Currently, the main industrial methods for obtaining β-arbutin include plant extraction and chemical synthesis. However, these methods generally suffer from low extraction efficiency, high resource dependence, complex processes, harsh reaction conditions, low product purity, and severe environmental pollution. With the increasing market demand for β-arbutin, existing production models are insufficient to meet industrialization needs. Therefore, utilizing microbial fermentation technology to produce β-arbutin is a promising alternative strategy.
[0003] Traditional free suspension culture is simple to operate, but it suffers from problems such as low cell density, poor stability, and the need for frequent inoculation, resulting in high costs and long cycles. Its limitations urgently need to be overcome. Summary of the Invention
[0004] Based on this, the present invention proposes a recombinant glutamate-producing Corynebacterium praecox, its construction method and application, aiming to provide a new technical path for the efficient and green biomanufacturing of this component.
[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:
[0006] In a first aspect, the present invention provides a recombinant Corynebacterium glutamicum that produces β-arbutin, which is derived from Corynebacterium glutamicum with an inactivated polyketide synthase gene. In addition, the gene for β-arbutin synthesis is expressed. It was obtained later.
[0007] Preferably, the *Corynebacterium glutamicum* is *Corynebacterium glutamicum* ATCC 13032; the polyketide synthase gene... Derived from Corynebacterium glutamicum ATCC 13032, its nucleotide sequence is shown in SEQ ID NO.1; the β-arbutin synthesis gene. It is derived from snake root wood, and its nucleotide sequence is shown in SEQ ID NO.2.
[0008] Secondly, the present invention provides a method for constructing recombinant Corynebacterium glutamicum that produces β-arbutin, comprising: firstly constructing a gene knockout plasmid pK18-mobsacB-Δpks13, electroporating the knockout plasmid into Corynebacterium glutamicum ATCC 13032, and screening to obtain polyketide synthase gene inactivated. The recombinant Corynebacterium glutamicum 13032-Δpks13 was obtained; then the expression plasmid pXMJ19-arbs was constructed, and the expression plasmid was transformed into the host 13032-Δpks13 and screened to obtain 13032-Δpks13-arbs.
[0009] Preferably, the method for constructing the gene knockout plasmid pK18-mobsacB-Δpks13 includes:
[0010] 1-1) Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR was performed to amplify the polyketide synthase gene. The upstream homologous arm fragment pks13-up and the downstream homologous arm fragment pks13-down have sequences shown in SEQ ID NO.3 and SEQ ID NO.4, respectively;
[0011] 1-2) The polyketide synthase gene obtained in step 1-1) The upstream and downstream homologous arm fragments were ligated using overlap extension PCR technology to obtain the insert fragment;
[0012] 1-3) Combine the insert obtained in step 1-2) with the restriction endonuclease-treated fragment. Linearized plasmid pK18-mobsacB was used for a one-step cloning and ligation reaction;
[0013] 1-4) Convert the ligation product obtained in steps 1-3) to In the competent state, the knockout plasmid pK18-mobsacB-Δpks13 was obtained through screening.
[0014] Preferably, the method for constructing the expression plasmid pXMJ19-arbs is as follows:
[0015] 2-1) PCR was performed using pUC57-arbs plasmid as a template to amplify the obtained... Gene;
[0016] 2-2) Obtain from step 2-1) Genes and restriction enzymes The processed pXMJ19 expression vector fragment was subjected to a one-step cloning and ligation reaction.
[0017] 2-3) Convert the product obtained in step 2-2) to In competent cells, the expression plasmid pXMJ19-arbs was obtained through screening.
[0018] Thirdly, the present invention provides the application of the above-mentioned recombinant Corynebacterium glutamicum producing β-arbutin or the recombinant Corynebacterium glutamicum obtained by the above construction method in microbial fermentation.
[0019] Preferably, the application includes inoculating a seed culture prepared from recombinant Corynebacterium glutamicum into a fermentation medium for fermentation to prepare β-arbutin.
[0020] More preferably, the fermentation is an adsorption-immobilization repeated batch fermentation, and the fermentation medium comprises a carbon source, a nitrogen source, inorganic salts, and cofactors, wherein:
[0021] The carbon source includes any one or a combination of glucose, molasses and sucrose;
[0022] The nitrogen source includes any one or a combination of several of ammonium sulfate and urea;
[0023] The inorganic salts mentioned include H2PO4. - K + Fe 2+ Mg 2+ and Mn 2+ Any combination of one or more of them;
[0024] The cofactors include any one or a combination of several of hydroquinone, calcium dextrorotatory pantothenate, nicotinamide, vitamin B1, and biotin;
[0025] The fermentation conditions in the fermentation medium were: temperature 28~34℃, pH 6~7, aeration rate 2~8 vvm, and initial stirring speed 400~700 rpm.
[0026] More preferably, the fermentation medium is formulated as follows: 40 g / L glucose, 5 g / L urea, 20 g / L ammonium sulfate, 0.4 g / L phosphoric acid, 0.53 g / L potassium chloride, 120 mg / L ferrous sulfate heptahydrate, 0.87 g / L magnesium sulfate heptahydrate, 100 mg / L manganese sulfate, 1.2 g / L hydroquinone, 6.3 mg / L calcium pantothenate, 42 mg / L nicotinamide, 6.3 mg / L vitamin B1 and 0.88 mg / L biotin.
[0027] More preferably, recombinant Corynebacterium glutamicum is activated on LBG plates until single clones are grown. These single clones are then picked and cultured in LBG medium, and then transferred to seed culture medium and cultured until the optical density (OD) reaches a certain level. 562Seed culture is obtained when the value reaches between 2 and 10. The seed culture is then inoculated into fermentation medium at an inoculation rate of 2% to 20% v / v (volume percentage) to obtain fermentation broth containing fermentation products. The seed culture medium is formulated as follows: 5 to 15 g / L peptone, 1 to 10 g / L yeast extract, 1 to 10 g / L ammonium sulfate, 0.1 to 2 g / L magnesium sulfate heptahydrate, 1 to 10 g / L potassium dihydrogen phosphate, 5 to 15 g / L dipotassium hydrogen phosphate, and 1 to 10 g / L urea, with water as the solvent. The culture conditions in the seed culture medium are: 28 to 34°C and 200 to 250 rpm for 4 to 8 hours.
[0028] Beneficial effects:
[0029] (1) The present invention inactivates the polyketide synthase gene in Corynebacterium glutamicum. A strain 13032-Δpks13 with enhanced biofilm formation ability was successfully obtained, which can effectively enhance the adsorption and growth ability of Corynebacterium glutamicum cells on various media surfaces.
[0030] (2) In this invention, the β-arbutin synthesis gene was overexpressed in strain 13032-Δpks13. As a fermentation strain, it enables the efficient production of β-arbutin through adsorption and immobilization. A solid carrier is used to promote cell adsorption and growth, allowing it to be continuously used in multiple batches of fermentation in an immobilized state, effectively achieving cell reuse.
[0031] (3) This invention improves the yield and efficiency of β-arbutin synthesis by Corynebacterium glutamicum by optimizing the fermentation medium (hydroquinone, carbon source, and nitrogen source). The average yield of β-arbutin produced continuously in a 6L bioreactor reached 30.62 g / L, which is 4.19 g / L higher than that of the control strain 13032-arbs, representing an increase of 15.9%. Attached Figure Description
[0032] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0033] Figure 1 The image shows the pK18-mobsacB-Δpks13 plasmid, which is 7617 kb in size.
[0034] Figure 2 The pXMJ19-arbs expression plasmid map is 7969kb in size.
[0035] Figure 3The diagram shows the autoagglutination rates of strains 13032 and 13032-Δpks13.
[0036] Figure 4 The image shows the agglutination phenomenon of strains 13032 and 13032-Δpks13 at 12 h.
[0037] Figure 5 The diagram shows the biofilm analysis of strains 13032 and 13032-Δpks13 after fermentation.
[0038] Figure 6 The diagram shows the biofilm formation phenomenon after fermentation of strains 13032 and 13032-Δpks13.
[0039] Figure 7 The HPLC chromatogram of β-arbutin in the fermentation broth of engineered strain 13032-Δpks13-arbs is shown.
[0040] Figure 8 A schematic diagram illustrating the optimization of hydroquinone concentration in the fermentation medium for β-arbutin production.
[0041] Figure 9 A schematic diagram illustrating the optimization of carbon source concentration in the fermentation medium for β-arbutin production.
[0042] Figure 10 A schematic diagram illustrating the optimization of nitrogen source concentration in the fermentation medium for β-arbutin production.
[0043] Figure 11 The graph shows the change in β-arbutin production over time by strains 13032-arbs and 13032-Δpks13-arbs after culture medium optimization.
[0044] Figure 12 Biofilm analysis diagram of strain 13032-arbs and engineered strain 13032-Δpks13-arbs.
[0045] Figure 13 The graph shows the change over time in the fermentation production of β-arbutin by strain 13032-arbs in a 6L bioreactor.
[0046] Figure 14 The graph shows the change over time in the fermentation production of β-arbutin by engineered strain 13032-Δpks13-arbs in a 6L bioreactor. Detailed Implementation
[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.
[0048] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.
[0049] In the following examples, plasmid pXMJ19 was purchased from Wuhan Miaoling Biotechnology Co., Ltd.
[0050] In the following examples, the host *Corynebacterium glutamicum* and the original strain of *Corynebacterium glutamicum* were both *Corynebacterium glutamicum* ATCC 13032.
[0051] In the following examples, the seed culture medium formulation is as follows: 10 g / L peptone, 5 g / L yeast extract, 5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 4 g / L potassium dihydrogen phosphate, 10.5 g / L dipotassium hydrogen phosphate, 3.5 g / L urea, and water as the solvent.
[0052] The LBG medium formula is: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 10 g / L glucose.
[0053] The fermentation basal medium formula is as follows: 20 g / L glucose, 5 g / L urea, 15 g / L ammonium sulfate, 0.4 g / L phosphoric acid, 0.53 g / L potassium chloride, 120 mg / L ferrous sulfate heptahydrate, 0.87 g / L magnesium sulfate heptahydrate, 100 mg / L manganese sulfate, 1 g / L hydroquinone, 6.3 mg / L calcium pantothenate, 42 mg / L nicotinamide, 6.3 mg / L vitamin B1 and 0.88 mg / L biotin.
[0054] Fermentation conditions: The seed culture was inoculated into a bioreactor containing fermentation medium. The culture temperature was controlled at 30℃, and the pH was maintained at 6.9 by adding ammonia. The initial aeration rate was 6 vvm, and the initial stirring speed was 550 rpm. When the glucose concentration in the fermentation broth was below 1 g / L, the stirring speed and aeration rate were adjusted to maintain dissolved oxygen at 30%, while sugar medium was added to stabilize the glucose concentration at 1-2 g / L. When the ammonium sulfate concentration was below 1 g / L, nitrogen medium was added to maintain its concentration at 1-1.5 g / L. The above feeding operations continued until the end of fermentation. The fermentation medium used has the following formulation: 40 g / L glucose, 5 g / L urea, 20 g / L ammonium sulfate, 0.4 g / L phosphoric acid, 0.53 g / L potassium chloride, 120 mg / L ferrous sulfate heptahydrate, 0.87 g / L magnesium sulfate heptahydrate, 100 mg / L manganese sulfate, 1.2 g / L hydroquinone, 6.3 mg / L calcium pantothenate, 42 mg / L nicotinamide, 6.3 mg / L vitamin B1, and 0.88 mg / L biotin. The sugar-supplemented medium has the following formulation: 600 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 1 g / L choline chloride, 0.1 g / L vitamin B1, 1 g / L potassium dihydrogen phosphate, and 1 mg / L biotin; the nitrogen-supplemented medium has the following formulation: 450 g / L ammonium sulfate.
[0055] This invention employs adsorption immobilization and repeated batch fermentation. The carrier medium can be any one or a combination of several of the following: cotton fiber, non-woven fabric, polyester fiber, polyvinyl alcohol fiber, zeolite, bacterial cellulose membrane, silk, bagasse, and corn stalk. The amount of carrier medium used is 5~40 g / L. In the following examples, cotton fiber is preferred, and the amount of carrier medium used is preferably 30 g / L.
[0056] Example 1: Construction of pK18-mobsacB-Δpks13 plasmid and pXMJ19-arbs plasmid
[0057] (1) Construction of pK18-mobsacB-Δpks13 plasmid:
[0058] (i) Genomic DNA was extracted from ATCC 13032 using a genomic DNA extraction kit according to the instructions of the ClonExpress II One Step Cloning Kit. Using the extracted genomic DNA as a template, appropriate PCR amplification primers were designed, and their sequences are shown in Table 1, where F represents the forward primer and R represents the reverse primer. PCR amplification was performed using primer pairs pks13-up-F / pks13-up-R and pks13-down-F / pks13-down-R to obtain upstream and downstream homologous arm fragments. The amplification products were verified by agarose gel electrophoresis and purified for later use. Subsequently, overlap extension PCR was used to ligate the upstream and downstream homologous arms through homologous recombination to construct a complete insert fragment. This was then verified by electrophoresis and purified for later use.
[0059] (ii) One-step cloning reaction: The resulting insert fragment and restriction endonuclease-mediated cloning reaction are combined. The linearized plasmid pK18-mobsacB was ligated in vitro using a one-step cloning kit, and all the ligation products were transformed into [the desired cloning agent]. Immediately after incubation, competent cells were placed on ice for 30 min, followed by heat shock at 42°C for 90 sec, and then immediately placed on ice for 3 min. Next, 900 μL of antibiotic-free LB medium was added to a clean bench, and the cells were cultured on a rotating shaker for 1 h. Afterward, the cells were plated on LB agar plates containing kanamycin (50 μg / mL) and incubated upside down at 37°C for 12–16 h. Once single colonies emerged, several colonies were selected and PCR was performed using primers Δpks13-F / Δpks13-R for verification. Agarose gel electrophoresis was used to check for the release of bands of the expected size. To ensure the complete correctness of the constructed plasmid sequence, the recombinant plasmid was sent to Beijing Qingke Biotechnology Co., Ltd. for full-length sequencing to confirm the correctness and sequence integrity of the inserted fragment. The final product was used to knock out the polyketide synthase gene. The recombinant plasmid pK18-mobscB-Δpks13, plasmid map is shown below. Figure 1 .
[0060] (2) Construction of pXMJ19-arbs plasmid:
[0061] Using pUC57-arbs plasmid synthesized by Qingke Biotechnology Co., Ltd. as a template, and primer pair arbs-F / arbs-R (Table 1) as upstream and downstream primers, the plasmid was amplified by PCR using 2× high-fidelity DNA polymerase premix (2× Phanta Max Master Mix). Gene. The PCR program was: 95℃ for 30 s, 55℃ for 15 s, 72℃ for 30 s / kb, 30 cycles. The PCR product was purified and verified by 1.5% agarose gel electrophoresis. The obtained PCR product was then reacted with restriction enzyme-modified... The processed pXMJ19 plasmid fragment was subjected to a one-step cloning and ligation reaction. After screening, the gene for expressing β-arbutin synthesis was obtained. The recombinant plasmid pXMJ19-arbs, plasmid map can be found in [link to plasmid image]. Figure 2 .
[0062] Table 1 Primer Sequences
[0063]
[0064] Example 2: Construction of recombinant Corynebacterium glutamicum
[0065] (1) Construction of Corynebacterium glutamicum 13032-arbs:
[0066] The expression plasmid pXMJ19-arbs constructed in Example 1 was introduced into Corynebacterium glutamicum ATCC 13032 using electroporation (1800 V, 25 μF, 200 Ω, 2 mm). Transformants were selected and cultured on LBG plates containing 25 μg / mL kanamycin sulfate for 2-3 days. The potential target strains were verified by colony PCR, and the PCR products were sent to a biotechnology company for sequencing analysis to further confirm the correct genome recombination, thus obtaining the recombinant Corynebacterium glutamicum strain 13032-arbs, which served as a control.
[0067] (2) Construction of Corynebacterium glutamicum 13032-Δpks13-arbs:
[0068] The pK18-mobsacBΔpks13 plasmid was introduced into ATCC 13032 via electroporation, and the target recombinant strain was obtained by sucrose negative screening. The specific experimental steps are as follows:
[0069] (i) Preparation and electroporation of competent cells: First, a single colony was picked from an LBG plate and inoculated into 5 mL of autoclaved LBG liquid medium. The plate was then incubated aerobically at 30°C and 220 rpm for 12 h to allow the cells to reach the logarithmic growth phase. Subsequently, the culture was transferred to a 500 mL shake flask containing 50 mL of LBG liquid medium (containing 3 mL of electroporation stock solution), and the initial OD was adjusted. 562 The value was 0.2, and the culture was continued with shaking for 3-6 hours until the OD value was 0.2. 562The plasmid concentration reached 0.8–1.0, and competent cells were then prepared according to the methods reported in the literature. Approximately 1 μg of plasmid was mixed with the competent cells and transferred to an electroporation cuvette for transformation at 1.8 kV, 25 μF, and 200 Ω. Immediately after electroporation, 1 mL of preheated LBG liquid medium (46 °C) was added, and the cells were incubated in a 46 °C water bath for 6 min to promote cell resuscitation. The resuscitated cells were then statically cultured at 30 °C for 2 h, followed by centrifugation to collect the cells, which were then plated onto LBG plates containing kanamycin and cultured until monoclonal colonies formed.
[0070] (ii) Screening of target strains: First, colony PCR was performed on the single clones grown on the plates to confirm that the pK18-mobsacB-Δpks13 plasmid was correctly integrated into the genome of *Corynebacterium glutamicum* via double crossover homologous recombination. Then, single clones that tested positive for colony PCR were picked and inoculated into antibiotic-free LBG liquid medium and cultured at 30°C and 220 rpm for 12 h. The culture was then spread onto LB plates containing 10% sucrose and cultured until single colonies formed. To further screen target strains, a spotting verification method was used: the same single clone was simultaneously spotted onto LB plates and LB+Kan plates. Strains that grew well on LB plates but not on LB plates containing Kan were selected as potential target strains. Finally, colony PCR was performed on the selected potential target strains, and the PCR products were sent to a biotechnology company for sequencing analysis to further confirm the correct genome recombination, ultimately yielding *Corynebacterium glutamicum* 13032-Δpks13.
[0071] (iii) The expression plasmid pXMJ19-arbs constructed in Example 1 was introduced into the Corynebacterium glutamicum 13032-Δpks13 constructed above by electroporation (1800 V, 25 μF, 200 Ω, 2 mm). After screening and culturing on LBG plates containing 25 μg / mL kanamycin sulfate for 2-3 days, transformants were picked out. The potential target strains were verified by colony PCR, and the PCR products were sent to a biotechnology company for sequencing analysis to further confirm the correct recombination of the genome, thus obtaining the recombinant Corynebacterium glutamicum strain 13032-Δpks13-arbs.
[0072] Example 3: Performance characterization of Corynebacterium glutamicum 13032-Δpks13
[0073] (1) Characterization of film-forming ability: Corynebacterium glutamicum 13032-Δpks13 obtained in Example 2 and the original strain ATCC 13032 were cultured in LBG medium at 30°C and 220 rpm for 22-24 h until single clones grew. Single clones were picked and inoculated into 50 mL centrifuge tubes containing 5 mL of LBG medium and cultured at 30°C and 220 rpm for 12 h. Then, they were inoculated into 500 mL shake flasks with baffles containing 50 mL of seed medium at a 1% v / v inoculation rate and cultured at 30°C and 220 rpm until OD. 562 The seed culture was obtained at approximately 8 μL. 300 mL of the seed culture was inoculated into a 6L bioreactor containing 1.8 L of fermentation medium to initiate fermentation. After each batch of fermentation, all fermentation broth was drained, and 2 L of fresh fermentation medium was added via a peristaltic pump. Subsequent batch fermentations with adsorption immobilization were repeated using this procedure. After the adsorption immobilization batch fermentations were completed, the cotton fiber carrier was removed from the bioreactor and gently washed in 1000 mL of PBS buffer to remove free cells from the carrier surface. The carrier was then transferred to 3000 mL of PBS buffer and repeatedly rubbed to elute as many biofilm cells attached to the fiber surface as possible into the PBS buffer. Next, the carrier was again placed in 1000 mL of PBS buffer and sonicated for 30 min (30℃, 40 kHz) to further elute the cells. Finally, all the eluent solutions were mixed, and the OD was measured. 562 The final OD 562 Converted to the number of cells contained in a 1000 mL volume.
[0074] (2) Characterization of flocculation ability: Wild-type strain ATCC 13032 and recombinant strain 13032-Δpks13 were cultured overnight in LBG medium. The bacterial cells were then collected by centrifugation and washed twice with PBS. Subsequently, the bacterial cells were resuspended in 10 mL of PBS and added to glass test tubes. The test tubes were placed vertically on a stable test tube rack and allowed to stand. Three replicates were prepared for each strain. From the start of the standing period, 1 mL of supernatant was taken from each test tube every 12 h, and the OD was measured using a spectrophotometer. 562 The self-agglomeration rate is calculated based on the agglomeration rate formula commonly used in the literature.
[0075] from Figure 3 , Figure 4 It can be seen that the culture medium of wild-type strain ATCC 13032 is homogeneous and transparent, while the culture medium of recombinant strain 13032-Δpks13 is significantly heterogeneous, with a large amount of flocculent precipitate. At 12 h, the recombinant strain 13032-Δpks13 has the strongest autoagglutination rate, which is 1.5 times that of wild-type strain ATCC 13032.
[0076] from Figure 5 , Figure 6 It can be seen that the recombinant strain 13032-Δpks13 forms a dense, visible biofilm structure on the vector, and the number of cells attached to the vector is 2.4 times that of the wild strain ATCC 13032.
[0077] Example 4: Detection of fermentation products by high performance liquid chromatography
[0078] (1) The constructed engineered strain 13032-Δpks13-arbs was streaked onto a plate and cultured in a 30℃ incubator for 24 h until single colonies grew. Single colonies were picked, activated, and inoculated into 5 mL LBG medium and cultured in a shaker at 30℃ and 220 rpm for 12 h. After activation, the inoculum was inoculated at a rate of 1% v / v into a 500 mL shake flask containing 50 mL of seed medium and cultured at 30℃ and 220 rpm until OD. 562 The ratio is 2~10, and seed liquid is obtained.
[0079] (2) The seed culture obtained in step (1) was inoculated at 10% v / v into a 500 mL shake flask with baffles containing 50 mL of fermentation medium. The flask was cultured at 30℃ and 220 rpm for 72 h to obtain the fermentation broth. Subsequently, the fermentation broth was centrifuged at 12,000 rpm for 5 min to collect the supernatant, which was then filtered through a 0.22 μm filter membrane to remove impurities for subsequent analysis. During the fermentation of the recombinant strain 13032-Δpks13-arbs, 6.5 μg / mL of chloramphenicol was added to the culture medium.
[0080] (3) HPLC analysis was performed using an Agilent 1260 Infinity II two-dimensional high-performance liquid chromatograph equipped with a UV detector (VWD). Quantitative analysis of β-arbutin was performed using an Agilent ZORBAX SB-Aq column (250 × 4.6 mm; Agilent, USA), with a mobile phase of 15% methanol and 85% ultrapure water, and a flow rate of 0.8 mL / min. Samples were sterilized by filtration through a 0.22 μm filter and stored at 4°C or injected for analysis. The sample injection volume was 20 μL, and detection was performed using a UV detector at a wavelength of 280 nm and a detection temperature of 30°C. The analysis time for each sample was 20 min. LC-MS / MS analysis was performed at Nanjing Jiangbei New Area Biomedical Public Service Platform Co., Ltd. (Nanjing, China).
[0081] from Figure 7The results showed that the elution position of the engineered strain 13032-Δpks13-arbs was consistent with that of the β-arbutin standard, while the control strain containing the empty vector showed no elution at the corresponding time. The results indicated that the β-arbutin yield of the engineered strain was 1.84 g / L. Subsequently, the fermentation conditions of the engineered strain 13032-Δpks13-arbs were optimized.
[0082] Example 5: Optimization of Fermentation Medium
[0083] Culture medium is an essential nutrient substrate for microbial growth, reproduction, and the synthesis of various metabolites. The components of the culture medium can significantly regulate the growth rate of microorganisms, the selectivity of metabolic pathways, and the yield of target metabolites. Therefore, this study systematically optimized the composition and culture conditions of the fermentation medium. Optimization was performed using a single-factor controlled variable method, with culture at 30℃ and 220 rpm for 72 h, and the yield of β-arbutin was determined.
[0084] (1) Adjust the final concentration of hydroquinone, the precursor added to the fermentation basal medium, to 0.5, 0.8, 1.0, 1.2, 1.5 and 2.0 g / L, while keeping other culture conditions unchanged. Each combination was replicated three times.
[0085] The results are as follows Figure 8 As shown, the yield of β-arbutin was highest when the concentration of the precursor hydroquinone was 1.2 g / L.
[0086] (2) Adjust the final concentration of the carbon source glucose / sucrose added to the fermentation basal medium (20 g / L, 40 g / L, 50 g / L and 60 g / L). Keep other culture conditions unchanged. Each combination was replicated three times.
[0087] The results are as follows Figure 9 As shown, the yield of β-arbutin was highest when the glucose concentration was 40 g / L.
[0088] (3) Further adjust the final concentration of the nitrogen source combination of urea and ammonium sulfate in the above-optimized fermentation medium (combinations of 2 g / L, 5 g / L, and 8 g / L urea with 15 g / L, 20 g / L, and 25 g / L ammonium sulfate concentrations, respectively). Keep all other culture conditions unchanged. Each combination is replicated three times.
[0089] The results are as follows Figure 10 As shown, the yield of β-arbutin was highest when the urea concentration was 5 g / L and the ammonium sulfate concentration was 20 g / L.
[0090] In summary, the optimal culture medium formula for the subsequent fermentation system was finally determined, which included 40 g / L glucose as a carbon source, 5 g / L urea and 20 g / L ammonium sulfate as nitrogen sources, and hydroquinone concentration set at 1.2 g / L.
[0091] The results are as follows Figure 11 As shown, in the optimized fermentation medium, the yields of 13032-arbs and 13032-Δpks13-arbs reached 2.18 g / L and 2.30 g / L, respectively, and the yield difference between the two in the free cell fermentation process was not significant.
[0092] Example 6: Continuous fermentation of engineered strains to produce β-arbutin in a 6L bioreactor
[0093] (1) The constructed engineered strain 13032-Δpks13-arbs and the control strain 13032-Δpks13 were spread on 90 mm diameter plates and cultured at 30℃ and 220 rpm for 22-24 h until single colonies grew. Single colonies were picked and inoculated into 50 mL centrifuge tubes containing 5 mL LBG medium and cultured at 30℃ and 220 rpm for 12 h. Then, they were inoculated at a rate of 1% v / v into 500 mL shake flasks with baffles containing 50 mL seed medium and cultured at 30℃ and 220 rpm until OD. 562 The seed culture was obtained at a concentration of approximately 8. 300 mL of the seed culture was inoculated into a 6L bioreactor containing 1.8 L of fermentation medium to initiate fermentation. After each batch of fermentation, all fermentation broth was drained, and 2 L of fresh fermentation medium was added using a peristaltic pump. Subsequent adsorption-immobilization and repeated batch fermentations were performed following this procedure.
[0094] (2) Glucose concentration: After fermentation, centrifuge the fermentation broth and take the supernatant for 100-fold serial dilution with pure water. Accurately draw 25 μL of the diluted glucose-lysine mixed standard solution using a syringe, insert it into the injection port of the SBA-40E biosensor analyzer, and quickly push it completely into the reaction chamber before withdrawing the syringe. Repeat the injection operation. Once the instrument displays that calibration is complete, the fermentation sample can be measured. The value displayed by the instrument corresponds to the glucose concentration in the sample and is recorded.
[0095] (3) After repeated batch fermentation of the immobilized adsorption, the cotton fiber carrier in the bioreactor was removed and gently washed in 1000 mL of PBS buffer to remove free cells from the carrier surface. The carrier was then transferred to 3000 mL of PBS buffer and repeatedly rubbed to elute as many biofilm cells attached to the fiber surface as possible into the PBS buffer. Next, the carrier was placed back into 1000 mL of PBS buffer and sonicated for 30 min (30℃, 40 kHz) to further elute the cells from the carrier. Finally, all the eluent solutions were mixed, and the OD was measured. 562 The final OD 562 Converted to the number of cells contained in a 1000 mL volume.
[0096] Carrier analysis results ( Figure 12 The results showed that the recombinant strain 13032-Δpks13-arbs formed a dense biofilm on the surface of the cotton fiber carrier, and the number of cells adhering to it was 2.2 times higher than that of the control strain 13032-arbs.
[0097] The results are as follows Figure 13 , Figure 14 As shown, the average yield of β-arbutin by recombinant strain 13032-Δpks13-arbs in a 6L bioreactor reached 30.62 g / L, which was 4.19 g / L higher than that of control strain 13032-arbs, representing an increase of 15.9%.
[0098] This invention provides a method for synthesizing β-arbutin using Corynebacterium glutamicum. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.
Claims
1. The application of a recombinant *Corynebacterium glutamicum* producing β-arbutin in microbial fermentation, the application comprising inoculating a seed culture prepared from the recombinant *Corynebacterium glutamicum* into a fermentation medium for fermentation to produce β-arbutin, wherein the fermentation is an adsorption-immobilization repeated batch fermentation, and the recombinant *Corynebacterium glutamicum* producing β-arbutin is obtained by inactivating the polyketide synthase gene in *Corynebacterium glutamicum*. In addition, the gene for β-arbutin synthesis is expressed. The subsequently obtained *Corynebacterium glutamicum* was *Corynebacterium glutamicum* ATCC 13032, and the polyketide synthase gene... Derived from Corynebacterium glutamicum ATCC13032, its nucleotide sequence is shown in SEQ ID NO.1; the β-arbutin synthesis gene. It is derived from snake root wood, and its nucleotide sequence is shown in SEQ ID NO.
2.
2. The application according to claim 1, characterized in that, The fermentation medium comprises a carbon source, a nitrogen source, inorganic salts, hydroquinone, and cofactors, wherein: The carbon source includes any one or a combination of glucose, molasses and sucrose; The nitrogen source includes any one or a combination of several of ammonium sulfate and urea; The inorganic salts mentioned include H2PO4. - K + Fe 2+ Mg 2+ and Mn 2+ Any combination of one or more of them; The cofactors include any one or a combination of several of the following: calcium pantothenate, nicotinamide, vitamin B1, and biotin. The fermentation conditions in the fermentation medium were: temperature 28~34℃, pH 6~7, aeration rate 2~8 vvm, and initial stirring speed 400~700 rpm.
3. The application according to claim 2, characterized in that, The fermentation medium is formulated as follows: 40 g / L glucose, 5 g / L urea, 20 g / L ammonium sulfate, 0.4 g / L phosphoric acid, 0.53 g / L potassium chloride, 120 mg / L ferrous sulfate heptahydrate, 0.87 g / L magnesium sulfate heptahydrate, 100 mg / L manganese sulfate, 1.2 g / L hydroquinone, 6.3 mg / L calcium pantothenate, 42 mg / L nicotinamide, 6.3 mg / L vitamin B1 and 0.88 mg / L biotin.
4. The application according to claim 1, characterized in that, Recombinant Corynebacterium glutamicum was activated on LBG plates until single colonies grew. These single colonies were then picked and cultured in LBG medium, and then transferred to seed culture medium and cultured until the optical density (OD) reached a certain level. 562 Seed culture is obtained when the value reaches between 2 and 10. The seed culture is then inoculated into fermentation medium at an inoculation rate of 2% to 20% v / v to obtain fermentation broth containing fermentation products. The seed culture medium is formulated as follows: 5 to 15 g / L peptone, 1 to 10 g / L yeast extract, 1 to 10 g / L ammonium sulfate, 0.1 to 2 g / L magnesium sulfate heptahydrate, 1 to 10 g / L potassium dihydrogen phosphate, 5 to 15 g / L dipotassium hydrogen phosphate, and 1 to 10 g / L urea, with water as the solvent. The culture conditions in the seed culture medium are: 28 to 34°C and 200 to 250 rpm for 4 to 8 hours.