Gluconobacter oxydans Strain Tolerant to High Concentrations of Mandelic Acid and Its Application

Abstract

The present invention discloses a Gluconobacter oxydans strain tolerant to high concentrations of mandelic acid and its application, belonging to the fields of microbial engineering technology and genetic engineering. In the present invention, Gluconobacter oxydoacetici 621H is used as the starting strain, and Gluconobacter oxydoacetici is subjected to adaptive evolution by gradually adding mandelic acid to the culture medium. The evolved strain has significantly improved tolerance to mandelic acid. Applying the strain Gluconobacter oxydans STA to catalyze the synthesis of R-mandelic acid from styrene oxide, both the yield and the conversion rate are significantly improved. Using the genetically engineered Gluconobacter oxydans strain constructed by the present invention to catalyze the synthesis of R-mandelic acid from styrene oxide, the final yield reaches 14.06 g / L, while that of the wild strain WT is only 10.26 g / L; the catalytic efficiency is also greatly improved, increasing from 0.366 g / L / h to 0.703 g / L / h.

Description

Technical Field

[0001] The present invention relates to a Gluconobacter oxydans strain tolerant to high concentrations of mandelic acid and its application, belonging to the technical fields of microbial engineering and genetic engineering. Background Art

[0002] Gluconobacter oxydans is an obligate aerobic Gram-negative bacterium with unique and powerful incomplete oxidation ability, and is widely used in the production of vitamin C, dihydroxyacetone, miglitol, etc. Its powerful redox ability mainly depends on a variety of membrane-bound dehydrogenases, which incompletely oxidize a variety of alcohols to generate corresponding sugars, aldehydes or acids, including catalyzing the synthesis of R-mandelic acid from 2-phenyl-1,2-ethanediol. R-mandelic acid is a highly valuable intermediate that can be converted into chiral drugs and pharmaceutical products, and can also be used to develop new chiral materials. The synthesis of R-mandelic acid by Gluconobacter oxydans has better stereospecificity and higher catalytic efficiency compared to general enzyme catalysis. However, Gluconobacter oxydans has poor tolerance to mandelic acid. R-mandelic acid not only strongly inhibits the growth of the strain, but also inhibits the oxidation reaction. Therefore, in order to further improve the catalytic efficiency and yield of R-mandelic acid synthesis by Gluconobacter oxydans, it is crucial to improve the tolerance of Gluconobacter oxydans to R-mandelic acid.

[0003] Adaptive evolution, as a remarkable method for strain improvement in microbial engineering, has been increasingly widely applied in obtaining special evolved strains with higher viability under extreme conditions, inhibitory metabolites, and toxic substrates or products. Adaptive evolution can enable the strain to improve one or some growth characteristics in a short time. Under specific selection pressure, the microorganism is continuously subcultured, and beneficial strains adapted to specific conditions are obtained under its own spontaneous mutation. Therefore, the method of adaptive evolution can also be used to improve the tolerance of Gluconobacter oxydans to R-mandelic acid. Summary of the Invention

[0004] The present invention obtains a Gluconobacter oxydans strain tolerant to high concentrations of mandelic acid and applies it to the efficient synthesis of R-mandelic acid, thereby improving the synthesis efficiency and final yield of R-mandelic acid; the starting strain of Gluconobacter oxydans used is Gluconobacter oxydans 621H.

[0005] The present invention provides a Gluconobacter oxydans STA, which was deposited at the China Center for Type Culture Collection on October 17, 2022, with the deposit number CCTCC NO: M 20221576.

[0006] The present invention also provides an adaptive evolution method for Gluconacetobacter oxydans tolerant to high concentrations of mandelic acid and its application, including the adaptive evolution method for Gluconacetobacter oxydans tolerant to R-mandelic acid and the application of the evolved strain in catalyzing styrene oxide to produce R-mandelic acid. The adaptive evolution method for tolerating high concentrations of mandelic acid includes domestication of Gluconacetobacter oxydans by gradient addition of R-mandelic acid in a culture medium. The R-mandelic acid production method includes heterologous expression of epoxide hydrolase in the evolved strain to obtain a recombinant strain, and the whole-cell catalysis is used to convert styrene oxide into R-mandelic acid.

[0007] The present invention also provides an adaptive evolution method for tolerating high concentrations of mandelic acid, including the following steps:

[0008] (1) Streak and activate the preserved strain, pick a single colony and inoculate it into a fresh sorbitol medium, and culture it to the logarithmic phase as the seed solution.

[0009] (2) Transfer the logarithmic-phase seed solution to a medium containing a low concentration of mandelic acid and continue culturing it to the logarithmic phase, and then transfer it to a medium with an increased concentration of mandelic acid and continue culturing. While continuously passaging and culturing in this way, gradually increase the concentration of mandelic acid in the medium until the concentration of mandelic acid reaches the domestication limit, that is, the strain no longer grows when the concentration of mandelic acid is increased.

[0010] (3) Dilute and spread the final domesticated bacterial solution on a solid medium containing the final concentration of mandelic acid, and isolate a single colony as the final evolved strain, which is named Gluconacetobacter oxydans STA.

[0011] (4) Conduct a spotting experiment with the evolved strain and the wild-type strain together to verify the tolerance to mandelic acid.

[0012] In an embodiment of the present invention, the starting strain for the adaptive evolution is Gluconacetobacter oxydans 621H.

[0013] In an embodiment of the present invention, in the step (2), the initial concentration of mandelic acid is 0.25 g / L, the concentration of mandelic acid is increased by 0.25 g / L each time, and the highest tolerance concentration of Gluconacetobacter oxydans is 6 g / L finally.

[0014] In an embodiment of the present invention, the culture conditions for the adaptive evolution strain are a culture temperature of 30 °C, a rotation speed of 200 rpm, and a culture time of 24 - 48 h.

[0015] The present invention also provides a microbial inoculum, which contains the above-mentioned Gluconacetobacter oxydans STA or its fermentation broth, or its extract.

[0016] In one embodiment of the present invention, in the microbial inoculum, the addition amount of Gluconacetobacter oxydans STA is at least: cell concentration OD 600 reaches 25.

[0017] The present invention also provides a product for synthesizing R-mandelic acid, and the product contains the above-mentioned Gluconacetobacter oxydans STA.

[0018] In one embodiment of the present invention, the product is a chemical.

[0019] The present invention also provides an application of the above-mentioned Gluconacetobacter oxydans STA in the preparation of a product capable of synthesizing R-mandelic acid.

[0020] The present invention provides a genetically engineered Gluconacetobacter oxydans bacterium for synthesizing R-mandelic acid. The genetically engineered Gluconacetobacter oxydans bacterium uses the above-mentioned Gluconacetobacter oxydans STA as a host cell and overexpresses an epoxide hydrolase derived from Sphingomonas.

[0021] In one embodiment of the present invention, P with a nucleotide sequence as shown in SEQ ID NO.1 is used to 12780 express epoxide hydrolase.

[0022] In one embodiment of the present invention, the nucleotide sequence encoding the epoxide hydrolase is as shown in SEQ ID NO.2.

[0023] In one embodiment of the present invention, the genetically engineered Gluconacetobacter oxydans bacterium uses the pBBR1MCS-2 plasmid as an expression vector.

[0024] The present invention also provides a method for preparing epoxide hydrolase. The method is to ferment and prepare using the above-mentioned genetically engineered Gluconacetobacter oxydans bacterium.

[0025] The present invention also provides a method for synthesizing R-mandelic acid. The method is to add the above-mentioned genetically engineered Gluconacetobacter oxydans bacterium to a reaction system containing styrene oxide for reaction to prepare R-mandelic acid.

[0026] In one embodiment of the present invention, in the reaction system, the addition amount of the genetically engineered Gluconacetobacter oxydans bacterium is: cell concentration OD 600 is 20 - 30.

[0027] In one embodiment of the present invention, in the reaction system, the addition amount of the genetically engineered Gluconacetobacter oxydans bacterium is cell concentration OD 600 = 25.

[0028] In one embodiment of the present invention, the addition amount of styrene oxide is 16 - 20 g / L.

[0029] In one embodiment of the present invention, the addition method of styrene oxide is as follows: the initial concentration of styrene oxide is 4 g / L, and then 3 - 4 g / L is added every 4 hours until it reaches 16 - 20 g / L.

[0030] In one embodiment of the present invention, the final addition amount of styrene oxide is 16 g / L.

[0031] In one embodiment of the present invention, the reaction conditions are: 25 - 30 °C, 180 - 220 rpm.

[0032] In one embodiment of the present invention, the reaction conditions are: 30 °C, 200 rpm, and the conversion is carried out for 28 - 40 hours.

[0033] Beneficial effects

[0034] (1) Through the adaptive evolution of continuous subculture of Gluconobacter oxydans with increased mandelic acid concentration in fresh medium, the present inventors successfully screened an evolved strain that can tolerate high concentrations of mandelic acid and applied it to catalyze the synthesis of R - mandelic acid from styrene oxide, resulting in significant improvements in both the conversion rate and the final yield.

[0035] (2) Using the genetically engineered strain of Gluconobacter oxydans constructed by the present invention to catalyze the synthesis of R - mandelic acid from styrene oxide, the final yield reaches 14.06 g / L, while that of the wild strain WT is only 10.26 g / L; the catalytic efficiency is also greatly improved, increasing from 0.366 g / L / h to 0.703 g / L / h.

[0036] Biological material preservation

[0037] A strain of Gluconobacter oxydans STA, taxonomically named Gluconobacter oxydans STA, was deposited at the China Center for Type Culture Collection on October 17, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20221576. Description of the drawings

[0038] Figure 1 It is a flow chart of adaptive evolution.

[0039] Figure 2 It is the spotting results of the evolved strain and the wild strain at different mandelic acid concentrations.

[0040] Figure 3Yield curve graph of the recombinant evolved strain and the wild strain for the synthesis of R-mandelic acid from styrene oxide Detailed implementation manners

[0041] The culture media described in the following examples are as follows:

[0042] Sorbitol liquid medium: 80 g / L sorbitol, 20 g / L yeast extract, 5 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 0.25 g / L anhydrous magnesium sulfate, 0.1 g / L glutamine

[0043] Sorbitol solid medium: Add 2% agar based on the sorbitol liquid medium

[0044] The mother liquor of R-mandelic acid added to the sorbitol medium is 100 g / L and is added to the liquid or solid medium according to the required concentration for use

[0045] Gluconacetobacter oxydans 621H involved in the following examples was purchased from the German DSMZ (DSMZ)

[0046] The preparation methods involved in the following examples are as follows:

[0047] Preparation of competent cells of Gluconacetobacter oxydans:

[0048] Inoculate a single colony of Gluconacetobacter oxydans into 10 mL of sorbitol liquid medium, culture at 30 °C and 200 rpm for 24 h, then inoculate at an inoculation amount of 2% into 50 mL of medium, culture at 30 °C and 200 rpm for 6 - 8 h, then centrifuge at 4 °C and 6500 rpm to remove the supernatant, wash three times with 10% glycerol, and finally aliquot 90 μL into each 1.5 mL centrifuge tube and store in a -80 °C refrigerator

[0049] Electrotransformation of Gluconacetobacter oxydans:

[0050] Add 3 μL of the recombinant plasmid to the competent cells and mix well. After standing for 30 minutes, add it to a pre-cooled electroporation cuvette, perform electroporation at 1.8 kV for 5 ms, immediately add 800 μL of liquid sorbitol medium, culture at 30 °C and 200 rpm for 4 h, centrifuge to remove most of the supernatant, mix the remaining part and spread it on a solid sorbitol medium plate containing 50 μg / mL kanamycin antibiotic, and single colonies will grow after culturing at 30 °C for 2 - 4 days

[0051] The detection methods involved in the following examples are as follows:

[0052] Detection of R-mandelic acid content:

[0053] Analysis was performed using high performance liquid chromatography (HPLC). The analytical column used was Aminex HPX-87H (Bio-Rad, 300×7.8 mm), the eluent was 5 mM H2SO4, the ultraviolet detection wavelength was 210 nm, and the detection temperature was 35°C.

[0054] Example 1: Preparation of Gluconobacter oxydans STA strain

[0055] The cryopreserved original strain of Gluconobacter oxydans 621H was streaked and activated on a solid plate. A single colony was picked and inoculated into 10 mL of sorbitol liquid medium, and cultured at 30°C and 200 rpm for 24 h until the logarithmic phase. It was then inoculated into sorbitol liquid medium containing 0.25 g / L R-mandelic acid at an inoculation amount of 5%, and after culturing for 24 h until the logarithmic phase, it was inoculated into sorbitol liquid medium containing 0.5 g / L R-mandelic acid at an inoculation amount of 5%. Subsequently, the logarithmic-phase bacterial liquid was continuously transferred to fresh medium containing a higher concentration of mandelic acid. It should be noted that as the concentration of R-mandelic acid increases, the time required to reach the logarithmic phase after transfer will increase. For concentrations with slow growth, the transfer can be repeated several times at the same concentration until the growth is relatively stable before increasing the concentration of R-mandelic acid. When the concentration of R-mandelic acid increased by 6 g / L, it was difficult for the bacteria to grow with an increase in the mandelic acid concentration. Therefore, 6 g / L was determined as the final concentration for adaptive evolution.

[0056] The finally domesticated bacterial liquid was diluted and spread on a sorbitol solid medium containing 6 g / L of R-mandelic acid, and cultured at a constant temperature of 30°C for 48 - 72 h. The single colony picked was the final evolved strain Gluconobacter oxydans STA, and the adaptive evolution process is as Figure 1 shown. The prepared Gluconobacter oxydans STA was deposited in: China Center for Type Culture Collection, deposit number: CCTCC NO: M 20221576.

[0057] Example 2: R-mandelic acid tolerance characteristics of Gluconobacter oxydans STA

[0058] The specific steps are as follows:

[0059] (1) Gluconobacter oxydans STA and the original strain 621H of Gluconobacter oxydans were separately inoculated into fresh sorbitol medium and cultured for 24 hours, controlling the initial growth OD 600 to be consistent (OD 600 was: 25); Bacterial suspensions of Gluconobacter oxydans STA and the original strain 621H of Gluconobacter oxydans were separately prepared;

[0060] (2) The bacterial suspensions of the above two strains were separately serially diluted 10-1 、 10 -2 、 10 -3 、 10 -4 、 10 -5 、 10 -6 times. Diluted bacterial suspensions and the bacterial suspensions before dilution were taken respectively, and 4 μL of each was spotted on sorbitol solid plates with R-mandelic acid concentrations of 0 g / L, 3 g / L, and 6 g / L, and cultured at 30 °C for 48 - 72 h. The growth status of the strains in the plates was detected. The results are as Figure 2 shown. From left to right, they are: undiluted bacterial suspension, bacterial suspension serially diluted 10 -1 、 10 -2 、 10 -3 、 10 -4 、 10 -5 、 10 -6 .

[0061] The results showed that the growth status of the two strains was almost the same on the 0 g / L plate. The wild strain 621H could hardly grow on the 3 g / L plate and could not grow at all on the 6 g / L plate, while the evolved strain could grow well on both the 3 g / L and 6 g / L plates, indicating that the ability of the evolved strain STA to tolerate R-mandelic acid was significantly improved after adaptive evolution.

[0062] Example 3: Construction of a recombinant expression strain of epoxide hydrolase of the evolved strain

[0063] The specific steps are as follows:

[0064] (1) Chemically synthesize the promoter P with the nucleotide sequence shown in SEQ ID NO.1 12780 fragment: Using the genome of Gluconacetobacter oxydans 621H as a template, amplify the promoter P 12780 gene fragment by PCR; The primers used for amplifying P 12780 are as follows:

[0065] PBB-P 12780 -F: 5’-GTACCGGGCCCCCCCTCGAGTGGAGAAATATCCGCAGTTTC.

[0066] P 12780 -R: 5’-GGATGTGTTCAACGTTCATTTCGTAATCCTTTTCATGTCAC.

[0067] (2) The nucleotide sequence encoding the epoxide hydrolase is shown in SEQ ID NO.2. Using the epoxide hydrolase SpEH gene stored in the laboratory as a template, amplify the SpEH gene fragment by PCR; The primers used for amplifying SpEH are as follows:

[0068] SpEH-F: 5’-GTGACATGAAAAGGATTACGAAATGAACGTTGAACACATCC。

[0069] PBB-SpEH-R: 5’-GGAATTCGATATCAAGCTTTTACAGGTCCATCTGAGCG。

[0070] (3) The two fragments obtained in steps (1) and (2) were ligated by homologous recombination with the plasmid pBBR1MCS-2 after digestion with Xho I and HindIII to prepare the recombinant vector pBBR1MCS-2-P 12780 -SpEH.

[0071] The recombinant expression vector of epoxide hydrolase was electrotransformed into Acetobacter gluconacetoxidans STA and Acetobacter gluconacetoxidans 621H, respectively, to prepare the recombinant strains: STA / pBBR1MCS-2-P 12780 -SpEH and 621H / pBBR1MCS-2-P 12780 -SpEH. Positive transformants were picked for PCR verification and then preserved.

[0072] Example 4: Whole-cell catalysis of styrene oxide by genetically engineered Acetobacter gluconacetoxidans to synthesize R-mandelic acid

[0073] The specific steps are as follows:

[0074] (1) The recombinant strains prepared in Example 3: STA / pBBR1MCS-2-P 12780 -SpEH and 621H / pBBR1MCS-2-P 12780 -SpEH were inoculated into 10 mL of sorbitol medium and cultured at 30 °C and 200 rpm for 24 h to prepare seed solutions;

[0075] The above seed solutions were inoculated into 100 mL of sorbitol medium at an inoculation amount of 1% (v / v) and cultured for 24 - 28 h. The cells were collected by a centrifuge (8000 rpm, 4 °C, 5 minutes) and washed twice, and then resuspended in PB buffer;

[0076] (2) Catalysis system: Whole-cell catalysis, controlling the cell concentration OD 600 to be 25, the initial concentration of styrene oxide was 4 g / L, and then 4 g / L was added every 4 h until it reached 20 g / L;

[0077] The reaction was carried out in a 50 mL conical flask at 30 °C and 200 rpm. 100 μL of the reaction solution was taken out every 4 h, and then the substrate was added again.

[0078] The reaction solution was centrifuged at 10,000 rpm for 1 minute. 50 μL of the supernatant was diluted with 950 μL of ultrapure water, and then filtered through a 0.22 μm filter membrane for HPLC analysis to determine the yield of R-mandelic acid. The results are as Figure 3 shown.

[0079] The results showed that the final yield of R-mandelic acid prepared by the genetically engineered strain of Gluconacetobacter oxydans constructed with the evolved strain Gluconacetobacter oxydans STA reached 14.06 g / L, while the final yield of R-mandelic acid prepared by Gluconacetobacter oxydans 621H was only 10.26 g / L;

[0080] Meanwhile, when using the genetically engineered strain of Gluconacetobacter oxydans constructed with the evolved strain Gluconacetobacter oxydans STA to prepare R-mandelic acid, the catalytic efficiency was also greatly improved, increasing from 0.366 g / L / h (the original strain Gluconacetobacter oxydans 621H) to 0.703 g / L / h.

[0081] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. An engineered Gluconacetobacter oxydans for synthesizing R-mandelic acid, characterized in that, The genetically engineered Gluconobacter oxydans bacterium uses Gluconobacter oxydans STA as the host cell and overexpresses an epoxide hydrolase derived from Sphingomonas sp. with a strong promoter having the nucleotide sequence shown in SEQ ID NO. 1; the Gluconobacter oxydans STA was deposited at the China Center for Type Culture Collection on October 17, 2022, with the deposit number CCTCC NO: M 20221576; the nucleotide sequence encoding the epoxide hydrolase is shown in SEQ ID NO.

2. 12780 The Gluconobacter oxydans STA was deposited at the China Center for Type Culture Collection on October 17, 2022, with the deposit number CCTCC NO: M 20221576; the nucleotide sequence encoding the epoxide hydrolase is shown in SEQ ID NO.

2.

2. The Gluconacetobacter oxydans genetically engineered bacterium according to claim 1, characterized in that, The Gluconacetobacter oxydans genetically engineered bacterium uses the pBBR1MCS-2 plasmid as an expression vector.

3. A method for synthesizing R-mandelic acid, characterized in that, The method is as follows: using the Gluconacetobacter oxydans genetically engineered bacterium described in claim 1 or 2 as a whole-cell catalyst, and using styrene oxide as a substrate to catalytically synthesize R-mandelic acid.

4. The method according to claim 3, characterized in that, In the reaction system, the addition amount of the genetically engineered bacteria of Gluconacetobacter oxydans is: the cell concentration OD 600 is 20 - 30.

5. The method according to claim 3, characterized in that The addition amount of the styrene oxide is 16-20 g / L.

6. The method according to claim 3, wherein The addition method of the styrene oxide is as follows: the initial concentration of the styrene oxide is 4 g / L, and then 3-4 g / L is added every 4 hours until it reaches 16-20 g / L.

7. The method according to claim 3, wherein The reaction conditions are: 25-30 °C, 180-220 rpm.

8. A method for preparing epoxide hydrolase, characterized in that, The method is as follows: obtained by fermenting the Gluconacetobacter oxydans genetically engineered bacterium described in claim 1 or 2.

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