Construction and application of a recombinant Gluconobacter oxydans

Abstract

The present invention discloses the construction and application of a recombinant Gluconobacter oxydans, belonging to the fields of genetic engineering and whole-cell catalysis. The present invention heterologously expresses an epoxide hydrolase from Sphingomonas in Gluconobacter oxydans, and simultaneously utilizes the alcohol and aldehyde dehydrogenases bound to the membrane of Gluconobacter oxydans itself to synthesize R-mandelic acid in a one-step process using styrene oxide as a substrate. The present invention combines the transcriptome data of Gluconobacter oxydans, screens 7 strong promoters using green fluorescent protein as a reporter gene, and determines the strongest promoter P for expressing the epoxide hydrolase gene. 12780 , ultimately significantly increasing the yield of R-mandelic acid.

Description

Technical Field

[0001] The invention relates to the construction and application of a recombinant Gluconobacter oxydans, and belongs to the technical field of genetic engineering and whole-cell catalysis. Background Art

[0002] (R)-Mandelic acid (R-MA) is an important intermediate in the preparation of many chiral drugs, including anti-tumor and anti-obesity agents, cephalosporins, and antibiotics such as semi-synthetic penicillins, and is crucial in the chemical and pharmaceutical industries. Currently, R-MA is mainly synthesized by chemical methods, which have harsh conditions, many by-products, and serious environmental pollution. In contrast, biosynthesis has the advantages of mild reaction conditions, high stereoselectivity, and environmental friendliness, making it a highly promising alternative method for the production of R-MA. However, the biological production of R-MA still faces many challenges, including the lack of suitable catalysts and host strains for efficient synthesis reactions.

[0003] The key to biomanufacturing high-value natural and non-natural compounds is to develop green, efficient and promising synthetic routes from inexpensive and readily available substrates. Epoxides are mainly derived from readily available petroleum-based byproducts and are widely used in the preparation of pharmaceuticals and fine chemicals. The production of R-MA using inexpensive styrene oxide as a substrate has great application potential. styrene oxide can be hydrolyzed to R-1-phenyl-1,2-ethylene glycol (R-PEG) by epoxide hydrolase, which is then further oxidized and dehydrogenated to produce R-MA. Li Zhi et al. reported that a sugar alcohol oxidase from Streptomyces coelicolor can directly oxidize R-PEG to R-MA, but the R-MA yield was only 2.43 g / L after 24 h of conversion. The low expression level and enzyme activity of the sugar alcohol oxidase may be the main reasons for the poor catalytic efficiency.

[0004] Gluconobacter oxydans is a Gram-negative bacterium belonging to the genus Acetobacter. As a food-safe strain, it is renowned for its ability to partially oxidize a variety of sugars and alcohols to their corresponding ketones or acids, conferred by its potent membrane-bound alcohol and aldehyde dehydrogenases. It has been successfully applied in the production of compounds such as vitamin C, dihydroxyacetone, gluconic acid, and miglitol. Furthermore, G. oxydans cells can be used to catalyze the oxidation of R-PEG to R-MA. In the catalytic process of G. oxydans cells, the reaction typically occurs in the periplasm using its membrane-bound dehydrogenase. Therefore, most substrate conversion and product release do not require entry or exit of the cell membrane, significantly improving biosynthetic efficiency. Therefore, we chose G. oxydans as the host cell to construct a catalytic system for the production of R-MA from styrene oxide, which required heterologous expression of the epoxide hydrolase in G. oxydans. In biosynthetic processes such as enzyme catalysis, promoters are crucial for controlling gene regulation and protein expression. However, the lack of a strong promoter remains a limiting factor in the genetic engineering of Gluconobacter oxydans 621H. Therefore, the development of an endogenous constitutive strong promoter is crucial for Gluconobacter oxydans. Summary of the Invention

[0005] The present invention aims to provide a method for constructing a recombinant Gluconobacter oxydans for synthesizing R-mandelic acid using styrene oxide, thereby solving the problems of poor catalytic efficiency and low yield in the prior art for synthesizing R-mandelic acid using styrene oxide as a substrate. Furthermore, a method for screening a strong endogenous constitutive promoter of Gluconobacter oxydans is provided to solve the defect that Gluconobacter oxydans lacks its own available genetic engineering means.

[0006] The present invention adopts the following technical solutions:

[0007] The present invention provides a promoter for increasing the expression of epoxide hydrolase, wherein the promoter is P 02805 、P 09400 、P 04650 、P 12780 、P 04000 、P 04750 or P 10190 ; The promoter P 02805 、P 09400 、P 04650 、P 12780 、P 04000 、P 04750 、P 10190The nucleotide sequences are shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0008] The present invention provides a method for screening the above-mentioned strong promoter, and the specific steps of the method are as follows:

[0009] (1) Transcriptome analysis was performed on Gluconobacter oxydans, and the nucleotide fragments about 500 bp before the start codons of genes with the top ten FPKM values ​​in the transcriptome results were selected as promoter regions.

[0010] (2) The promoter sequence was fused to green fluorescent protein by PCR and connected to the pBBR1MCS-2 plasmid to construct the recombinant vector pBBR1MCS-2-Promoter-eGFP.

[0011] (3) The recombinant plasmid was transformed into Gluconobacter oxydans and the fluorescence intensity was measured. The intensity was higher than that of P on the pBBR1MCS-2 plasmid. lac The promoter was identified as a strong promoter.

[0012] (4) The strong promoter sequence was fused with the epoxide hydrolase gene fragment and connected to the pBBR1MCS-2 plasmid to construct the recombinant vector pBBR1MCS-2-Promoter-SpEH.

[0013] (5) The above recombinant plasmid was electroporated into Gluconobacter oxydans, and the expression level and enzyme activity of epoxide hydrolase were measured to obtain a strong promoter that is optimal for the expression of epoxide hydrolase.

[0014] The present invention also provides a genetically engineered bacterium of Gluconobacter oxydans for synthesizing R-mandelic acid, wherein the genetically engineered bacterium of Gluconobacter oxydans uses Gluconobacter oxydans 621H as a host cell and adopts the promoter P 02805 、P 09400 、P 04650 、P 12780 、P 04000 、P 04750 or P 10190 Overexpression of epoxide hydrolase from Sphingomonas; the promoter P 02805 、P 09400 、P 04650 、P 12780 、P 04000 、P 04750 、P 10190The nucleotide sequences are shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0015] In one embodiment of the present invention, the promoter is P 12780 .

[0016] In one embodiment of the present invention, the nucleotide sequence encoding the epoxide hydrolase is shown as SEQ ID NO.1.

[0017] In one embodiment of the present invention, the genetically engineered bacterium Gluconobacter oxydans uses the pBBR1MCS-2 plasmid as an expression vector.

[0018] The present invention also provides a method for preparing epoxide hydrolase, which comprises inoculating the genetically engineered bacterium Gluconobacter oxydans into a culture medium and performing fermentation to prepare the epoxide hydrolase.

[0019] The present invention also provides a method for synthesizing R-mandelic acid, wherein the method comprises adding the genetically engineered bacterium Gluconobacter oxydans to a reaction system containing styrene oxide to carry out a reaction, thereby preparing R-mandelic acid.

[0020] In one embodiment of the present invention, in the reaction system, the amount of the genetically engineered bacteria of Gluconobacter oxydans is: cell concentration OD 600 It is 20 to 30.

[0021] In one embodiment of the present invention, in the reaction system, the amount of the genetically engineered bacteria of Gluconobacter oxydans is 0.05477kJ / mL. 600 =25.

[0022] In one embodiment of the present invention, the added amount of the styrene oxide is 16 to 20 g / L.

[0023] In one embodiment of the present invention, the amount of styrene oxide added is: the initial concentration of styrene oxide is 4 g / L, and then 3 to 4 g / L is added every 4 hours until it reaches 20 g / L.

[0024] In one embodiment of the present invention, the reaction conditions are: 25-30° C., 180-220 rpm.

[0025] In one embodiment of the present invention, the reaction conditions are: 30° C., 200 rpm, and conversion for 28 to 40 hours.

[0026] The present invention also provides the use of the genetically engineered bacterium Gluconobacter oxydans in preparing epoxide hydrolase, or in preparing R-mandelic acid and products containing R-mandelic acid.

[0027] Beneficial effects

[0028] The present invention successfully constructed a catalytic strain for synthesizing R-mandelic acid using styrene oxide as a substrate by expressing epoxide hydrolase in Gluconobacter oxydans. Through transcriptome data, seven endogenous constitutive strong promoters in Gluconobacter oxydans were screened. When applied to heterologous expression of epoxide hydrolase, promoter P 12780 It has the highest promoter activity, and the expression level in Gluconobacter oxydans is 3.76 times higher than that of the original promoter. At the same time, a genetically engineered Gluconobacter oxydans that can efficiently synthesize R-mandelic acid using styrene oxide as a substrate was obtained.

[0029] In addition, the present invention also provides a direct and efficient method for screening endogenous strong promoters, which provides a new idea for genetic engineering transformation of Gluconobacter oxydans. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 These are the top 10 genes with the highest FPKM values ​​in Gluconobacter oxydans.

[0031] Figure 2 is the fluorescence intensity of different promoters.

[0032] Figure 3 The enzymatic activities of epoxide hydrolases under 7 strong promoters.

[0033] Figure 4 The SDS-PAGE results of the expression levels of epoxide hydrolases under 7 strong promoters are shown.

[0034] Figure 5 This is the result of epoxide hydrolase synthesizing R-mandelic acid in the recombinant strain under the optimal strong promoter and the original promoter of the plasmid. DETAILED DESCRIPTION

[0035] The Gluconobacter oxydans 621H involved in the following examples was purchased from DSM Zürich (DSMZ), Germany. The styrene oxide substrate solution was purchased from Aladdin Reagents, CAS No. 96-09-3.

[0036] The culture medium involved in the following examples is as follows:

[0037] 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, and 0.1 g / L glutamine.

[0038] Sorbitol solid medium: Add 2% agar to the sorbitol liquid medium.

[0039] The preparation methods involved in the following examples are as follows:

[0040] Preparation of competent cells of Gluconobacter oxydans:

[0041] A single colony of Gluconobacter oxydans was inoculated into 10 mL of sorbitol liquid culture medium, cultured at 30°C, 200 rpm for 24 h, then inoculated into 50 mL of culture medium at a 2% inoculum size, cultured at 30°C, 200 rpm for 6-8 h, and centrifuged at 4°C, 6500 rpm to remove the supernatant. The supernatant was washed three times with 10% glycerol, and finally 90 μL per tube was dispensed into 1.5 mL centrifuge tubes and stored in a -80°C refrigerator.

[0042] Electrotransformation of Gluconobacter oxydans:

[0043] Add 3 μL of recombinant plasmid to the competent medium and mix well. After standing for 30 minutes, add it to a pre-cooled electroporation cup and shock it at 1.8 kV for 5 ms. Immediately add 800 μL of liquid sorbitol medium and culture it at 30°C and 200 rpm for 4 hours. 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. Culture it at 30°C for 2-4 days to grow single colonies.

[0044] The detection methods involved in the following embodiments are:

[0045] Epoxide hydrolase activity detection:

[0046] A 0.2 M ethylene oxide substrate solution was prepared in methanol; 850 μL of PB buffer was mixed with 100 μL of crude enzyme solution and incubated at 35°C for 5 minutes; the reaction was initiated by adding 50 μL of substrate solution and incubated for 10 minutes; the reaction was terminated by treatment in a boiling water bath for 10 minutes; the enzymatic activity of SpEH was analyzed using high-performance liquid chromatography (HPLC); one unit of SpEH activity was defined as the amount of enzyme required to hydrolyze ethylene oxide to produce 1 μmol of 1-phenyl-1,2-ethanediol per minute.

[0047] Detection of R-mandelic acid content:

[0048] High performance liquid chromatography (HPLC) was used for analysis. The analytical column used was Aminex HPX-87H (Bio-Rad, 300×7.8 mm), the eluent was 5 mM H 2 SO 4 , the UV detection wavelength was 210 nm, and the detection temperature was 35° C.

[0049] Example 1: Construction of plasmid pBBR1MCS-2-SpEH

[0050] The specific steps are as follows:

[0051] The epoxide hydrolase is derived from Sphingomonas, and the nucleotide sequence encoding the epoxide hydrolase is shown in SEQ ID NO.1. Using the SpEH gene preserved in the laboratory as a template, the SpEH gene fragment was amplified by PCR, and the fragment was ligated with the plasmid pBBR1MCS-2 after being digested with Xho I and Hind III to prepare the recombinant vector pBBR1MCS-2-SpEH.

[0052] The primers used to amplify SpEH are as follows:

[0053] pBB-SpEH-F:5'-ACCGGGCCCCCC CTCGAG AATGAACGTTGAACACATCCG.

[0054] pBB-SpEH-R:5'-GGAATTCGATATC AAGCTT TTACAGGTCCATCTGAGCG.

[0055] Example 2: Promoter screening

[0056] The FPKM value of each gene's transcription level in bacterial transcriptome analysis can, to a certain extent, represent the strength of its corresponding promoter. Transcriptome analysis was performed on the logarithmic-growth strain Gluconobacter oxydans 621H, and the 500 bp preceding the ATG start codon of the top 10 genes with the highest FPKM values ​​were selected as the corresponding promoter regions.

[0057] The specific steps are as follows:

[0058] (1) Transcriptome analysis was performed on Gluconobacter oxydans 621H, and the nucleotide fragments about 500 bp before the start codons of genes with the top ten FPKM values ​​in the transcriptome results were selected as promoter regions ( Figure 1 );Select promoter P tufB and P dnak As a control, the strong promoter P 14120 、P 10775 、P 05500 、P 02805 、P 09400 、P 04650 、P 12780 、P 04000 、P 04750 、P 10190 ;

[0059] (2) Using the genome of Gluconobacter oxydans 621H as a template, the nucleotide fragment of the promoter obtained in step (1) was cloned by PCR. At the same time, the eGFP gene fragment corresponding to each promoter was cloned by PCR for fusion PCR (the eGFP nucleotide sequence is shown in SEQ ID NO. 12). The fragments were digested with XhoI and HindIII and then ligated with pBBR1MCS-2 to prepare the recombinant vector pBBR1MCS-2-Promoter-eGFP;

[0060] (3) The above recombinant vectors were transformed into Gluconobacter oxydans 621H respectively, and the positive transformants were picked for PCR verification. A single colony of the constructed recombinant strain was inoculated into 10 mL of culture medium and cultured at 30°C, 200 rpm for 24 hours, and then inoculated into 50 mL of culture medium at a 1% (v / v) inoculation amount. After culturing for 20 to 24 hours, 2 mL of bacterial solution was centrifuged to remove the supernatant, washed twice with 0.2MPB buffer (pH 7.0), and resuspended in PB buffer. Cell fluorescence was measured using a black 96-well ELISA plate with excitation at 488 nm and emission at 509 nm using Gen5 data analysis software (BioTek, USA). The normalized activity of eGFP is defined as the ratio of fluorescence units (RFU) divided by the absorbance at 600 nm ( Figure 2 ). Measure the fluorescence intensity; the intensity is higher than that of P on pBBR1MCS-2 plasmid lac The promoter of P was identified as a strong promoter; 02805 、P 09400 、P 04650 、P 12780 、P 04000 、P 04750 and P 10190 The fluorescence intensity of the promoters was stronger than that of the original promoter of pBBR1MCS-2 plasmid, and the results are shown in Table 1 (P 14120 、P 10775 、P 05500 The nucleotide sequences are shown in SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11).

[0061] Table 1: Fluorescence intensity / OD under different promoters 600 ratio

[0062]

[0063]

[0064] The strong promoter finally screened was P 02805 、P 09400 、P 04650 、P 12780 、P04000 、P 04750 、P 10190 ; The promoter P 02805 、P 09400 、P 04650 、P 12780 、P 04000 、P 04750 、P 10190 The nucleotide sequences are shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0065] Example 3: Construction of recombinant plasmid pBBR1MCS-2-Promoter-SpEH

[0066] The specific steps are as follows:

[0067] (1) Using the recombinant plasmid in Example 2 as a template, PCR was performed to clone the nucleotide fragment of the strong promoter (P 02805 、P 09400 、P 04650 、P 12780 、P 04000 、P 04750 and P 10190 , the nucleotide sequences of the promoters are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively);

[0068] (2) At the same time, using the recombinant plasmid pBBR1MCS-2-SpEH in Example 1 as a template, PCR cloning of the corresponding epoxide hydrolase SpEH gene fragment was performed by fusion PCR, and the fragment was ligated with pBBR1MCS-2 after being digested with Xho I and Hind III to prepare the recombinant vector pBBR1MCS-2-Promoter-SpEH containing the promoter in step (1).

[0069] Example 4: Construction of Epoxide Hydrolase Recombinant Expression Strain and Epoxide Hydrolase Activity Assay

[0070] (1) The epoxide hydrolase cloning expression vector pBBR1MCS-2-Promoter-SpEH prepared in Example 3 was electroporated into Gluconobacter oxydans 621H to prepare the recombinant strain: 621H / pBBR1MCS-2-Promoter-SpEH, and the positive transformants were selected for PCR verification.

[0071] (2) A single colony of the recombinant strain constructed in step (1) was inoculated into 10 mL of sorbitol medium, cultured at 30°C, 200 rpm for 24 h, and then inoculated into 50 mL of sorbitol medium at a 1% (v / v) inoculation rate. After culture for 24-28 h, the cells were collected by centrifugation at 4°C, 8000 rpm for 5 minutes, washed twice with PB buffer, and resuspended in 5 mL of PB buffer. After ultrasonic disruption, the cells were centrifuged at 12000 rpm for 10 minutes. The supernatant was the crude enzyme solution. The growth and cell disruption concentrations of the recombinant expression strains with different promoters were controlled to be the same.

[0072] According to the above method, P tufB and P dnak The promoter served as a control.

[0073] Take 80 μL of crude enzyme solution and add 20 μL of loading buffer for SDS-PAGE ( Figure 4 ).

[0074] The results showed that P 12780 The expression level of epoxide hydrolase under the promoter was much higher than that under the original promoter P lac , and compared with P tufB and P dnak The expression level of epoxide hydrolase from the promoter was also significantly increased.

[0075] (3) Enzyme activity detection, the results are as follows Figure 3 As shown in Table 2.

[0076] Table 2: Epoxide hydrolase activity expressed by strains with different promoters

[0077]

[0078] The results showed that the final promoter P 12780 The expressed epoxide hydrolase had the highest activity, reaching 21.41 U / mL, respectively. tufB and P dnak 1.69 and 1.67 times.

[0079] Example 5: Synthesis of R-mandelic acid by whole cells of genetically engineered Gluconobacter oxydans catalyzed by styrene oxide

[0080] The specific steps are as follows:

[0081] (1) According to the method of Example 4, the recombinant strain 621H / pBBR1MCS-2-P was prepared. 12780 -SpEH (named GO-P 12780 -SpEH); at the same time, the recombinant strain 621H / pBBR1MCS-2-SpEH (named GO-P lac-SpEH) as the control strain, P lac is the promoter on the original vector.

[0082] (2) The above recombinant strains were inoculated into 10 mL of sorbitol medium, and cultured at 30°C and 200 rpm for 24 h to prepare seed liquid; the above seed liquid was inoculated into 100 mL of sorbitol medium at an inoculum amount of 1% (v / v), and cultured for 24-28 h. The cells were collected by centrifuge (8000 rpm, 4°C, 5 minutes), washed twice, and then resuspended in PB buffer.

[0083] (3) In the catalytic system, whole-cell catalysis controls the cell concentration OD 600 The initial concentration of styrene oxide is 4 g / L, and then 4 g / L is added every 4 hours until it reaches 20 g / L;

[0084] The reaction was carried out in a 50 mL conical flask at 30°C and 200 rpm. 100 μL of the reaction solution was removed every 4 hours and then the substrate was added.

[0085] The reaction solution was centrifuged at 10000 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 shown in Table 3 and Figure 5 shown.

[0086] Table 3: Yields under different reaction times and substrate concentrations

[0087]

[0088] The results showed that the promoter P 12780 The expression strain catalyzed the synthesis of R-almond styrene oxide with a final yield of 10.26 g / L, while the strain expressing the original promoter of the plasmid was only 7.36 g / L.

[0089] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A promoter P for increasing the expression of epoxide hydrolase 12780 , characterized in that, The nucleotide sequence is shown in SEQ ID NO.

5.

2. A genetically engineered bacterium of Gluconobacter oxydans for synthesizing R-mandelic acid, characterized in that: The genetically engineered bacterium Gluconobacter oxidans uses Gluconobacter oxidans 621H as a host cell and adopts a promoter P shown in SEQ ID NO.

5. 12780 The epoxide hydrolase gene whose nucleotide sequence is shown in SEQ ID NO.1 is overexpressed.

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

4. A method for preparing epoxide hydrolase, characterized in that: The method comprises the following steps: inoculating the genetically engineered bacterium Gluconobacter oxydans according to claim 2 or 3 into a culture medium for fermentation to prepare the epoxide hydrolase.

5. A method for synthesizing R-mandelic acid, characterized in that, The method comprises adding the genetically engineered bacterium Gluconobacter oxydans according to claim 2 or 3 to a reaction system containing styrene oxide to carry out a reaction and prepare R-mandelic acid.

6. The method according to claim 5, characterized in that In the reaction system, the amount of the genetically engineered bacteria of Gluconobacter oxidans added is: cell concentration OD 600 The amount of epoxy styrene added is 16 to 20 g / L.

7. The method according to claim 5, characterized in that The reaction conditions are: 25-30°C, 180-220 rpm, and conversion time of 28-40 hours.

8. Use of the genetically engineered bacterium Gluconobacter oxydans according to claim 2 or 3 in the preparation of epoxide hydrolase, or in the preparation of R-mandelic acid and products containing R-mandelic acid.

Images