CN116286569A - A method for transforming whole cells of recombinant Bacillus licheniformis to produce (R)-citramalic acid - Google Patents

Abstract

The invention discloses a method for producing (R) -citramalic acid by whole cell transformation of recombinant bacillus licheniformis, belonging to the technical field of bioengineering. According to the invention, the (R) -citramalate synthase is heterologously expressed in bacillus licheniformis, a synthetic path of (R) -citramalate is built, glucose is used as a substrate, conditions for producing (R) -citramalate by whole cell transformation of a recombinant strain are established and optimized, synthesis and accumulation of (R) -citramalate are realized, the transformation time is 120 hours, 8.57g/L of (R) -citramalate can be produced, the transformation rate is 142.84mg/g glucose, and the method has the advantages of simplicity in preparation, low production cost, strong environmental pressure resistance, few byproducts, high yield and the like.

Description

A method for transforming whole cells of recombinant Bacillus licheniformis to produce (R)-citramalic acid

technical field

The invention relates to a method for transforming whole cells of recombinant bacillus licheniformis to produce (R)-citramalic acid, which belongs to the technical field of bioengineering.

Background technique

(R)-citramalate (R-citramalate) is a five-carbon hydroxydicarboxylic acid widely present in the metabolic pathways of some bacteria, such as Methanococcus jannaschii, Geobactersulfurreducens ), the biosynthetic pathway of isoleucine in Chlorobaculum tepidum; the anaerobic metabolism of glutamate in Clostridium tetanomorphum, etc., all involve the synthesis of (R)-citramalate. (R)-citramalic acid is an important multifunctional organic acid. As a chemical synthesis precursor of the polymer intermediate methacrylic acid (MAA), it can not only be used in the resin production industry, but also as an acidifying agent and wrinkle removing agent. It plays an important role in food processing, beauty, medical and other fields. Therefore, (R)-citramalic acid has broad application and market prospects.

The synthesis method of citramalic acid is mainly a chemical method, which is synthesized by condensation reaction with hydrogen cyanide and ethyl acetyl ester as substrates. However, due to the toxicity of precursor substances and the high cost they bring, it is difficult to meet the needs of sustainable development, which greatly limits the application of this method. In recent years, with the development of synthetic biology, the biosynthesis of citramalate has become a more attractive alternative: using pyruvate and acetyl-CoA as precursors, under the catalysis of citramalate synthase Specific condensation forms citramalic acid.

In 2018, Wu Xianghao et al. Heterologously expressed citramalate synthase - CimA3.7 in Escherichia coli (the enzyme was derived from Methanococcus janesii and was obtained by ATSUMI et al. after screening and directed evolution Enzymes with higher stability), thus successfully introducing the citramalate synthesis pathway, and obtaining recombinant E. coli strains with high citramalate production. In this study, glucose was used as the substrate, and 2.9g/L citramalate was finally produced at the shake flask fermentation level. Although the genetic manipulation of Escherichia coli is simple, there is still no effective solution to the problem of phage infection in the industrial fermentation process; moreover, for the process of using recombinant Escherichia coli to produce citramalic acid, due to the restriction of the substrate glucose by Escherichia coli Tolerance and product accumulation lead to a decrease in the pH value of the environment, resulting in impaired cell growth and metabolism, which is not conducive to the accumulation of high concentrations of citramalic acid.

At present, the research on the biological production of citramalic acid mainly focuses on the traditional fermentation method, and the production process of whole cell transformation has not been proposed for product synthesis.

Contents of the invention

The object of the present invention is to provide a method for transforming whole cells of recombinant Bacillus licheniformis to produce (R)-citramalic acid, aiming to solve technical problems such as low conversion rate and waste of resources in the conventional fermentation method for synthesizing citramalic acid in the prior art .

To achieve the above object, the present invention takes the following technical measures:

The invention provides a recombinant Bacillus licheniformis capable of transforming and producing (R)-citramalic acid, using Bacillus licheniformis (Bacillus licheniformis) CICIM B1391 as a host; the amino acid sequence of the citramalate synthase is shown in SEQ ID NO.2 shown.

In one embodiment, the CICIM B1391 is disclosed in the paper "Identification of Strong Constitutive Promoter of Bacillus licheniformis and Verification of Its Expression Effect", and the applicant promises to release the strain to the public through legal channels within 20 years from the date of application .

In one embodiment, the gene encoding citramalate synthase is shown in SEQ ID NO.1.

In one embodiment, the plasmid pHY-PLK300 is used as the expression vector.

The invention also provides a cell catalyst containing the bacillus licheniformis.

In one embodiment, the preparation method of the cell catalyst is as follows: culturing the recombinant Bacillus licheniformis in a culture medium for a period of time, and collecting the cells as the whole cell catalyst.

In one embodiment, the medium is a CMC fermentation medium, containing: sucrose 100g/L, cottonseed protein 30g/L, K ₂ HPO ₄ ·3H ₂ O 9.12g/L, KH ₂ PO ₄ 1.36g/L L. (NH ₄ ) ₂ HPO ₄ 10g/L, pH 7.5.

In one embodiment, the culturing is at 37°C for at least 48 hours.

The invention also provides a method for transforming whole cells of the recombinant Bacillus licheniformis to produce (R)-citramalic acid.

In one embodiment, in the whole cell transformation system, the bacterial cells have an OD ₆₀₀ of 70-80 based on the final concentration.

In one embodiment, in the whole cell transformation system, the glucose concentration is 50-150 g/L.

In one embodiment, the initial pH of the whole cell transformation system is 4-9.5, or 6.5-7.5, or 7.

In one embodiment, the whole cell transformation is carried out at 30-37° C. and 150-250 rpm.

In one embodiment, the whole cell transformation reaction time is ≥24h, or ≥48h, or ≥72h.

In one embodiment, the method is to transform for 120 h under the conditions of 37°C, pH 7.0, substrate concentration 100 g/L, cell concentration OD ₆₀₀ 70, and shaker speed 250 r/min.

The present invention also claims the application of the recombinant bacteria, the cell catalyst or the method in the production of products containing (R)-citramalic acid.

Beneficial effect:

(1) The present invention uses Bacillus licheniformis with strong stress resistance and rich enzyme system as the chassis cell, introduces heterologous citramalate synthase to build a citramalate synthesis pathway, and solves the process of industrially producing citramalate by using Escherichia coli problem of bacteriophage infection.

(2) The present invention applies the Bacillus licheniformis whole-cell transformation system to the synthesis of citramalic acid for the first time. By optimizing the conditions for the transformation of whole cells of recombinant strains to produce citramalic acid, the yield and conversion rate of citramalic acid can be significantly improved, and the transformation takes 120 hours It can generate (R)-citramalate 8.57g/L, and the conversion rate is 142.84mg/g glucose, which is the highest level of (R)-citramalate synthesis in shake flask culture reported so far.

Description of drawings

Figure 1: The citramalate synthesis pathway in Bacillus licheniformis.

Figure 2: Construction and verification of the recombinant plasmid pHY-P _Shuttle09 -cimA3.7.

Figure 3: Condition optimization for the preparation of whole-cell catalysts; where, a: selection of fermentation medium; b: fermentation results of recombinant Bacillus licheniformis.

Figure 4: Liquid chromatograms of standards (glucose, R-citramalate) and samples.

Figure 5: Optimization of conditions for the transformation of recombinant Bacillus licheniformis whole cells to produce (R)-citramalic acid a: cell concentration; b: shaker speed; c: temperature; d: glucose concentration; e: pH; f: transformation time .

Detailed ways

Medium:

LB medium: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L (solid medium plus 1.5% agar powder).

CMA fermentation medium: peptone FP321 20g/L, yeast powder FM408 10g/L, glucose 100g/L, corn steep liquor powder 5g/L, K ₂ HPO ₄ 3H ₂ O 9.12g/L, KH ₂ PO ₄ 1.36g/L L, CaCl ₂ 0.5g/L, MgSO ₄ ·7H ₂ O 0.5g/L, (NH ₄ ) ₂ HPO ₄ 10g/L.

MB fermentation medium: peptone FP321 20g/L, yeast powder FM408 10g/L, glucose 200g/L, corn steep liquor powder 5g/L, K ₂ HPO ₄ 3H ₂ O 9.12g/L, KH ₂ PO ₄ 1.36g/L L, CaCl ₂ 0.5g/L, MgSO ₄ ·7H ₂ O 0.5g/L, (NH ₄ ) ₂ HPO ₄ 10g/L.

CMC fermentation medium: sucrose 100g/L, cottonseed protein 30g/L, K ₂ HPO ₄ 3H ₂ O 9.12g/L, KH ₂ PO ₄ 1.36g/L, (NH ₄ ) ₂ HPO ₄ 10g/L (pH 7.5).

TB transformation medium: peptone 12g/L, yeast powder 24g/L, K ₂ HPO ₄ 12.54g/L, KH ₂ PO ₄ 2.31g/L, glycerol 5g/L (pH 7.0).

Product detection and identification: centrifuge the fermentation broth at 12 000 r/min for 20 min, take the supernatant and add an equal volume of 95% (volume fraction) ethanol solution to precipitate protein at 4°C for 8-12 h. After centrifugation, the supernatant is filtered through a 0.22 μm organic membrane; the detection conditions of high performance liquid chromatography (HPLC) are as follows: the mobile phase is (0.05%, V/V) sulfuric acid-water; 0.8mL/min; differential detector; liquid chromatography column is Dikma CarboPac H+ (300mm×8.0mm, 6μm); column temperature 50°C; injection volume 10μL.

The construction of embodiment 1 recombinant plasmid pHY-P _Shuttle09 -cimA3.7

Synthesize the gene of coding citramalate synthase—CimA3.7 shown in SEQ ID NO.1 (synthesized by Shanghai Sangong), take this gene as template, use forward primer CimA-F and reverse primer CimA-R Perform PCR amplification, and purify and recover the target fragment cimA3.7. Using the plasmid PHE11 (disclosed in "Identification of Strong Constitutive Promoter of Bacillus licheniformis and Verification of its Expression Effect") as a template, use forward primer pHY-S09-F and reverse primer pHY-S09-R for reverse PCR amplification The expression vector pHY-Pshuttle09 was obtained. Restriction sites Xho I and EcoR I were introduced at both ends of the primers, and T4 ligase was used to ligate the target fragment cimA3.7 and the expression vector pHY-Pshuttle09 to form the recombinant plasmid pHY-P _Shuttle09 -cimA3.7 after double digestion. The ligation product was transformed into E.coli JM109, the positive transformants with correct sequencing were selected, the plasmid was extracted and electroporated into Bacillus licheniformis CICIM B1391 cells to obtain recombinant Bacillus licheniformis BLA.

CimA-F: CCGCTCGAGATGATGGTCAGAATCTTTGATACGACACT

imA-R: CCGGAATTCATCGACCGGGCCGAC

pHY-S09-F: GTCGACGGATCCCCGGGAATTCCTG

pHY-S09-R: CCGCTCGAGGGATCCCACTTTATGGACGCCG

The optimization of embodiment 2 recombinant Bacillus licheniformis fermentation medium

Three different fermentation media, CMA, CMB, and CMC, were selected to ferment the recombinant Bacillus licheniformis, and their effects on the growth of the bacteria and the output of citramalic acid were explored. The specific steps were as follows:

1) Seed activation: Streak the recombinant Bacillus licheniformis BLA constructed in Example 1 stored in a glycerol tube at -70°C onto a solid LB plate with tetracycline resistance, and culture at 37°C for 12-16 hours. Pick a single colony from the plate, inoculate it into an Erlenmeyer flask filled with 15 mL of LB medium (containing tetracycline at a final mass concentration of 20 μg/mL), and culture it in a shaker at 37 °C and 250 r/min for 24 h to obtain a seed solution .

2) Shake flask culture: the seed solution was transferred to different fermentation media with a liquid volume of 30 mL at an inoculum size of 10%, and cultured at 37° C. and 250 r/min. Take 1mL of fermentation broth every 24h, and record the optical density _OD600 of the strain; centrifuge the fermentation broth at 12 000r/min for 20min, and the supernatant is used for HPLC analysis to detect the content of citramalic acid and components in the sample solution.

The experimental results are shown in Figure 3a. After 168 hours of fermentation, when the fermentation medium was CMC, the yield of citramalic acid was the highest and the growth of the bacteria was the best. The final cell concentration OD ₆₀₀ reached 29.13, and the yield of citramalic acid could reach 29.13. up to 1.49g/L, indicating that citramalate synthase can be better expressed under this condition. Combined with the growth curve (3b) of the recombinant bacteria in the fermentation process, it can be obtained that the rate of citramalic acid production is the fastest after 36-48 hours of fermentation. Considering the production of citramalic acid and the time cost, the bacteria after 48 hours of fermentation in CMC medium were selected. body as a whole-cell catalyst.

Embodiment 3 Preparation of recombinant bacillus licheniformis whole cell catalyst

Prepare recombinant Bacillus licheniformis whole-cell catalyst according to the following steps:

1) Seed activation: Streak the recombinant Bacillus licheniformis BLA constructed in Example 1 stored in a glycerol tube at -70°C onto a solid LB plate with tetracycline resistance, and culture at 37°C for 12-16 hours. Pick a single colony from the plate, inoculate it into an Erlenmeyer flask filled with 15 mL of LB medium (containing tetracycline at a final mass concentration of 20 μg/mL), and culture it in a shaker at 37 °C and 250 r/min for 24 h to obtain a seed solution .

2) Shake flask culture: the seed solution was transferred to 30 mL of CMC medium with a 10% inoculum size, and cultured at 37° C. and 250 r/min for 48 hours;

3) Cell collection: centrifuge the cell culture solution in step 2) at 9000r/min, 4°C for 15min, discard the supernatant, collect the bacteria, and obtain the recombinant Bacillus licheniformis whole-cell catalyst.

Example 4 Transformation of whole cells to produce (R)-citramalic acid

Prepare the whole cell catalyst according to the method of Example 3, wash the bacteria twice with pre-cooled TB solution, then resuspend the bacteria in the TB solution to form a cell suspension, control the concentration of the bacteria by adjusting the _OD600 , and add the final mass Glucose at a concentration of 100 g/L and tetracycline at 20 μg/mL. The whole cell transformation reaction system with a total system of 25mL was reacted in a 250mL shake flask at 37°C and 250r/min on a constant temperature shaker for 24h.

The fermented sample was subjected to HPLC to detect the peak of the product. The liquid phase results are shown in Figure 4. Glucose was eluted at 9.50min, and the product was eluted at 10.29min, and the peak time of the product and the standard sample of citramalic acid Consistent, thus judging that the product is citric acid.

Example 5 Recombinant bacteria with different thalline concentrations transformed into whole cells to produce citramalic acid

The specific implementation method is the same as in Example 4, the difference is that the _OD600 of the control bacterial cell concentration is 40, 50, 60, 70, and 80 respectively, adding glucose with a final concentration of 100g/L and tetracycline at 20 μg/mL, and then at 37°C and 250r After 24 hours of reaction on a constant temperature shaker, the content of citric acid was determined. The results are shown in Figure 5a. When the OD ₆₀₀ is 70-80, the yield of citramalic acid is higher. When the OD ₆₀₀ is 70, the yield of citramalic acid is 3.48g/L, and the conversion rate is 69.51mg/g glucose.

Example 6 Transformation of whole cells of recombinant bacteria under different rotating speeds to produce citramalic acid

The specific implementation method is the same as in Example 4, the difference is that the _OD600 of the bacterial cell concentration is controlled to be 70, the rotating speed of the shaker is controlled to be 150, 200, 250, and 270 r/min respectively, and glucose with a final concentration of 100 g/L and 20 μg/mL of Tetracycline, after reacting at 37°C for 24h, the content of citramalic acid was determined. The results are shown in Figure 5b. With the increase of the rotational speed, the yield and conversion rate of citramalic acid gradually increased. At 250r/min, the yield of citramalic acid could reach 3.35g/L, and the conversion rate was 76.60mg/g glucose. .

Example 7 Transformation of whole cells of recombinant bacteria to produce citramalic acid at different transformation temperatures

The specific implementation method is the same as in Example 4, the difference is that the _OD600 of the bacterial cell concentration is controlled to be 70, the transformation temperature is controlled to be 30, 37, 42, and 50°C respectively, and glucose with a final concentration of 100g/L and tetracycline of 20μg/mL are added, The content of citramalic acid was determined after reacting on a shaker at 250r/min for 24h. The results are shown in Figure 5c, the yield of citramalic acid can reach more than 4g/L at 30-37°C, 4.78g/L at 37°C, and the conversion rate is 74.13mg/g glucose.

Example 8 Transformation of whole cells of recombinant bacteria to produce citramalic acid under different glucose concentrations

The specific embodiment is the same as in Example 4, the difference is that the OD ₆₀₀ of the control bacterium concentration is 70, the control substrate glucose concentration is 50, 100, 150, 200 g/L respectively, adding tetracycline with a final mass concentration of 20 μg/mL, at 37 The content of citramalic acid was determined after reacting for 24 hours under the condition of ℃ and 250r/min. The results were shown in Figure 5d, when the glucose concentration was too high or too low, it was not conducive to the production of citramalic acid. When the glucose concentration was 100g/L, the yield of citramalic acid was 4.55g/L, and the conversion rate was 78.85mg/g glucose.

Example 9 Transformation of whole cells of recombinant bacteria under different initial pHs to produce citramalic acid

The specific implementation is the same as in Example 4, the difference is that the _OD600 of the control bacterium concentration is 70, the initial pH is controlled to be acidic, neutral and alkaline respectively, and the acidity is to use 0.2M PB buffer solution, and hydrochloric acid is added to adjust the pH to 4～5.5 ; Neutral is to use 0.2MPB buffer to adjust the pH to 6.5-7.5; alkaline is to use 0.2M PB buffer and add sodium hydroxide to adjust the pH to 8-9.5. Glucose with a final mass concentration of 100 g/L and tetracycline at 20 μg/mL were then added, and the content of citramalic acid was determined after reacting on a shaking table at 37°C and 250 r/min for 24 hours. As shown in Figure 5e, in the range of neutral pH 6.5-7.5, the yield of citramalic acid can reach more than 4g/L, and the yield of citramalic acid is 4.62g/L when the initial pH is 7.0, and the conversion rate reaches 79.81mg/g glucose .

Example 10 Recombinant Bacteria Whole Cell Transformation Produces Citramalic Acid under Different Transformation Times

The specific implementation is the same as in Example 4, the difference is that the OD ₆₀₀ of the control bacterium concentration is 70, the control conversion time is 24, 48, 72, 96, 120, and 132 h respectively, and the final mass concentration is 100 g/L of glucose and 20 μg/L mL of tetracycline was reacted on a shaker at 37°C and 250 r/min, and then the content of citramalic acid was determined. The results are shown in Figure 5f. After 72 hours of conversion reaction, the yield of citramalic acid can reach more than 4.35 g/L; after 120 hours of conversion, the yield of citramalic acid reaches 8.57 g/L, and the conversion rate is 142.84 mg/g glucose. Continue to extend the fermentation time. The output of citric malic acid does not increase anymore, considering the problem of time cost, the conversion time should be selected as 120h.

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 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 defined by the claims.

Claims (10)

1. A recombinant bacillus licheniformis capable of producing (R) -citramalic acid in a converting way is characterized in that a bacillus licheniformis is taken as a host, and a heterologous citramalic acid synthase is introduced to construct a (R) -citramalic acid synthesis way; the amino acid sequence of the citramalate synthase is shown as SEQ ID NO. 2.

2. The recombinant bacillus licheniformis according to claim 1, wherein the coding gene of the citramalate synthase is shown in SEQ ID No. 1.

3. The recombinant bacillus licheniformis according to claim 1 or 2, characterized in that bacillus licheniformis (Bacillus licheniformis) cic im B1391 is used as host and plasmid pHY-PLK300 is used as expression vector.

4. A cell catalyst comprising the bacillus licheniformis of any of claims 1-3.

5. A method for preparing the cell catalyst according to claim 4, wherein the recombinant Bacillus licheniformis according to any of claims 1 to 4 is cultured in a medium for a period of time, and the cells are collected as a whole cell catalyst.

6. The method of claim 5, wherein the medium comprises sucrose as a carbon source and cottonseed protein as a nitrogen source; the culturing is at 37 ℃ for at least 48 hours.

7. Use of the recombinant bacillus licheniformis of any of claims 1-3 for the whole cell conversion of (R) -citramalate production.

8. A method for producing (R) -citramalic acid by whole cell transformation, which is characterized in that the recombinant bacillus licheniformis according to any of claims 1-4 is used as a cell catalyst, or the cell catalyst according to claim 5 is used, and glucose is used as a substrate for reaction for at least 24 hours.

9. The method of claim 8, wherein the recombinant bacillus licheniformis OD in a whole cell transformation system ₆₀₀ 70-80 g/L, glucose concentration of 50-150 g/L and initial pH of 4-9.5.

10. Use of a recombinant bacterium according to any one of claims 1 to 3, or a cell catalyst according to claim 4, or a method according to any one of claims 8 to 9, for the production of a (R) -citramalic acid containing product.

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