A method for increasing the production of 2,5-dimethylpyrazine by expressing key polyhydroxybutyrate biosynthesis genes

Abstract

The application provides a method for improving 2,5-dimethylpyrazine yield by expressing polyhydroxybutyrate (PHB) biosynthesis key genes, comprising the following steps: introducing an expression vector containing polyhydroxybutyric acid synthesis module encoding genes and an expression vector containing threonine dehydrogenase genes and NADH oxidase genes into host cells to obtain engineering bacteria; the engineering bacteria are subjected to fermentation culture, and then lactose is added for induction, and the method is completed. The method for high-yield 2,5-dimethylpyrazine provided by the application introduces a PHB biosynthesis pathway into E. coli, and cooperatively expresses threonine dehydrogenase and NADH oxidase to construct an engineering strain for biosynthesis of 2,5-dimethylpyrazine; the fermentation result shows that after introducing the PHB biosynthesis pathway, the fermentation yield of 2,5-dimethylpyrazine of the engineering strain is significantly improved, and the yield of 2,5-dimethylpyrazine of the engineering strain EC-3 reaches 2097.68 mg / L after 60 h of shake flask fermentation.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, and in particular relates to a method for increasing the yield of 2,5-dimethylpyrazine by expressing key genes for the biosynthesis of polyhydroxybutyrate. Background Technology

[0002] 2,5-Dimethylpyrazine is a nitrogen-containing six-membered heterocyclic compound, a colorless or slightly yellow liquid at room temperature, widely found in various foods, and possesses a strong baked aroma. Due to its extremely low odor threshold, only 1-2 μg / kg is needed to significantly enhance food flavor, making it widely used as a natural flavoring agent in the food industry. Furthermore, 2,5-Dimethylpyrazine also has certain medicinal value, serving as an important raw material for the synthesis of pharmaceutical intermediates such as amoxicillin, glipizide, and methyl 5-methylpyrazine-2-carboxylate.

[0003] Currently, the industrial production of 2,5-dimethylpyrazine mainly relies on chemical synthesis methods, such as the reaction of ethyl acetoacetate with ammonia and the condensation of aldehyde compounds. However, these traditional chemical synthesis routes typically suffer from harsh catalytic conditions, complex byproducts, difficulties in product separation, and severe environmental pollution, making it difficult to meet the demands of green and sustainable production.

[0004] With the development of synthetic biology, the biosynthesis of 2,5-dimethylpyrazine using microbial cell factories has become a research hotspot. Compared with chemical synthesis, biosynthesis offers milder and more environmentally friendly conditions, making it more suitable for the production of food-grade additives. However, existing studies have shown that 2,5-dimethylpyrazine itself has certain toxicity to host cells, and its intracellular accumulation can inhibit cell growth and metabolism, thus limiting efficient synthesis.

[0005] Existing research indicates that the polyhydroxybutyric acid (PHB) biosynthesis pathway can enhance the tolerance of some microorganisms to environmental stresses such as organic solvents, oxidative stress, and salt stress. However, there are no reports of using the PHB biosynthesis pathway to improve the fermentation production capacity of 2,5-dimethylpyrazine, and it remains unclear whether it can improve the toxicity of 2,5-dimethylpyrazine and increase fermentation yield. Summary of the Invention

[0006] In view of this, the present invention aims to overcome the deficiencies in the prior art and proposes a method for high-yield production of 2,5-dimethylpyrazine by expressing PHB synthase.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows: This invention provides a method for high-yield production of 2,5-dimethylpyrazine by expressing PHB synthase, comprising the following steps: (1) An expression vector containing a polyhydroxybutyrate synthesis module encoding gene and an expression vector containing a threonine dehydrogenase (TDH) gene and a NADH oxidase gene were introduced into a host cell to obtain engineered bacteria; (2) The engineered bacteria are fermented and cultured, and lactose is added to induce fermentation.

[0008] Step (1) further includes the following steps: (a) with Ralstonia eutropha H16 genome serves as template for gene amplification. phaA , phaB and phaC ;by Escherichia coli K12 genome serves as a template for gene amplification. tdh ;by Streptococcus pneumoniae Genome as template for gene amplification nox Alternatively, codon optimization can be performed using the gene-encoded proteins described above to obtain the target gene sequence; (b) The expression vectors pRSFDuet-1 and pACYCDuet-1 were digested with enzymes and linearized fragments were obtained by gel recovery; (c) The linearized vector pRSFDuet-1 described above is combined with the target gene. tdh Seamless cloning and ligation were performed to obtain the recombinant plasmid pRSFDuet-1 / tdh Furthermore, the target gene nox With pRSFDuet-1 / tdh Ligation was performed to obtain the recombinant plasmid pRSFDuet-1 / tdh - nox At the same time, the target gene phaCBA Ligated with the linearized vector pACYCDuet-1, the recombinant plasmid pACYCDuet-1 / was obtained. phaCBA The recombinant plasmid pRSFDuet-1 / tdh and pRSFDuet-1 / tdh - nox An engineered strain producing 2,5-dimethylpyrazine was obtained through screening with kanamycin-resistant plates.

[0009] Furthermore, the polyhydroxybutyric acid (PHB) synthesis module encoding gene in step (1) includes phaA Gene, phaB Genes and phaC Gene.

[0010] Furthermore, the engineered bacteria in step (1) is Escherichia coli BL21.

[0011] Furthermore, the fermentation step in step (2) uses a liquid culture medium.

[0012] Furthermore, the liquid culture medium comprises the following raw materials in parts by weight: 0.4-2 parts glycerol, 0.5-2 parts tryptone, 0.5-4 parts yeast extract, 0.01-0.3 parts potassium dihydrogen phosphate, 0.1-1.5 parts dipotassium hydrogen phosphate, 0.01-2 parts lactose, and 0.5-3 parts threonine.

[0013] Furthermore, the lactose in step (2) has an OD value of 600 When the concentration is 0.4-0.8, the lactose is added, and the final concentration of the lactose is 0.05-0.3 w / v.

[0014] The present invention also provides an engineered bacterium, wherein the engineered bacterium contains an expression phaA Gene, phaB Genes and phaC Recombinant expression vectors and expression of genes tdh Genes and nox Recombinant gene expression vectors.

[0015] The present invention also provides an application of the engineered bacteria in the production of 2,5-dimethylpyrazine.

[0016] Compared with the prior art, the present invention has the following advantages: The method for high-yield 2,5-dimethylpyrazine described in this invention involves introducing the PHB biosynthetic pathway into *Escherichia coli* and co-expressing it with threonine dehydrogenase (TDH) and NADH oxidase to construct an engineered strain for the biosynthesis of 2,5-dimethylpyrazine. Fermentation results show that the introduction of the PHB biosynthetic pathway significantly increases the 2,5-dimethylpyrazine yield of the engineered strain. After 60 h of shake-flask fermentation, the 2,5-dimethylpyrazine yield of engineered strain EC-3 reached 2097.68 mg / L. Furthermore, the introduction of the PHB biosynthetic pathway helps improve the tolerance of the engineered strain to 2,5-dimethylpyrazine during fermentation, thereby improving fermentation performance and increasing the yield of the target product.

[0017] The method for high-yield 2,5-dimethylpyrazine described in this invention introduces the polyhydroxybutyric acid (PHB) biosynthetic pathway into the 2,5-dimethylpyrazine production system for the first time, improving the host cell's tolerance to 2,5-dimethylpyrazine through intracellular PHB accumulation; and through synergistic expression... tdh , nox and phaCBA The gene improved the biosynthesis efficiency of 2,5-dimethylpyrazine; the use of lactose instead of IPTG as an inducer reduced production costs and made it more suitable for industrial applications. Attached Figure Description

[0018] Figure 1The heterologous expression gene described in the embodiments of the present invention tdh A schematic diagram of plasmid construction; Figure 2 The heterologous expression gene described in the embodiments of the present invention tdh , nox A schematic diagram of plasmid construction; Figure 3 The heterologous expression gene described in the embodiments of the present invention phaCBA A schematic diagram of plasmid construction; Figure 4 This is a schematic diagram illustrating the 2,5-dimethylpyrazine tolerance of *E. coli* cells according to an embodiment of the present invention: Figure a shows the growth of the engineered strain under different concentrations of 2,5-dimethylpyrazine; Figure b shows the expression... phaCBA Transmission electron microscopy (TEM) images of intracellular PHB particles in engineered strains; Figure 5 This is a schematic diagram of the 2,5-dimethylpyrazine fermentation process of each strain described in the embodiments of the present invention; Figure 6 This is a schematic diagram of the fermentation of the recombinant strain after optimizing the carbon source and inducer according to an embodiment of the present invention. Detailed Implementation

[0019] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0020] The Escherichia coli BL21(DE3) used in the various embodiments of the present invention is a commercial strain in the field and can be obtained commercially.

[0021] The present invention will be described in detail below with reference to embodiments.

[0022] Example 1: Liquid culture of engineered Escherichia coli This embodiment examines heterologous gene expression. tdh , tdh - nox , tdh - nox / phaCBA The effect on the production of 2,5-dimethylpyrazine in an Escherichia coli liquid fermentation system. Using the E. coli plasmids pRSFDuet-1 and pACYCDuet-1 as expression vectors, the linearized vector pRSFDuet-1 was used to express the target gene. tdh Seamless cloning and ligation were performed to obtain the recombinant plasmid pRSFDuet-1 / tdh ( Figure 1 Furthermore, the target gene noxWith pRSFDuet-1 / tdh Ligation was performed to obtain the recombinant plasmid pRSFDuet-1 / tdh - nox ( Figure 2 ); at the same time, the target gene phaCBA Ligated with the linearized vector pACYCDuet-1, the recombinant plasmid pACYCDuet-1 / was obtained. phaCBA ( Figure 3 plasmid pRSFDuet-1- tdh Recombinant strain EC-1 was obtained by transformation into Escherichia coli BL21, plasmid pRSFDuet-1- tdh - nox Recombinant strain EC-2 was obtained by transformation into Escherichia coli BL21, plasmid pRSFDuet-1- tdh - nox and pACYCDuet-1- phaCBA Co-transformation into Escherichia coli BL21 yielded recombinant strain EC-3. Single colonies were picked and inoculated into 5 / 50 mL centrifuge tubes and cultured overnight. The colonies were then transferred to liquid fermentation medium at a 1% (v / v) inoculation rate.

[0023] The liquid fermentation medium consisted of: 1% (w / v) glucose, 1.2% (w / v) tryptone, 2.4% (w / v) yeast extract, 0.22% (w / v) potassium dihydrogen phosphate, 0.94% (w / v) dipotassium hydrogen phosphate, 1 mM IPTG, and 0.1% (w / v) threonine, with the pH adjusted to 7.2.

[0024] When fermentation broth OD 600 When the concentration reaches 0.6, add 1 mM IPTG for induction. The volume of liquid in the shake flask is 50 mL / 250 mL, and the shaking speed is 200 rpm.

[0025] Example 2: Analysis of extracellular 2,5-dimethylpyrazine fermentation yield by engineered strains With recombinant plasmid pRSFDuet-1- tdh Using Escherichia coli BL21(DE3) as the control group, liquid fermentation experiments were conducted on engineered strains EC-2 and EC-3.

[0026] The content of 2,5-dimethylpyrazine in the fermentation broth was determined by Fuli LC5190 low-pressure ultra-high performance liquid chromatography. The detection conditions were: UV detector wavelength 275 nm, column temperature 40℃, injection volume 10 μL, mobile phase 50 mM sodium acetate solution and methanol (1:1), and column POSITISIL ODS-P (10×250 mm).

[0027] Example 3: Effect of exogenous addition of 2,5-dimethylpyrazine on cell growth Experimental results are as follows Figure 4 As shown. Figure 4 (a) shows that the growth of both strains was inhibited with increasing concentration of 2,5-dimethylpyrazine in the culture medium; however, the expression of... phaCBA The engineered strains maintained high OD values ​​under all concentration conditions. 600 Especially under conditions of 1.0% (v / v) or higher 2,5-dimethylpyrazine, the experimental group still showed significantly better growth ability than the control group, indicating that the PHB biosynthetic pathway can improve the tolerance of engineered strains to 2,5-dimethylpyrazine.

[0028] Figure 4 (b) Further observation using transmission electron microscopy revealed that the expression phaCBA The engineered strain formed a large number of spherical PHB particles intracellularly, indicating that the constructed PHB biosynthetic pathway can effectively synthesize and accumulate PHB within the host cell, providing a material basis for improving the tolerance of the engineered strain. These results demonstrate that the PHB biosynthetic pathway not only forms PHB particles intracellularly in the engineered strain but also enhances the strain's tolerance to 2,5-dimethylpyrazine, providing favorable conditions for increasing the fermentation yield of 2,5-dimethylpyrazine.

[0029] Example 4: Comparison of fermentation yields of engineered strains Following the culture conditions described in Example 1, engineered strains EC-1, EC-2, and EC-3 were subjected to shake-flask fermentation. After fermentation, the content of 2,5-dimethylpyrazine in the fermentation broth of each strain was determined according to the method described in Example 2, and the highest yield achieved by each strain was compared.

[0030] Experimental results are as follows Figure 5 As shown. Only expresses... tdh The engineered strain EC-1 is capable of synthesizing 2,5-dimethylpyrazine; based on this, co-expression tdh and nox The engineered strain EC-2 further improved the yield of 2,5-dimethylpyrazine; and a PHB biosynthesis module was further introduced. phaCBA The engineered strain EC-3 (344.36 mg / L) showed a significantly higher yield of 2,5-dimethylpyrazine compared to EC-1 (7.98 mg / L) and EC-2 (24.81 mg / L).

[0031] The above results indicate that, tdh and noxBased on the expression, the introduction of the PHB biosynthesis module can further promote the biosynthesis of 2,5-dimethylpyrazine and improve the fermentation production capacity of the engineered strain, indicating that the PHB biosynthesis pathway provided by the present invention has the effect of increasing the fermentation yield of 2,5-dimethylpyrazine.

[0032] Example 5: Optimization of Carbon Source and Inducer Replacement The fermentation medium was optimized according to the culture conditions described in Example 1. Glucose (10 g / L) was replaced with glycerol (5 g / L), the inducer was replaced with lactose (2 g / L) instead of IPTG, and the threonine concentration was increased to 20 g / L, while other culture conditions remained unchanged. After fermentation, the 2,5-dimethylpyrazine content in the fermentation broth was determined according to the method described in Example 2. The experimental results are as follows: Figure 6 As shown, after using lactose induction and optimizing the carbon source, the yields of 2,5-dimethylpyrazine from both engineered strains EC-2 and EC-3 were further improved, with EC-2 reaching a yield of 1489.18 mg / L and EC-3 reaching a yield of 2097.68 mg / L. This further demonstrates that the PHB biosynthesis module constructed in this invention, combined with the optimized fermentation process, can significantly improve the fermentation production level of 2,5-dimethylpyrazine.

[0033] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for increasing the yield of 2,5-dimethylpyrazine by expressing key genes in PHB biosynthesis, characterized in that: Includes the following steps: (1) An expression vector containing a polyhydroxybutyric acid synthesis module encoding gene and an expression vector containing a threonine dehydrogenase gene and a NADH oxidase gene were introduced into a host cell to obtain engineered bacteria; (2) The engineered bacteria are fermented and cultured, and lactose is added to induce fermentation.

2. The method for increasing the yield of 2,5-dimethylpyrazine by expressing key genes in PHB biosynthesis according to claim 1, characterized in that: The polyhydroxybutyric acid (PHB) synthesis module encoding gene in step (1) includes phaA Gene, phaB Genes and phaC Gene.

3. The method for increasing the yield of 2,5-dimethylpyrazine by expressing key genes in PHB biosynthesis according to claim 1, characterized in that: The engineered bacteria in step (1) is Escherichia coli BL21.

4. The method for increasing the yield of 2,5-dimethylpyrazine by expressing key genes in PHB biosynthesis according to claim 1, characterized in that: The fermentation step in step (2) uses a liquid culture medium.

5. The method for increasing the yield of 2,5-dimethylpyrazine by expressing key genes in PHB biosynthesis according to claim 4, characterized in that: The liquid culture medium comprises the following raw materials in parts by weight: 0.4-2 parts glycerol, 0.5-2 parts tryptone, 0.5-4 parts yeast extract, 0.01-0.3 parts potassium dihydrogen phosphate, 0.1-1.5 parts dipotassium hydrogen phosphate, 0.01-2 parts lactose, and 0.5-3 parts threonine.

6. The method for increasing the yield of 2,5-dimethylpyrazine by expressing key genes in PHB biosynthesis according to claim 1, characterized in that: The lactose in step (2) mentioned above has an OD value of 600 When the concentration is 0.4-0.8, the lactose is added, and the final concentration of the lactose is 0.05-0.3 w / v.

7. An engineered bacterium, characterized in that: The engineered bacteria contain expression phaA Gene, phaB Genes and phaC Recombinant expression vectors and expression of genes tdh Genes and nox Recombinant gene expression vectors.

8. The application of the engineered bacteria according to claim 7, characterized in that: The application of the engineered bacteria in the production of 2,5-dimethylpyrazine.

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