Method for improving pantoic acid production capacity of engineering strain

Abstract

The invention discloses a method for improving pantoic acid production capacity of an engineering strain. The invention provides a method for improving the pantoic acid yield of pantoic acid engineering bacteria. The method comprises the following step: adding amino acid into a fermentation system. Specifically, the amino acids are serine, alanine and glycine. By adding amino acid, the pantoic acid yield of the pantoic acid engineering bacteria is greatly increased, and the method has great application and popularization value for industrial production of pantoic acid and downstream products thereof.

Description

Technical Field

[0001] The present invention belongs to the field of microbial fermentation and relates to a method for improving the production capacity of pantothenic acid by engineering strains. Background Art

[0002] The rapid development of synthetic biology and the social demand for green and environment-friendly economic development have led to great attention and rapid development of related research on the production of bulk chemicals, fine chemicals, natural products, etc. by metabolically engineering microorganisms in recent years. At present, a variety of compounds, such as amino acids, vitamins, and organic acids, have been successfully produced by biological manufacturing. With the rapid development of synthetic biology, biological manufacturing through microbial fermentation has become an important foundation for replacing petrochemical-based manufacturing.

[0003] Fermentation is one of the most critical links in biological manufacturing, in addition to the creation of engineering strains, and is also an essential step to achieve the production of target compounds by engineering strains. The fermentation process directly determines whether the production potential of engineering strains can be exerted and is also a test standard for the production capacity of engineering strains. Therefore, the research on fermentation technology plays a crucial role in the realization of biological manufacturing.

[0004] Vitamin B5, also known as calcium pantothenate, as one of the 13 essential vitamins for the human body, can only be synthesized in microorganisms and plants. Vitamin B5 is also an important precursor for the synthesis of coenzyme A and an essential vitamin for maintaining normal physiological functions of organisms. At the same time, vitamin B5 also has a wide range of applications as a food additive and pharmaceutical raw material, etc. Currently, vitamin B5 is mainly obtained by reacting high-purity D-pantolactone obtained by biochemically resolving DL-pantolactone with β-alanine. The process of preparing pantolactone by petrochemical technology involves highly toxic or strong acid / alkali reagents, and a large amount of wastewater / gas is generated during the production process, so it has a huge impact on the environment. In addition, the high cost of chiral resolution of DL-pantolactone, etc., makes pantothenic acid calcium synthesis enterprises often subject to environmental inspections, and the price of pantothenic acid calcium ranges from tens of thousands to hundreds of thousands of yuan per ton. D-pantothenic acid, as a key intermediate for the synthesis of vitamin B5, its biological manufacturing is the key factor restricting the full biological synthesis of vitamin B5. Achieving the synthesis of pantothenic acid with low cost, high chiral purity, and low pollution through biological manufacturing will play a great role in solving problems such as heavy pollution and expensive raw materials in the manufacturing process of vitamin B5. Therefore, providing an efficient biological fermentation process for pantothenic acid is crucial for the industrialization of biological manufacturing of pantothenic acid and vitamin B5. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for improving the production capacity of pantothenic acid by engineering strains.

[0006] The present invention provides a method for increasing the pantothenic acid yield of pantothenic acid engineering bacteria, comprising the following steps: adding an amino acid to a fermentation system.

[0007] Specifically, the amino acid is serine.

[0008] Specifically, the amino acid is alanine.

[0009] Specifically, the amino acids are serine and alanine.

[0010] Specifically, the amino acid is glycine.

[0011] Specifically, the amino acids are serine and glycine.

[0012] Exemplarily, the fermentation system consists of pantothenic acid engineering bacteria and a fermentation medium.

[0013] As an example, the composition of the fermentation medium is: containing 50 g / L of glucose, 3.5 g / L of (NH4)2HPO4, 3.91 g / L of KH2PO4, 4.48 g / L of K2HPO4, 0.18 g / L of MgSO4·7H2O, 0.15 g / L of betaine-HCl, 1.5 μg / L of FeCl3·6H2O, 0.1 μg / L of CoCl2·6H2O, 0.1 μg / L of CuCl2·2H2O, 0.1 μg / L of ZnCl2, 0.1 μg / L of Na2MoO4·2H2O, 0.2 μg / L of MnCl2·4H2O, 0.05 μg / L of H3BO3, and the balance is water.

[0014] As an example, the initial OD 550nm value of the fermentation system is 0.05 - 0.5.

[0015] As an example, the initial OD 550nm value of the fermentation system is 0.1.

[0016] As an example, after adding the amino acid to the fermentation system, the culture is carried out under the following conditions: culturing at 37 °C with shaking at 250 rpm for 72 hours.

[0017] The present invention also protects the application of an amino acid in increasing the pantothenic acid yield of pantothenic acid engineering bacteria.

[0018] In the said application, the amino acid is added to the fermentation system of the pantothenic acid engineering bacteria.

[0019] Specifically, the amino acid is serine.

[0020] Specifically, the amino acid is alanine.

[0021] Specifically, the amino acids are serine and alanine.

[0022] Specifically, the amino acid is glycine.

[0023] Specifically, the amino acids are serine and glycine.

[0024] Exemplarily, the fermentation system consists of a pantothenic acid engineering bacterium and a fermentation medium.

[0025] As an example, the composition of the fermentation medium is as follows: containing 50 g / L of glucose, 3.5 g / L of (NH4)2HPO4, 3.91 g / L of KH2PO4, 4.48 g / L of K2HPO4, 0.18 g / L of MgSO4·7H2O, 0.15 g / L of betaine-HCl, 1.5 μg / L of FeCl3·6H2O, 0.1 μg / L of CoCl2·6H2O, 0.1 μg / L of CuCl2·2H2O, 0.1 μg / L of ZnCl2, 0.1 μg / L of Na2MoO4·2H2O, 0.2 μg / L of MnCl2·4H2O, 0.05 μg / L of H3BO3, and the balance is water.

[0026] As an example, the initial OD 550nm value of the fermentation system is 0.05 - 0.5.

[0027] As an example, the initial OD 550nm value of the fermentation system is 0.1.

[0028] As an example, after adding amino acids to the fermentation system, it is cultured under the following conditions: 37°C, shaking culture at 250 rpm for 72 hours.

[0029] In the said application, the usage concentration of serine is 0.1 - 50 g / L.

[0030] In the said application, the usage concentration of serine is 1 - 25 g / L.

[0031] In the said application, the usage concentration of serine is 5 - 15 g / L.

[0032] In the said application, the usage concentration of serine is 8 - 12 g / L.

[0033] In the said application, the usage concentration of serine is 10 g / L.

[0034] In the said application, the usage concentration of alanine is 0.1 - 10 g / L.

[0035] In the said application, the usage concentration of alanine is 0.5 - 5 g / L.

[0036] In the said application, the usage concentration of alanine is 1 - 3 g / L.

[0037] In the said application, the usage concentration of alanine is 2 g / L.

[0038] In the said application, the usage concentration of glycine is 0.1 - 10 g / L.

[0039] In the said application, the usage concentration of glycine is 0.5 - 5 g / L.

[0040] In the said application, the usage concentration of glycine is 1 - 3 g / L.

[0041] In the said application, the usage concentration of glycine is 2 g / L.

[0042] Specifically, the concentration of serine in the fermentation system is 0.1 - 50 g / L.

[0043] Specifically, the concentration of serine in the fermentation system is 1 - 25 g / L.

[0044] Specifically, the concentration of serine in the fermentation system is 5 - 15 g / L.

[0045] Specifically, the concentration of serine in the fermentation system is 8 - 12 g / L.

[0046] Specifically, the concentration of serine in the fermentation system is 10 g / L.

[0047] Specifically, the concentration of alanine in the fermentation system is 0.1 - 10 g / L.

[0048] Specifically, the concentration of alanine in the fermentation system is 0.5 - 5 g / L.

[0049] Specifically, the concentration of alanine in the fermentation system is 1 - 3 g / L.

[0050] Specifically, the concentration of alanine in the fermentation system is 2 g / L.

[0051] Specifically, the concentration of glycine in the fermentation system is 0.1 - 10 g / L.

[0052] Specifically, the concentration of glycine in the fermentation system is 0.5 - 5 g / L.

[0053] Specifically, the concentration of glycine in the fermentation system is 1 - 3 g / L.

[0054] Specifically, the concentration of glycine in the fermentation system is 2 g / L.

[0055] Specifically, any of the above-mentioned amino acids is an L - amino acid.

[0056] Specifically, the pantolactone engineering bacterium is Escherichia coli with the ability to produce pantolactone.

[0057] Specifically, the pantolactone engineering bacterium is Escherichia coli with the ability to produce pantolactone using glucose as a substrate.

[0058] Specifically, the pantolactone engineering bacterium is recombinant Escherichia coli with the ability to produce pantolactone.

[0059] Specifically, the pantolactone engineering bacterium is recombinant Escherichia coli with the ability to produce pantolactone using glucose as a substrate.

[0060] Exemplarily, the pantolactone engineering bacterium is Escherichia coli Span050.

[0061] Escherichia coli Span050, with a preservation registration number of CGMCC No. 21699.

[0062] The present invention has achieved a substantial increase in the pantolactone yield of the pantolactone engineering bacterium by adding amino acids, and has great application and promotion value for the industrial production of pantolactone and its downstream products. Detailed Embodiments

[0063] The present invention will be further described in detail below in conjunction with the detailed embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0064] The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels. Unless otherwise specified, the quantitative tests in the following embodiments are all set with three repeated experiments, and the results are averaged. Unless otherwise specified, the pH of the culture medium in the embodiments is the natural pH. In the embodiments, the amino acids used are all L-amino acids.

[0065] Escherichia coli Span050 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on January 22, 2021, with the deposit registration number CGMCC No. 21699. Escherichia coli Span050 is described in the patent application with the application number 202110391896.0 (publication number CN 115109736 A).

[0066] Example 1

[0067] I. Preparation of culture media

[0068] Seed culture medium: containing 20 g / L of glucose, 3.5 g / L of (NH4)2HPO4, 3.91 g / L of KH2PO4, 4.48 g / L of K2HPO4, 0.18 g / L of MgSO4·7H2O, 0.15 g / L of betaine-HCl, 1.5 μg / L of FeCl3·6H2O, 0.1 μg / L of CoCl2·6H2O, 0.1 μg / L of CuCl2·2H2O, 0.1 μg / L of ZnCl2, 0.1 μg / L of Na2MoO4·2H2O, 0.2 μg / L of MnCl2·4H2O, 0.05 μg / L of H3BO3, and the balance being water.

[0069] Fermentation culture medium 1: containing 50 g / L of glucose, 3.5 g / L of (NH4)2HPO4, 3.91 g / L of KH2PO4, 4.48 g / L of K2HPO4, 0.18 g / L of MgSO4·7H2O, 0.15 g / L of betaine-HCl, 1.5 μg / L of FeCl3·6H2O, 0.1 μg / L of CoCl2·6H2O, 0.1 μg / L of CuCl2·2H2O, 0.1 μg / L of ZnCl2, 0.1 μg / L of Na2MoO4·2H2O, 0.2 μg / L of MnCl2·4H2O, 0.05 μg / L of H3BO3, and the balance being water.

[0070] Fermentation culture medium 2: differing from fermentation culture medium 1 only in that serine is added, and the concentration of serine in the culture medium is 10 g / L.

[0071] II. Cultivation and fermentation

[0072] Seed culture: Inoculate Escherichia coli Span050 into 4 ml of the seed medium prepared in Step 1, and culture it overnight at 37 °C with shaking at 250 rpm; then, transfer it to 30 ml of the seed medium prepared in Step 1 according to an inoculation amount of 2% (volume ratio), and culture it at 37 °C with shaking at 250 rpm for 12 hours to obtain a seed solution.

[0073] Fermentation culture: Inoculate the seed solution obtained from the seed culture into a 250 ml Erlenmeyer flask containing 25 ml of the fermentation medium (the OD value of the initial system is 0.1), and culture it at 37 °C with shaking at 250 rpm for 72 hours to obtain a fermentation broth. The fermentation medium is respectively the fermentation medium 1 or fermentation medium 2 prepared in Step 1. 550nm Value is 0.1), culture it at 37 °C with shaking at 250 rpm for 72 hours to obtain a fermentation broth. The fermentation medium is respectively the fermentation medium 1 or fermentation medium 2 prepared in Step 1.

[0074] Analysis and detection: Centrifuge the fermentation broth obtained from the fermentation culture and collect the supernatant, then filter and collect the filtrate; then, analyze the filtrate using an Agilent (Agilent-1260) high performance liquid chromatography instrument, and the pantothenic acid concentration in the fermentation broth is detected and calculated using an Aminex HPX–87H organic acid analysis column from Biorad company.

[0075] The results are shown in Table 1. Adding serine can significantly improve the production capacity of the pantothenic acid engineering strain. In the fermentation broth with added serine, under the same fermentation conditions, the pantothenic acid yield can be increased by 4.36 times.

[0076] Example 2

[0077] I. Preparation of the medium

[0078] Seed medium: The same as the seed medium in Step 1 of Example 1.

[0079] Fermentation medium 3-1: The difference from the fermentation medium 2 in Step 1 of Example 1 is only that threonine is added, and the concentration of threonine in the medium is 2 g / L.

[0080] Fermentation medium 3-2: The difference from the fermentation medium 2 in Step 1 of Example 1 is only that leucine is added, and the concentration of leucine in the medium is 2 g / L.

[0081] Fermentation medium 3-3: The difference from the fermentation medium 2 in Step 1 of Example 1 is only that isoleucine is added, and the concentration of isoleucine in the medium is 2 g / L.

[0082] Fermentation medium 3-4: The difference from the fermentation medium 2 in Step 1 of Example 1 is only that glycine is added, and the concentration of glycine in the medium is 2 g / L.

[0083] Fermentation medium 3-5: It is only different from the fermentation medium 2 in Step 1 of Example 1 in that lysine is added, and the concentration of lysine in the medium is 2 g / L.

[0084] Fermentation medium 3-6: It is only different from the fermentation medium 2 in Step 1 of Example 1 in that alanine is added, and the concentration of alanine in the medium is 2 g / L.

[0085] II. Cultivation and fermentation

[0086] The same as Step 2 of Example 1.

[0087] The fermentation media used are respectively the fermentation medium 3-1, fermentation medium 3-2, fermentation medium 3-3, fermentation medium 3-4, fermentation medium 3-5 or fermentation medium 3-6 prepared in Step 1.

[0088] The results are shown in Table 1. Adding different types of amino acids to the fermentation system will have completely different effects on the production of pantothenic acid by the engineered strain. Among them, the promoting effect of adding alanine is the most significant. The yield of pantothenic acid has increased by 49% compared to the fermentation with only serine added, and the concentration of pantothenic acid has increased by 696% compared to the sample without any amino acid added.

[0089] Table 1

[0090]

[0091]

[0092] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application and are made by using conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A method for increasing the pantothenic acid yield of engineered bacteria producing pantothenic acid, comprising the following steps: adding an amino acid to a fermentation system.

2. The method according to claim 1, wherein: The amino acid is serine.

3. The method according to claim 1, wherein: The amino acid is alanine.

4. The method according to claim 1, characterized in that: The amino acids are serine and alanine.

5. The method according to claim 1, wherein: The amino acid is glycine.

6. The method according to claim 1, characterized in that: The amino acids are serine and glycine.

7. Use of an amino acid in increasing the pantothenic acid yield of engineered bacteria producing pantothenic acid.

8. The application according to claim 7, wherein: The amino acid is serine or alanine or glycine.

9. The application according to claim 7, characterized in that: The amino acids are serine and alanine.

10. The application according to claim 7, wherein: The amino acids are serine and glycine.

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