阅读说明：本技术 二氢硫辛酸脱氢酶突变体P213R及其在地衣芽胞杆菌的聚γ-谷氨酸合成中的应用 (Dihydrolipoic dehydrogenase mutant P213R and application thereof in poly-gamma-glutamic acid synthesis of bacillus licheniformis ) 是由 陈守文 张蒙 蔡冬波 杨帆 陈耀中 张清 马昕 于 2019-12-05 设计创作，主要内容包括：本发明属于基因工程和酶工程技术领域,公开了二氢硫辛酸脱氢酶突变体P213R在地衣芽胞杆菌聚γ-谷氨酸合成中的应用。本发明通过基因组上定点突变的方式,将来源于地衣芽胞杆菌WX-02(<I>Baclicus lincheniformis</I> WX-02)二氢硫辛酸脱氢酶PdhD的第213位脯氨酸编码的碱基CCA改为CGG,即213位由脯氨酸改成精氨酸,显著提高了聚γ-谷氨酸合成水平,突变菌株聚γ-谷氨酸产量相较于对照菌株至少提高了21%以上。本发明为聚γ-谷氨酸的高效生产提供了一种新策略。(The invention belongs to the technical field of genetic engineering and enzyme engineering, and discloses application of a dihydrolipoic dehydrogenase mutant P213R in synthesis of bacillus licheniformis poly gamma-glutamic acid. The invention uses the mode of site-directed mutagenesis on genome to lead the bacillus licheniformis WX-02 (B) Baclicus lincheniformis WX-02) dihydrolipoic dehydrogenase PdhD, wherein the base CCA coded by the 213 th proline of PdhD is changed into CGG, namely the 213 th proline is changed into arginine, so that the synthesis level of poly-gamma-glutamic acid is obviously improved, and the yield of the mutant strain poly-gamma-glutamic acid is compared with that of the mutant strain poly-gamma-glutamic acidThe control strain is at least improved by more than 21 percent. The invention provides a new strategy for the efficient production of poly-gamma-glutamic acid.)

1. The amino acid sequence of the mutant is shown in SEQ ID NO. 1.

2. A nucleotide sequence encoding the mutant of claim 1.

3. The nucleotide sequence of claim 2, which is represented by SEQ ID NO. 2.

4. Use of a mutant as claimed in claim 1 or a nucleotide sequence as claimed in claim 2 in the fermentative production of poly-gamma-glutamic acid using bacillus licheniformis.

5. The use of claim 4, wherein the Bacillus licheniformis is Bacillus licheniformis (Bnfillus licheniformis)Bacilluslicheniformis) WX-02。

6. The use according to claim 4, wherein the fermentation medium used is formulated as follows:

30-90g/L glucose, 0-30 g/L sodium glutamate, 0-10 g/L sodium citrate and NaNO₃0~10 g/L，NH₄Cl0-10g/L，K₂HPO₄·3H₂O 0.5-1 g/L，MgSO₄·7H₂O 0.5-1 g/L，ZnSO₄·7H₂O 0-1 g/L，MnSO₄·H₂O 0-0.15 g/L，CaCl₂0-1 g/L; the sodium glutamate, the sodium citrate and the NaNO₃、NH₄Cl、ZnSO₄·7H₂O、MnSO₄·H₂O and CaCl₂In each group of formulas, at most one component is zero at a time;

or 20-60g/L of glycerin, 25-35g/L of sodium glutamate, 8-12g/L of sodium citrate and NaNO₃8-12 g/L，NH₄Cl 8-12 g/L，K₂HPO₄·3H₂O 0.7-1.2 g/L，MgSO₄·7H₂O 0.8-1.1 g/L，ZnSO₄·7H₂O 0.8-1.2 g/L，MnSO₄·H₂O 0.1-0.25 g/L，CaCl₂0.7-1.3g/L。

7. Use according to claim 5, characterized in that: 30-90g/L of glucose, 0-30 g/L of sodium glutamate, 9-10g/L of sodium citrate and NaNO₃9-10g/L，NH₄Cl 9-10g/L，K₂HPO₄·3H₂O 0.9-1 g/L，MgSO₄·7H₂O0.9-1g/L，ZnSO₄·7H₂O 0.9-1 g/L，MnSO₄·H₂O 0.12-0.15 g/L，CaCl₂0.9-1 g/L;

Or

20-40g/L of glycerin, 25-35g/L of sodium glutamate, 8-12g/L of sodium citrate and NaNO₃8-12 g/L，NH₄Cl 8-12g/L，K₂HPO₄·3H₂O 0.7-1.2 g/L，MgSO₄·7H₂O 0.8-1.1 g/L，ZnSO₄·7H₂O 0.8-1.2 g/L，MnSO₄·H₂O 0.1-0.25 g/L，CaCl₂0.7-1.3g/L。

Technical Field

The invention belongs to the technical field of enzyme engineering and genetic engineering, and particularly relates to a dihydrolipoic dehydrogenase mutant P213R and application thereof in the synthesis of poly-gamma-glutamic acid of bacillus licheniformis.

Background

The poly-gamma-glutamic acid (gamma-PGA) is an anionic biopolymer, the acting force of intermolecular hydrogen bonds is provided by carboxyl, the poly-gamma-glutamic acid (gamma-PGA) is a good environment-friendly polymer material, has excellent properties of adsorptivity, water solubility, biodegradability and the like, can be used as a water-retaining agent, a heavy metal ion adsorbent, a flocculating agent, a slow release agent of pesticide and fertilizer and the like, and has great application value in industries of food processing, environmental management, cosmetics, medical treatment, agriculture and the like.

Currently, the commercial production of poly-gamma-glutamic acid mainly depends on a microbial fermentation method, but the conversion rate of glucose into poly-gamma-glutamic acid is low due to the addition of poly-gamma-glutamic acid synthesis precursors and excessive fermentation byproducts. From the current reports, the commercial production strains of poly-gamma-glutamic acid are almost completely dependent on bacillus, such as bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis and the like.

The dihydrolipoic dehydrogenase PdhD is a subunit of the pyruvate dehydrogenase complex, and the pyruvate dehydrogenase is a key enzyme catalyzing the reaction from pyruvate to acetyl CoA. At present, the expression level and the relation between the expression activity of pyruvate dehydrogenase and dihydrolipoic dehydrogenase and the synthesis level of poly-gamma-glutamic acid are not researched, and whether the synthesis level of the poly-gamma-glutamic acid can be improved by carrying out site-specific modification on the dihydrolipoic dehydrogenase is unpredictable. The research improves the synthesis level of poly-gamma-glutamic acid by carrying out fixed-point modification on the dihydrolipoic acid dehydrogenase. The invention provides a technical strategy for obviously synthesizing poly-gamma-glutamic acid, and provides theoretical guidance and technical support for realizing the industrial production of gamma-PGA.

Disclosure of Invention

The invention aims to provide a dihydrolipoic dehydrogenase PdhD mutant P213R, wherein the amino acid sequence of the dihydrolipoic dehydrogenase mutant P213R is shown in SEQ ID NO. 1.

Another object of the present invention is to provide the use of poly-gamma-glutamic acid in the synthesis of Bacillus licheniformis.

In order to achieve the purpose, the invention adopts the following technical measures:

the amino acid sequence of the dihydrolipoic dehydrogenase PdhD mutant P213R is shown in SEQ ID NO. 1.

The application of the dihydrolipoic dehydrogenase mutant P213R in the synthesis of poly-gamma-glutamic acid of Bacillus licheniformis comprises changing the 213 th position of the dihydrolipoic dehydrogenase PdhD in the Bacillus licheniformis from proline to arginine, and then fermenting and producing the poly-gamma-glutamic acid by using the obtained recombinant strain, wherein the amino acid sequence of the mutant P213R is shown in SEQ ID NO. 1.

In the above-mentioned application, preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO. 2.

In the above application, preferably, the bacillus licheniformis is bacillus licheniformis capable of producing poly-gamma-glutamic acid.

In the above application, preferably, the bacillus licheniformis is bacillus licheniformis (bacillus licheniformis) WX-02.

In the above application, in the application process, the formula of the fermentation medium used in the fermentation is as follows:

30-90g/L glucose, 0-30 g/L sodium glutamate, 0-10 g/L sodium citrate and NaNO₃0～10g/L，NH₄Cl0-10g/L，K₂HPO₄·3H₂O 0.5-1g/L，MgSO₄·7H₂O 0.5-1g/L，ZnSO₄·7H₂O 0-1g/L，MnSO₄·H₂O 0-0.15g/L，CaCl₂0-1 g/L; the sodium glutamate, the sodium citrate and the NaNO₃、NH₄Cl、ZnSO₄·7H₂O、MnSO₄·H₂O and CaCl₂In each set of formulations, at most one component at a time is zero.

Or 20-60g/L of glycerin, 25-35g/L of sodium glutamate, 8-12g/L of sodium citrate and NaNO₃8-12g/L，NH₄Cl 8-12g/L，K₂HPO₄·3H₂O 0.7-1.2g/L，MgSO₄·7H₂O 0.8-1.1g/L，ZnSO₄·7H₂O 0.8-1.2g/ L，MnSO₄·H₂O 0.1-0.25g/L，CaCl₂0.7-1.3g/L。

In the above application, the formulation of the fermentation medium is preferably:

30-90g/L of glucose, 0-30 g/L of sodium glutamate, 9-10g/L of sodium citrate and NaNO₃9-10g/L，NH₄Cl 9-10g/L，K₂HPO₄·3H₂O 0.9-1g/L，MgSO₄·7H₂O 0.9-1g/L，ZnSO₄·7H₂O 0.9-1g/L，MnS O₄·H₂O 0.12-0.15g/L，CaCl₂0.9-1g/L。

Or

20-40g/L of glycerin, 25-35g/L of sodium glutamate, 8-12g/L of sodium citrate and NaNO₃8-12g/L，NH₄Cl 8 -12g/L，K₂HPO₄·3H₂O 0.7-1.2g/L，MgSO₄·7H₂O 0.8-1.1g/L，ZnSO₄·7H₂O 0.8-1.2g/L，MnSO₄·H₂O 0.1-0.25g/L，CaCl₂0.7-1.3g/L。

Compared with the prior art, the invention has the following advantages:

the invention changes the base CCA coded by the 213 th proline of the dihydrolipoic dehydrogenase PdhD into the base CGG coded by arginine by a genome site-directed mutagenesis strategy to obtain the dihydrolipoic dehydrogenase PdhD mutant P213R, and the enzyme obviously improves the synthesis level of poly-gamma-glutamic acid in the synthesis application of the poly-gamma-glutamic acid, and compared with a control strain, the synthesis level of the poly-gamma-glutamic acid is obviously improved.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof. The technical scheme of the invention is a conventional scheme in the field if not specifically stated; the reagents or materials, if not specifically mentioned, are commercially available.

Test materials and reagents

1. The strain is Bacillus licheniformis (Bacillus licheniformis) WX-02 with the preservation number of CCTCC NO. M208065, and the strain E.coli DH5 α is purchased from Beijing all-purpose gold biotechnology limited.

2. Enzymes and other biochemical reagents: high fidelity Taq enzyme was purchased from Wuhan Pongziaceae Biotechnology Ltd. The bacterial genome DNA extraction kit is purchased from Tiangen company, molecular biological reagents such as T4DNA ligase, restriction endonuclease and the like are purchased from Nanjing Novophilia Biotech Co., Ltd, and other reagents are all made in China (all can be purchased from common biochemical reagents).

3. Culture medium:

the LB culture medium formula is: 10g/L tryptone, 5g/L yeast powder, 10g/L sodium chloride, pH 7.0-7.2, sterilizing at 121 ℃ for 20min and using.