阅读说明：本技术 一种提高苏氨酸发酵效率的工艺 (Process for improving threonine fermentation efficiency ) 是由 赵春晓 曹博超 张婷婷 罗玮 于 2019-12-04 设计创作，主要内容包括：本发明属于发酵技术领域,公开了一种提高苏氨酸发酵效率的方法,其包括如下步骤：在发酵过程中添加了N-甲基天冬氨酸和甲硫氨酸,提高了苏氨酸的发酵效率,发酵周期缩短,节约了成本。(The invention belongs to the technical field of fermentation, and discloses a method for improving threonine fermentation efficiency, which comprises the following steps: n-methyl aspartic acid and methionine are added in the fermentation process, so that the fermentation efficiency of threonine is improved, the fermentation period is shortened, and the cost is saved.)

1. A method for improving threonine fermentation efficiency, which comprises the following steps: n-methyl aspartic acid and methionine are added in the fermentation process.

2. Method according to claim 1, characterized in that it comprises the following steps:

step 1) preparing a fermentation medium: 40-60g/L of glucose, 20-25g/L of corn steep liquor, 0.5-0.7g/L of monopotassium phosphate, 0.5-0.7g/L of dipotassium phosphate, 0.10-0.15g/L of magnesium sulfate, 50-100mg/L of N-methyl aspartic acid, 20-30mg/L of methionine, 10-15mg/L of ferrous sulfate heptahydrate, 10-15mg/L of manganese sulfate monohydrate, and the pH value is 6.5-7.0;

step 2) preparing threonine by fermentation: inoculating the threonine-producing Brevibacterium flavum seed solution into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 8-10% for fermentation, stopping fermentation when the fermentation time is 48-60h, and collecting the fermentation liquid.

3. The method as claimed in claim 2, wherein the sugar content of the fermentation broth is controlled to 0.5% by feeding a nutrient solution having a glucose concentration of 200-300g/L and the pH of the fermentation broth is controlled to 6.5 by feeding ammonia water throughout the fermentation process.

4. The method of claim 2, wherein the fermentation conditions are: the temperature is 30-32 ℃, the tank pressure is 0.03-0.04MPa, the ventilation volume is 0.5-0.6vvm, and the rotating speed is 50-100 rpm.

5. The method of claim 3, wherein the nutrient solution comprises 3-5g/L N-methylaspartic acid and 1-2g/L methionine.

6. Threonine products prepared according to the process of claims 1-5.

Technical Field

The invention belongs to the technical field of fermentation, and particularly relates to a process for improving threonine fermentation efficiency.

Background

Threonine (Threonine, abbreviated as Thr) has a chemical name of α -amino- β -hydroxybutyric acid, which is a substance first isolated from fibrin hydrolysate in 1935 by w.c. rose, in 1936, Meger found a spatial structure similar to the structure of threose, named Threonine, which has 4 isomers, L-Threonine, D-Threonine, L-allo-Threonine and D-allo-Threonine, L-Threonine being the only naturally occurring substance and having a certain physiological effect.

Threonine plays an increasingly important role in human life as one of amino acids essential to the human body. With the development of the breeding industry and the rapid increase of the livestock and poultry feed demand, threonine plays a role in the nutritional ingredients which must be taken from the outside and is more and more emphasized. Has wide application in medicine, food, feed and other fields. Threonine belongs to one of products of industrial fermentation, and according to data statistics, the global threonine supply in 2017 reaches 68.5 ten thousand tons, the equivalent increase is 15.5 percent, or the global threonine supply increases 9.2 ten thousand tons, and the increase is 80 percent from China. In 2017, the supply of Chinese threonine reaches 53.5 ten thousand tons, and the increase is 15.6 percent on the same scale, which accounts for 78 percent of the global market. China threonine production enterprises in 2017 mainly use plum blossom, Fufeng, Yipin and Chengfu, and supplement Dacheng and Xijie; international enterprises mainly use ajinomoto and ADM. In 2017, the domestic threonine is exported 37.4 ten thousand tons, accounting for 69.9 percent of the yield, and the domestic supply is 16.1 ten thousand tons, and the domestic demand is 13 ten thousand tons.

The production method of L-threonine includes protein hydrolysis, chemical synthesis and microbial fermentation. Because the protein hydrolysis method and the chemical synthesis method have the defects of complex process, low yield, large environmental pollution, high cost and the like, the method is difficult to be applied to industrial production and is basically not used any more. The microbial fermentation method has the characteristics of low production cost and small environmental pollution, and is a main method for industrially producing the L-threonine. Microbial fermentation refers to a process of converting a raw material into a target product through a specific metabolic pathway by using a microorganism under appropriate conditions. The production level of microbial fermentation depends mainly on the genetic characteristics of the species itself and the culture conditions. The strain is reformed by using modern genetic engineering technology, the biosynthesis of byproducts is reduced, and the gene expression of products is improved, so that the yield of the L-threonine is improved. With the development of genetic engineering technology and the increase of information content of industrial microorganisms, particularly the successful construction of an industrial biological vector system, researchers in the Soviet Union before the last 70 th century began to construct threonine engineering bacteria by using the genetic engineering technology, and reliable technical support is provided for screening excellent L-threonine producing bacteria and improving the acid production level of strains.

The microorganisms used for L-threonine fermentation are mainly of the genera Escherichia, Brevibacterium, Corynebacterium, Proteus, and the biosynthetic pathways in different microbial species are approximately the same. The glycolytic pathway (EMP), the tricarboxylic acid cycle (TCA), the pentose phosphate pathway (HMP), the salvage pathway and the phosphotransferase system (PTS) have been reported in literature. The HMP pathway can provide a large amount of NADPH for amino acid synthesis, and has important significance. During the fermentation of L-threonine, glucose synthesizes oxaloacetate through glycolysis, the tricarboxylic acid cycle, and oxaloacetate is an important intermediate product and is an important precursor substance for L-threonine synthesis. Research shows that glyoxylate cycle does not appear in fermentation culture using glucose as a substrate in escherichia coli, which means that TCA cycle is the main oxidation mode in the fermentation process of escherichia coli; meanwhile, phosphoenolpyruvate carboxylase (PPC) -catalyzed reactions are the major anaplerotic reaction of the TCA cycle.

For the research on threonine fermentation culture medium, synthetic pathway and the like, the applicant's prior patent technology has been extensively elucidated, the research on the threonine synthesis mechanism is further carried out, and culture medium, culture conditions and the like are optimized, so as to further improve the fermentation efficiency of threonine and reduce the synthesis of metabolic byproducts.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a process for improving threonine fermentation efficiency. The invention optimizes the culture medium, improves the fermentation efficiency and has wide application prospect by researching the threonine synthesis mechanism.

The invention is realized by the following technical scheme:

a method for improving threonine fermentation efficiency, which comprises the following steps: n-methyl aspartic acid and methionine are added in the fermentation process.

Further, the method comprises the steps of:

step 1) preparing a fermentation medium: 40-60g/L of glucose, 20-25g/L of corn steep liquor, 0.5-0.7g/L of monopotassium phosphate, 0.5-0.7g/L of dipotassium phosphate, 0.10-0.15g/L of magnesium sulfate, 50-100mg/L of N-methyl aspartic acid, 20-30mg/L of methionine, 10-15mg/L of ferrous sulfate heptahydrate, 10-15mg/L of manganese sulfate monohydrate, and the pH value is 6.5-7.0;

step 2) preparing threonine by fermentation: inoculating the threonine-producing Brevibacterium flavum seed solution into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 8-10% for fermentation, stopping fermentation when the fermentation time is 48-60h, and collecting the fermentation liquid.

Further, in the whole fermentation process, the sugar content in the fermentation liquor is controlled to be 0.5% by feeding the nutrient solution with the glucose concentration of 200-300g/L, and the pH value of the fermentation liquor is controlled to be 6.5 by feeding ammonia water.

Further, the fermentation conditions are: the temperature is 30-32 ℃, the tank pressure is 0.03-0.04MPa, the ventilation volume is 0.5-0.6vvm, and the rotating speed is 50-100 rpm.

Furthermore, the nutrient solution contains 3-5g/L of N-methyl aspartic acid and 1-2g/L of methionine.

The invention also claims the threonine product prepared by the above method.

The starting point and the beneficial effects of the research of the invention mainly comprise but are not limited to the following aspects:

in the threonine synthesis mechanism, by increasing the intracellular concentration of methionine to increase the amount of homoserine, which is an intermediate product in the pathway of L-aspartate to threonine synthesis, the accumulation of S-adenosylmethionine, which is a metabolite of methionine, inhibits succinyl homoserine synthase, thereby promoting L-threonine synthesis;

n-methyl aspartic acid can improve methyl for threonine synthesis, and can also be used as an intermediate product for threonine synthesis, thereby improving L-threonine synthesis;

the N-methyl aspartic acid and the methionine carry out dual regulation and control on the threonine synthesis way, the synergistic performance is good, the acid production efficiency is improved, the fermentation time is shortened, and the cost is saved.

Experimental data show that the yield of threonine can be improved by both N-methyl aspartic acid and methionine, and the yield of threonine can be improved by more than 20 percent by the synergistic effect of N-methyl aspartic acid and methionine compared with the yield of threonine without adding N-methyl aspartic acid and methionine.

Drawings

FIG. 1: the influence of N-methyl aspartic acid and methionine on the growth of thallus;

FIG. 2: the effect of N-methylaspartic acid and methionine on threonine production;

FIG. 3: influence of the addition of N-methylaspartic acid in the fermentation medium on the yield of threonine;

FIG. 4: influence of the amount of methionine added to the fermentation medium on the threonine production.

Detailed Description

Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.