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Method for increasing yield of L-histidine

A histidine and amino acid technology, applied in the field of metabolic engineering, can solve problems such as difficulty in consumer acceptance and approval

Active Publication Date: 2021-05-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Wherein hydrolyzing protein is the most traditional method of histidine production, but this method mainly depends on the availability of resources rich in natural protein (such as blood meal or soybean), which is difficult to meet the increasing demand of people for histidine; and The production of histidine by chemical synthesis tends to produce racemic mixtures, which are generally considered "unnatural" compounds, which are difficult to obtain approval from the Food and Drug Administration and are difficult to be accepted by consumers; microbial fermentation is the current method for the production of L -The mainstream method of histidine, so the breeding of L-histidine high-yielding bacteria has become a current research hotspot

Method used

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  • Method for increasing yield of L-histidine
  • Method for increasing yield of L-histidine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Determination of the critical lethal 6-MP concentration of S. marcescens ATCC 31026

[0033] Inoculate a single colony of S.marcescens ATCC 31026 before mutagenesis into 2mL LB medium (yeast powder 5g / L, tryptone 10g / L, NaCl 10g / L), culture at 37°C, 220r / min, for 10-12h, Centrifuge the bacteria, wash 2-3 times with sterile normal saline, then dilute 100,000-fold with sterile normal saline, and then take 100 μL of the bacteria solution and spread them respectively to 1.5 mg / mL MET and different concentrations of 6-MP Resistance plate (glucose 5.0g / L, beef extract 10g / L, peptone 10g / L, yeast extract 5.0g / L, sodium chloride 2.5g / L, agar 20g / L, pH adjusted to 7.0, extinguished at 121°C Bacteria 15min), the critical lethal concentration of 6-MP was determined to be 1.5mg / mL, so 1.5mg / mL was selected as the experimental concentration. As shown in table 2.

[0034] Table 2

[0035]

[0036] Note: ++ means more single colonies; + means a few single colonies; - ...

Embodiment 2

[0037] Example 2: Mutagenesis of S. marcescens ATCC 31026 by ARTP

[0038] Select S.marcescens ATCC 31026 for ARTP mutagenesis. Firstly, prepare the bacterial suspension, put the single colony of S.marcescensATCC 31026 in LB medium into a 14mL shaking tube, 37°C, 220r / min, after overnight culture, use 1% inoculum Transfer to LB shaker flask, 37°C, 220r / min, and cultivate for 4-6h. Collect the cultured bacteria by centrifugation, wash 2-3 times with sterile normal saline, and then dilute to OD with sterile normal saline. 600 For the bacterial suspension with a value of 0.6-0.8, take 10 μL of the bacterial suspension and apply it on a glass slide for processing. The parameters of the ARTP mutagenesis treatment were as follows: the slide was positioned 2mm from the airflow port, the power was 120W, the airflow was 10SLM, and the action time was 35s.

[0039] Spread the bacterial suspension treated with ARTP mutagenesis on MET (1.5 mg / mL) and 6-MP (1.5 mg / mL) resistant plates, and...

Embodiment 3

[0040] Embodiment 3: Fermentative synthesis of L-histidine

[0041] Pick a single colony of the thirty mutagenized strains of S.marcescens P1--30 obtained and inoculate them into a 500mL Erlenmeyer flask with a liquid volume of 500mL, 30°C, 220r / min, and cultivate for 18h. The formulation of the seed medium For: glucose 25g / L, corn steep liquor 20g / L, urea 1.25g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 0.5g / L, pH adjusted to 7.0, sterilized at 121°C for 15min, glucose was sterilized separately Bacteria (115°C, 15min); draw 1.5mL of seed culture medium with 10% inoculum volume into a 250mL Erlenmeyer flask, fill liquid volume 15mL, 30°C, 220r / min, cultivate for 72h, the formula of the fermentation medium is: glucose 130g / L, ammonium sulfate 35g / L, corn steep liquor 15g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 0.5g / L, calcium carbonate 20g / L (disinfection), pH adjusted to 7.0, extinguished at 115°C Bacteria, 15min.

[0042] Liquid phase an...

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Abstract

The invention relates to a method for increasing the yield of L-histidine. By using an ARTP mutagenesis method which is safe and mild in operation condition and high in controllability, a mutation library with the higher mutation rate is obtained; in combination with L-histidine structural analogue screening, a stable L-histidine high-yield mutant strain is obtained through ten times of passage; the mutation condition of L-histidine synthesis related genes of the mutagenic high-yield strain is further analyzed; 5-phosphoribose-1-pyrophosphate synthesis related gene Prs and ATP phosphoribosyl transferase encoding gene hisG mutants which can promote the increase of the yield of the L-histidine are obtained through comparison and screening; and one or two of the genes Prs and hisG are expressed in host bacteria for producing the L-histidine, so that the yield of the L-histidine can be effectively increased. A foundation is laid for producing the L-histidine by further transforming serratia marcescens or other strains in metabolic engineering.

Description

technical field [0001] The invention relates to the technical field of metabolic engineering, in particular to a method for increasing the production of L-histidine. Background technique [0002] L-histidine, also known as α-amino-β-imidazolylpropionic acid, is a basic amino acid with an imidazole nucleus in its molecule. In the field of nutrition, histidine is considered an essential amino acid for infants and young children. L-histidine has a variety of physiological functions, and plays an important role in growth, tissue repair, and the treatment of ulcers and hyperacidity. It can also be used as an additive to treat diseases such as allergies, rheumatoid arthritis, and anemia. Therefore, It is widely used in industries such as medicine and food. [0003] The methods for producing histidine mainly include proteolysis, chemical synthesis and microbial fermentation. Wherein hydrolyzing protein is the most traditional method of histidine production, but this method mainl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N9/12C12N9/10C12N15/70C12N15/74C12P13/24C12R1/19C12R1/43
CPCC12N9/1235C12N9/1077C12N15/70C12N15/74C12P13/24C12Y207/06001C12Y204/02017
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰李梦莹
Owner JIANGNAN UNIV
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