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Acid-resistant high-yield epsilon-polylysine mutant strain and application thereof

A technology of Streptomyces and microbial strains, applied in mutant preparation, climate change adaptation, bacteria, etc., can solve problems such as low yield and inability to meet industrial production, achieve high transformation efficiency, good passage stability, and improve positive mutation rate effect

Active Publication Date: 2021-02-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The yield of wild-type ε-polylysine-producing bacteria is generally very low, which cannot meet industrial production

Method used

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  • Acid-resistant high-yield epsilon-polylysine mutant strain and application thereof
  • Acid-resistant high-yield epsilon-polylysine mutant strain and application thereof
  • Acid-resistant high-yield epsilon-polylysine mutant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Construction and screening of embodiment 1 recombinant strain

[0053] First, the integrative plasmid pSET152 was selected, and a recombinant plasmid containing the promoter SF14p (shown in SEQ ID NO.1) was constructed. Using the Pls gene (SEQ ID NO.2) of bacterial strain BNCC 18622 as a template, the Pls gene was amplified by primers pls-F2 (SEQ ID NO.3) and pls-R (SEQ ID NO.4), and Nde I and Xba I Design degenerate primers for the enzyme cutting site, double-enzyme cut the Pls gene, and simultaneously cut the integrated plasmid pSET152 (carrying the promoter SF14p), connect the cut Pls gene to the plasmid, and transform it into E. coli competent cells. The positive transformants were verified by DNA sequencing, and the target plasmid was extracted and transformed into E.coli ET12567 / pUZ8002. pUZ8002 is a plasmid containing the tra gene, which encodes the transfer protein Tra, allowing the transformation of plasmid DNA from Escherichia coli to Streptomyces. E.coli ET...

Embodiment 2

[0064] The mensuration of embodiment 2 bacterial strain passage stability

[0065] The mutant strain PL-2-AH66 was subcultured, and the 1st, 3rd, 5th, 7th, and 9th generation strains were tested for shake flask fermentation. image 3 It is a genetic stability curve, and it can be seen that the curve is relatively gentle, indicating that the variation range of ε-PL yield is low. It can be seen from Table 2 that the yield of ε-PL in each generation has little difference (less than 0.05g / L), and the yield of the ninth generation is 2.51±0.01g / L, which is basically the same as that of the first generation, which shows its genetic stability better.

[0066] Table 2 PL-2-AH66 genetic stability test

[0067]

Embodiment 3

[0068] Example 3 Bacterial Strain Two-Stage Batch Fermentation

[0069] The bacterial strain PL-2-AH66 that embodiment 1 obtains is fermented in batches of pH two stages in 5L fermentor:

[0070] The seed solution was cultured in a 500mL Erlenmeyer flask with a volume of 100mL, the culture condition was 30°C, and the time was 24-26h at 200rpm.

[0071] The inoculum was inoculated into a fermenter containing 2.76L of fermentation medium (initial pH 6.8) according to the volume ratio of 8%, the initial rotation speed was 200rpm, and the ventilation rate was 1vvm. During the fermentation process, when the pH drops to 2.8±0.1, maintain this pH value until the bacterial biomass begins to decline, and adjust the pH to 3.8±0.1 by feeding ammonia water until the end of fermentation. During the fermentation process, the dissolved oxygen (DO) was maintained at 15%–25% by changing the rotational speed and ventilation. Depend on Figure 4 It can be seen that the entire fermentation pro...

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Abstract

The invention discloses an acid-resistant high-yield epsilon-polylysine mutant strain and application thereof, and belongs to the field of microbial breeding. According to the invention, streptomycesalbulus is subjected to genetic engineering modification and mutation breeding, and a high-yield strain PL-2-AH66 is obtained. Compared with an original strain, the yield of epsilon-polylysine fermented by the strain at a shake flask level is increased by 150%, the yield of epsilon-polylysine at a 5L fermentation tank level can reach 42.69 g / L, and the production strength is 7.115 g / L / d. The high-yield strain PL-2-AH66 has good acid resistance and antibiotic tolerance, still has a higher survival rate when the pH is 2.0-3.0, and is good in genetic stability, and the yield of epsilon-polylysineafter passage 9 is basically consistent with the yield of primary generation. The fermentation process of the strain is simple, the process is easy to control, the yield of epsilon-polylysine is greatly increased, and a solid foundation is laid for industrial production and application of epsilon-polylysine.

Description

technical field [0001] The invention relates to an acid-resistant and high-yield ε-polylysine mutant strain and its application, belonging to the field of microbial breeding. Background technique [0002] ε-polylysine is a natural polymer, which is mainly produced by Streptomyces. Generally 25–35. It has a wide range of antibacterial properties and can inhibit most Gram-positive bacteria, Gram-negative bacteria, yeast and some viruses, so it is widely used in the food preservation industry. Up to now, the United States, Japan, South Korea, China and other countries have successively approved the application of ε-polylysine as a food preservative in food. In addition, ε-polylysine has the advantages of good water solubility, strong thermal stability, and high safety performance, and has good application potential in the fields of medicine and pesticides. [0003] The yield of wild-type ε-polylysine-producing bacteria is generally very low, which cannot meet industrial prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/76C12N15/01C12N13/00C12P13/02C12R1/465
CPCC12N9/93C12N15/76C12N15/01C12N13/00C12P13/02Y02A40/90
Inventor 张荣珍徐岩李利宏
Owner JIANGNAN UNIV
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