A mutant strain with acid resistance and high production of ε-polylysine and its application

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

Active Publication Date: 2022-08-09
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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  • A mutant strain with acid resistance and high production of ε-polylysine and its application
  • A mutant strain with acid resistance and high production of ε-polylysine and its application
  • A mutant strain with acid resistance and high production of ε-polylysine and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Construction and screening of recombinant strains

[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 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), with Nde I and Xba I Degenerate primers were designed for the restriction site, the Pls gene was double digested, and the integrated plasmid pSET152 (carrying the promoter SF14p) was cut at the same time, the cut Pls gene was connected to the plasmid, and transformed 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, enabling the transformation of plasmid DNA from E. coli to Streptomyces. E.coli ET12...

Embodiment 2

[0064] Embodiment 2 Determination of strain passage stability

[0065] The mutant strain PL-2-AH66 was subcultured, and the 1st, 3rd, 5th, 7th, and 9th generation strains were subjected to shake flask fermentation tests. image 3 For the genetic stability curve, it can be seen that the curve is relatively flat, 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 is not much different (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 Genetic stability test of PL-2-AH66

[0067]

Embodiment 3

[0068] Example 3 Bacterial two-stage batch fermentation

[0069] The strain PL-2-AH66 obtained in Example 1 was fermented in two-stage batches at pH in a 5L fermentor:

[0070] The seed solution was cultured in a 500mL conical flask with a volume of 100mL, and the culture conditions were 30°C and 200rpm for 24–26h.

[0071] According to the inoculum volume of 8%, it was inoculated into a fermenter containing 2.76L of fermentation medium (initial pH 6.8), the initial rotation speed was 200rpm, and the aeration rate was 1vvm. During the fermentation process, when the pH dropped to 2.8±0.1, the pH value was maintained until the biomass of the bacteria began to decrease, and the pH was adjusted to 3.8±0.1 by adding ammonia water until the fermentation was over. The fermentation process maintains dissolved oxygen (DO) at 15%–25% through changes in rotational speed and aeration. Depend on Figure 4 It can be seen that the whole fermentation process lasted for 54h, and the content...

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Abstract

The invention discloses an acid-resistant and high-yield ε-polylysine mutant strain and an application thereof, belonging to the field of microbial breeding. The present invention obtains a high-yielding strain PL-2-AH66 by performing genetic engineering on Streptomyces and combining with mutation breeding. Compared with the starting strain, the ε-polylysine fermented by the strain at the shake flask level is increased by 150%, and the ε-polylysine production at the level of the 5L fermenter can reach 42.69 g / L, and the production intensity is 7.115 g / L / d. The high-yielding strain PL-2-AH66 of the present invention has good acid resistance and antibiotic resistance, still has a high survival rate at pH 2.0 to 3.0, and has good genetic stability. The yield of lysine was basically the same as that of the first passage. The fermentation process of the strain is simple, the process is easy to control, and the yield of ε-polylysine is greatly improved, which lays a solid foundation for the industrial production and application of ε-polylysine.

Description

technical field [0001] The invention relates to a mutant strain with acid resistance and high production of ε-polylysine and its application, belonging to the field of microbial breeding. Background technique [0002] ε-Polylysine is a natural polymer mainly produced by Streptomyces sp. It is a secondary metabolite formed by the dehydration condensation of α-amino group and ε-carboxyl group on L-lysine. Typically 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 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 y...

Claims

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Application Information

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Patent Type & Authority Patents(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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