A promoter nucleic acid sequence of sdaa gene, recombinant strain containing the nucleic acid sequence and application thereof

A technology for recombining bacterial strains and nucleotide sequences, which is applied in the fields of genetic engineering and microorganisms, can solve problems such as lack of directionality, poor predictability of strain performance, and difficulty in controlling gene mutation sites, so as to facilitate popularization and application and increase production.

Active Publication Date: 2020-11-10
HEILONGJIANG EPPEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutation breeding refers to the stimulation of ultraviolet radiation or other external conditions to induce strains to undergo unspecific gene mutations, and then obtain high-yield strains through screening. This method lacks directionality, and the gene mutation sites are difficult to control. Poor predictability

Method used

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  • A promoter nucleic acid sequence of sdaa gene, recombinant strain containing the nucleic acid sequence and application thereof
  • A promoter nucleic acid sequence of sdaa gene, recombinant strain containing the nucleic acid sequence and application thereof
  • A promoter nucleic acid sequence of sdaa gene, recombinant strain containing the nucleic acid sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Contains point mutations sdaA Gene promoter region transformation vector pK18-PsdaA A(170)C build

[0047] According to the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers for amplifying the sdaA gene promoter region fragment were synthesized, by replacing the sdaA gene promoter region (SEQID NO: 1) in the strain YP097158 background with alleles Introducing point mutations. The primers were designed as follows (synthesized by Shanghai Invitrogen Company):

[0048] P1: 5' CCGGAATTC ATTCGCGGATCTCCGTTTA AG 3' (EcoR I) (SEQ ID NO: 3)

[0049] P2: 5' CTTTACAGGG GCTGGGTCAT GTCTTG 3' (SEQ ID NO: 4)

[0050] P3: 5' CAAGAC ATGACCCAGC CCCTGTAAAG 3' (SEQ ID NO: 5)

[0051] P4: 5' ACATGCATGC AACGTCAGGG AATACGACAC 3' (Sph I) (SEQ ID NO: 6)

[0052] Using Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was carried out with primers P1 / P2 and P3 / P4 respectively. PCR system: 10× Ex Taq Buffer 5 μL, dN...

Embodiment 2

[0053] Embodiment 2 comprises the PsdaA of point mutation A(170)C Construction of the strain

[0054] Allelic replacement plasmid pK18-PsdaA A(170)C Electrotransformation into the patented strain YP97158 of L-lysine producing bacteria (for its construction method, please refer to WO2014121669A1; it was confirmed by sequencing that the strain retained the wild-type sdaA Gene promoter), the single colonies produced by culture were identified by primers P1 / M13F, and the strains that could amplify a band with a size of 1300bp were positive strains. The positive strains were cultured on a medium containing 15% sucrose, and the single colonies produced by the culture were respectively cultured on a medium containing kanamycin and a medium without kanamycin, and cultured on a medium without kanamycin The strains that grow on culture medium but do not grow on medium containing kanamycin are further identified by PCR using the following primers (synthesized by Shanghai Invitrogen Co...

Embodiment 3

[0058] Example 3 L-lysine fermentation experiment

[0059] The bacterial strain YPL-4-005 constructed in Example 2 and the original bacterial strain YP97158 were used in the BLBIO-5GC-4-H type fermenter (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the medium and table shown in Table 1. The control process shown in 2 was used for fermentation experiments. Each strain was repeated three times, and the results are shown in Table 3.

[0060] Table 1 Fermentation medium formula

[0061]

[0062] Table 2 Fermentation control process

[0063]

[0064] Table 3 L-lysine fermentation experiment results

[0065]

[0066] The results are shown in Table 3. Point mutation PsdaA was carried out in the sdaA gene promoter in Corynebacterium glutamicum A(170)C , contribute to the improvement of L-lysine production.

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Abstract

The invention provides a promoter nucleic acid sequence of sdaA gene, a recombinant strain containing the nucleic acid sequence and an application of the recombinant strain. The promoter is obtained by introducing a point mutation into a wild-type promoter sequence of sdaA gene. The recombinant strain is used for fermenting and producing L-lysine to further improve the yield of L-lysine, has no conflict with modification sites of other existing modified strains producing L-lysine, and realizes a new mode for improving the fermentation yield of L-lysine, and thus popularization and applicationare facilitated.

Description

technical field [0001] The invention belongs to the field of genetic engineering and microorganism technology, and specifically relates to a promoter nucleic acid sequence of sdaA gene, a recombinant bacterial strain containing the nucleic acid sequence and application thereof. Background technique [0002] L-Lysine is one of the eight amino acids necessary for human and animal life activities. L-lysine has a variety of physiological functions, such as regulating metabolic balance, promoting human development, promoting the body's absorption of cereal protein and other amino acids, enhancing immune function, etc., and is widely used in food additives, animal feed, drug synthesis, etc. aspect. [0003] The production methods of L-lysine mainly include proteolysis and microbial fermentation. Among them, the microbial fermentation method has become the most commonly used method for industrially producing L-lysine because of the advantages of easy control of the production pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/70C12N15/77C12N1/21C12P13/08C12R1/19C12R1/15
CPCC07K14/34C12N15/70C12N15/77C12P13/08
Inventor 马风勇魏爱英孟刚周晓群杨立鹏苏厚波贾慧萍郭小炜
Owner HEILONGJIANG EPPEN BIOTECH CO LTD
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