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High-nucleoside-yield bacillus subtilis engineering bacterium as well as construction method and application thereof

A technology of Bacillus subtilis and engineering bacteria, applied in the field of microorganisms, can solve the problems of low conversion rate and high cost, and achieve the effects of long fermentation cycle, reduced fermentation cost and increased production

Active Publication Date: 2020-12-29
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of the fermentation method are high cost and low conversion rate.

Method used

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  • High-nucleoside-yield bacillus subtilis engineering bacterium as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 engineering strain B.subtilis A9 (gltA A1181V ) construction

[0070] Using primers gltA-1f / 1r and gltA-2f / 2r, using the B. subtilis A5 genome as a template, use pfu high-fidelity DNA polymerase to amplify the upper and downstream homology arms of gltA respectively; use primer gltA-1f / 2r Fusion upstream and downstream fragments with glnA-1f / 2r to obtain gltA* homologous fragments (containing the A1181V mutation, the nucleotide sequence of the original gltA gene is shown in SEQ ID NO: 1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO: 2), the fragment and the pKSU plasmid were subjected to SalI / PstI double digestion, ligation, transformation and other operations to obtain the plasmid pKSU-gltA. Transform into B. subtilis A5 by electrochemical transformation, use LB plates containing 2.5 μg / mL chloramphenicol to select transformants at 30°C, transfer the obtained transformants into 5ml LB liquid medium, and culture at 42°C at 200rpm fo...

Embodiment 2

[0071] The construction of embodiment 2 engineering strain B.subtilis A10 (P43-gltA), B.subtilis A11 (P43-glnA)

[0072] Using primers P43-gltA-1f / 1r and P43-gltA-2f / 2r, P43-glnA-1f / 1r and P43-glnA-2f / 2r, using the B. subtilis A5 genome as a template, using pfu high-fidelity DNA polymerase Amplify the upper and lower homology arms of gltA and glnA respectively; use primers P43-f / r to amplify the plasmid as a template to obtain the P43 promoter; use primers gltA-1f / 2r and glnA-1f / 2r to fuse the P43 promoter and the upstream and downstream fragments to obtain gltA homologous fragments (the nucleotide sequence of the complete gltA gene is shown in SEQ ID NO: 1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO: 2) and glnA homologous fragments ( The nucleotide sequence of the complete glnA gene is shown in SEQ ID NO: 3, and the amino acid sequence of its encoded protein is shown in SEQ ID NO: 4), and the two fragments were digested with pKSU plasmid by SalI...

Embodiment 3

[0073] Example 3 Construction of engineering strains B.subtilis A12 and B.subtilis A13 (introducing P43-glnA)

[0074] The plasmid pKSU-P43-glnA was transformed into B.subtilis A9 and B.subtilis A10 bacterial strains respectively to obtain engineering bacteria B.subtilis A12 (gltA A1181V &P43-glnA), B. subtilis A13 (P43-gltA&P43-glnA), the screening method is the same as in Example 1.

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Abstract

The invention provides high-nucleoside-yield bacillus subtilis engineering bacterium as well as a construction method and application thereof The invention also provides a method for producing nucleoside by fermentation. The method comprises the following steps of (1) enhancing a glutamate synthase gene gltA for encoding an NCBI reference sequence WP-009967365.1 on a bacillus subtilis chromosome;and / or enhancing a glutamine synthetase gene glnA for encoding an NCBI reference sequence WP-003231737.1 on the chromosome of the bacillus subtilis; and (2) applying the gene-enhanced strain obtainedin the step (1) to fermentation production of nucleoside. The bacillus subtilis engineering bacterium provided by the invention is a high-nucleoside-yield strain, can effectively accumulate nucleoside, improves the yield of nucleoside, and lays a foundation for industrial production of nucleoside.

Description

technical field [0001] The invention relates to the field of microorganisms and the technical field of bioengineering, in particular to a high-yield nucleoside-producing bacillus subtilis engineering bacterium, a construction method and application thereof. Background technique [0002] Nucleoside is a general term for a class of glycosides. Nucleosides are the building blocks of nucleic acids and nucleotides. Nucleosides are formed by condensation of D-ribose or D-Z-deoxyribose with pyrimidine base or purine base. Nucleosides are generally colorless crystals, insoluble in common organic solvents, easily soluble in hot water, with a melting point of 160-240°C. The nucleosides generated from D-ribose are called ribonucleosides, which participate in the composition of RNA, and the nucleosides generated from D-α-deoxyribose are called deoxyribonucleosides, which participate in the composition of DNA. D-ribose is condensed with adenine, guanine, cytosine, thymine or uracil to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/90C12N15/67C12N15/52C12N15/60C12P19/40C12N1/21C12R1/125
CPCC12N15/75C12N15/902C12N15/67C12N9/93C12N9/88C12P19/40C12Y603/01002C12Y401/03
Inventor 孙莹莹胡丹白立宽吴涛
Owner MEIHUA BIOTECH LANGFANG CO LTD
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