Nucleoside high-yielding strain as well as construction method and application thereof

A technology for high nucleoside yield and nucleoside, 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 yield

Active Publication Date: 2020-12-25
MEIHUA BIOTECH LANGFANG CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the disadvantages of the fermentation

Method used

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  • Nucleoside high-yielding strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Mutagenesis screening to obtain adenosine high-yielding strain

[0061] In the early stage, B. subtilis 168 was used as the starting strain. After multiple rounds of mutagenesis screening, the B. subtilis MHA strain was obtained. This strain can grow on the 8-azaguanine medium containing 1g / L. The adenosine-producing level is 10g / L, and the conversion rate is 14%. It is a high-yielding adenosine strain.

[0062] B. subtilis MHA contains glyA by comparative genomics analysis E146K , pgm A110V Point mutations of two key genes, the mutations of these two sites may be the factors that promote the high production of adenosine. Therefore, the mutations at these two sites were introduced into the B. subtilis A5 strain for verification.

Embodiment 2

[0063] Example 2 engineering strain B.subtilis A6 (glyA E146K ), B. subtilis A7 (pgm A110V ) construction

[0064] Using primers glyA-1f / 1r and glyA-2f / 2r and pgm-1f / 1r and pgm-2f / 2r, using the B. subtilis A5 genome as a template, use pfu high-fidelity DNA polymerase to amplify glyA and pgm respectively Upper and lower homology arms; use primers glyA-1f / 2r and pgm-1f / 2r to fuse the upper and lower fragments respectively to obtain glyA* homologous fragments (containing the E146K mutation, the nucleotide sequence of the original glyA gene is as SEQ ID NO: 1, the amino acid sequence of its encoded protein is shown in SEQ ID NO: 2) and the pgm* homologous fragment (containing A110V mutation, the nucleotide sequence of the original pgm gene is shown in SEQ ID NO: 3, its encoded protein The amino acid sequence is shown in SEQ ID NO: 4), the two fragments and the pKSU plasmid were subjected to SalI / PstI double digestion, ligation, transformation and other operations to obtain plasm...

Embodiment 3

[0065] Construction of embodiment 3 engineering bacterial strain B.subtilis A8 (introducing glyA E146K and pgm A110V mutation)

[0066] Transform the plasmid pKSU-pgm* into the B.subtilis A6 strain to obtain the engineering bacteria B.subtilis A8 (glyA E146K &pgm A110V mutation), the screening method is the same as in Example 2.

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Abstract

The invention provides a nucleoside high-yielding strain 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 serine hydroxymethyltransferase gene glyA, as shown in SEQ ID NO:2, of bacillus subtilis; and/or (2) weakening a 2,3-diphosphoglyceric acid independent phosphoglyceric acid mutant enzyme gene pgm of a coded NCBI reference sequence WP_003228330.1 on a chromosome of the bacillus subtilis; and (3) applying the strain obtained in the step (1) and/or the step (2) to fermentation production of the nucleoside. The engineered strain bacillus subtilis provided by the invention is the nucleoside high-yielding strain, can effectively accumulate the nucleoside, improves the yield of the nucleoside, and lays a foundation for industrial production of the nucleoside.

Description

technical field [0001] The present invention relates to the field of microorganisms and the technical field of bioengineering, in particular to a high-nucleoside-producing bacterium and its construction method and application. 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 produce the corresponding aden...

Claims

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Application Information

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IPC IPC(8): C12P19/40C12P19/38C12N9/10C12N9/90C12N1/21C12N15/54C12N15/61C12R1/125
CPCC12P19/40C12P19/385C12N9/1014C12N9/90C12Y504/02001
Inventor 孙莹莹胡丹白立宽吴涛
Owner MEIHUA BIOTECH LANGFANG CO LTD
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