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Phosphopentose mutase mutant and application thereof in construction of high-nucleoside-yield bacillus subtilis

A Bacillus subtilis and pentose phosphate technology, applied in the field of bioengineering, can solve problems such as promoter deletion, deletion, and no further site information, and achieve the effect of increasing yield

Active Publication Date: 2021-07-23
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the prior art, although CN106906174A mentioned a recombinant bacterium producing inosine and its preparation method and application, it weakened or inactivated the pentose phosphate mutase, and the modification method was promoter deletion or gene deletion , without further providing information on sites with a high degree of correlation with the enzyme effect

Method used

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  • Phosphopentose mutase mutant and application thereof in construction of high-nucleoside-yield bacillus subtilis
  • Phosphopentose mutase mutant and application thereof in construction of high-nucleoside-yield bacillus subtilis
  • Phosphopentose mutase mutant and application thereof in construction of high-nucleoside-yield bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Obtaining adenosine high-yielding strains by mutagenesis screening

[0049] After multiple rounds of mutagenesis screening, the B.subtilis MHA strain was obtained, which can grow on 1g / L 8-azaguanine medium, and the adenosine production level of B.subtilis MHA shake flask is 10g / L. The conversion rate was 14%, and it was a high-yielding adenosine strain.

[0050] Through comparative genomics analysis, the pentose phosphate mutase (encoded by the drm gene) of B. subtilis MHA has two mutations, S159F and H336Y, which may be the factors that promote the high production of adenosine. Therefore, the present invention introduces the mutations at these two sites into the B. subtilis A5 strain for verification.

Embodiment 2

[0051] Embodiment 2: engineering bacterial strain B.subtilisA14 (drm S159F ), A15 (drm H336Y ) construction

[0052] Using primers drm-1f / 1r, drm-2f / 2r and drm-3f / 3r, drm-4f / 4r to use the B. subtilis A5 genome as a template, use pfu high-fidelity DNA polymerase to amplify the upstream and downstream synchronisms of drm respectively Source arm; use primers drm-1f / 2r and drm-3f / 4r to fuse upstream and downstream fragments respectively to obtain drm* homologous fragments (containing S159F and H336Y mutations, the nucleotide sequence of the complete drm* gene is shown in SEQ ID No.2 It shows a total of 1185bp, and the amino acid sequence is as shown in SEQ ID No.1, a total of 394 amino acid sequences), and the two fragments and the pKSU (tool vector) plasmid are subjected to SalI / PstI double enzyme digestion, connection, transformation and other operations to obtain the plasmid pKSU- drm* 1 and pKSU-drm* 2 . Through electrochemical transformation into B.subtilisA5, use LB pla...

Embodiment 3

[0053] Embodiment 3: construction of engineering bacterial strain B.subtilis A16 (introducing drm S159F，H336Y mutation)

[0054] The plasmid pKSU-drm* 1 Transform into the B.subtilis A15 bacterial strain, obtain engineering bacterium B.subtilis A16 (drm S159F，H336Y ), the screening method is the same as in Example 2.

[0055] The formula of LB liquid medium is: 10g / L peptone, 5g / L yeast extract, 10g / L NaCl, adjust the pH to 7.2, and sterilize under 0.15Mpa pressure for 20min. The formula of LB solid medium is: add agar powder (final concentration 18g / L) to LB liquid medium, and sterilize at 121°C for 20min.

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Abstract

The invention relates to the field of bioengineering, in particular to a phosphopentose mutase mutant and application thereof in construction of high-nucleoside-yield bacillus subtilis. According to the invention, mutation of the 159th position (serine mutated into phenylalanine) and the 336th position (histidine mutated into tyrosine) of phosphopentose mutase plays a main role in nucleoside accumulation, so that the high-nucleoside-yield bacillus subtilis can be constructed and screened, the yield of nucleoside can be increased, and a foundation is laid for industrial production of nucleoside.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a pentose phosphate mutase mutant and its application in constructing high-yielding nucleoside-producing Bacillus subtilis. Background technique [0002] Genetically engineered bacteria have obvious advantages of using bacteria as host bacteria, such as short fermentation cycle, simple raw material requirements, and mature genetic engineering technology. At present, microbial fermentation is the main method for producing nucleosides, and the production bacteria used are mainly Bacillus species, including Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus pumilus. As the starting strain of nucleosides, Bacillus has the advantages of relatively active pentose phosphate pathway and low activity of purine nucleoside phosphorylase. Among the Bacillus genus, many strains including Bacillus subtilis have a reliable safety profile. Traditional strain breeding found that the mutant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N1/21C12P19/38C12R1/125
CPCC12N9/90C12P19/38C12Y504/02007
Inventor 孙莹莹胡丹齐丹丹李岩
Owner MEIHUA BIOTECH LANGFANG CO LTD