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Recombinant bacterium for fermentation production of purine nucleoside as well as construction method and application thereof

A technology of recombinant strains and purines, applied in the field of microbial fermentation, can solve problems such as changes in metabolic flux, lack of global regulation and engineering strategies for metabolic networks of target microorganisms, and inability to understand organism functions and phenotypes as a whole

Pending Publication Date: 2021-11-02
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the existing research cannot fully understand the function and phenotype of organisms, resulting in the lack of global regulation and engineering strategies for the metabolic network of target microorganisms, which has a certain degree of blindness and often fails to achieve the expected effect of metabolic flux changes

Method used

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  • Recombinant bacterium for fermentation production of purine nucleoside as well as construction method and application thereof
  • Recombinant bacterium for fermentation production of purine nucleoside as well as construction method and application thereof
  • Recombinant bacterium for fermentation production of purine nucleoside as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0170] Example 1. Prediction of the transformation target of purine nucleosides based on the genome metabolic network model

[0171] (1) Correlation analysis between IMP and biomass

[0172] Based on the Bacillus subtilis genome metabolic network model iBsu1103V3, using flux balance analysis (flux balance analysis, hereinafter referred to as FBA) to calculate and analyze the maximum theoretical growth rate of cells is 0.26; the maximum theoretical synthesis flux of IMP is 1.52mmol· wxya -1 h -1 , the maximum theoretical conversion of IMP is 0.84mol / mol (IMP / glucose). The interaction between synthetic IMP metabolic flux and biomass growth was further exploited. like figure 1 As shown, the production rate of IMP decreases with the continuous increase of cell growth rate, indicating that the production and growth of IMP in the wild-type strain are in a competitive relationship, and the result of optimal growth of the strain will make the production of IMP zero. Therefore, it...

Embodiment 2

[0175] Embodiment 2. Construction and shake flask fermentation of purine nucleoside universal engineering strain

[0176] In order to enhance the purine synthesis pathway, the present invention transforms the expression control elements of the purine operon in the wild type W168. Four constitutive promoters with different strengths were selected (P 43 ,P veg ,P ctc and P gsiB ) to replace the promoter P of the purine operon pur .

[0177] First, primers P1-P16 were designed according to the genome sequence of Bacillus subtilis 168 in Genbank to amplify the promoters respectively; then, the amplified fragments were combined with P pur Overlap PCR (SOE-PCR) was performed on the upstream and downstream fragments of the fragments; finally, the recovered PCR product was digested and purified with NheI, and ligated with the gene editing vector pWYE486, and the ligated product was transformed into Escherichia coli EC135. Transformants were screened on LB plates containing 100 μg / ...

Embodiment 3

[0199] Embodiment 3. Construction and shake flask fermentation of the inosine engineering strain IR-9 enhanced by the purine operon

[0200] In order to increase the inosine accumulation of inosine engineering bacteria, the purine operon promoter of the engineering strain IR-4 was replaced by the screened P veg . Specifically: the pWYE486-P veg Transformed into IR-4, on the LB plate containing 1% xylose and 20 μg / mL erythromycin, the colony with the recombinant plasmid integrated into the chromosome was obtained by forward selection; by reverse selection, the second homologous Recombinant positive colonies were identified by PCR amplification of the strains. At the same time, the PCR product was purified for sequence determination and analysis to further confirm the correctness of the recombinant, which was named IR-9.

[0201] The 500mL shake flask fermentation test was carried out on the strains IR-6 and IR-9, and the shake flask fermentation results showed that P pur re...

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Abstract

The invention discloses a recombinant bacterium for producing purine nucleoside through fermentation as well as a construction method and application thereof. The invention relates to the field of microbial fermentation, in particular to recombinant bacteria for producing purine nucleoside as well as a construction method and application thereof. Compared with an original strain, the recombinant strain for producing purine nucleoside has a mutated purine operon regulation region, inactivated aldose transferase, enhanced 6-phosphate glucose dehydrogenase activity and weakened 6-phosphate glucose isomerase. The inosine yield of the recombinant bacteria provided by the invention is obviously improved. The invention develops a novel method for improving the fermentation yield of purine nucleoside, so that the method can be used for producing inosine, guanosine and adenosine by bacterial fermentation in practice.

Description

technical field [0001] The invention relates to the field of microbial fermentation, in particular to a recombinant bacterium for fermenting and producing purine nucleosides and its construction method and application. Background technique [0002] Purine nucleosides are composed of purines and nucleosides, including adenosine (adenosine), guanosine (guanosine), inosine (inosine) and xanthine (xanthosine) ). Purine nucleosides are important metabolites in organisms, have important physiological functions, participate in many important cellular metabolic processes such as nucleic acid synthesis, energy supply, and amino acid synthesis, and play an important role in maintaining the physiological metabolic functions of cells. They can be used as coenzyme Drugs, antiviral drugs and food freshness enhancers, etc., have a very wide range of application values ​​in the fields of medical treatment, food and health care. [0003] The production method of purine nucleosides is mainl...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N15/53C12N15/61C12N15/54C12N9/04C12N9/92C12N9/10C12P19/40C12R1/125
CPCC12N15/52C12N9/0006C12N9/92C12N9/1048C12N15/75C12P19/40C12Y101/01049C12Y503/01009
Inventor 邓爱华温廷益陈振翔王珺玥刘树文
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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