High-temperature riboflavin-producing engineering bacteria and its construction method and application

The technology of riboflavin and engineering bacteria is applied in the fields of genetic engineering and fermentation engineering, which can solve the problem of high energy consumption, achieve the effects of simple operation, saving production cost, and shortening the period of fermentation to produce riboflavin.

Active Publication Date: 2021-11-05
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, Bacillus subtilis has become the main riboflavin industrial production bacterium through genetic transformation, but its production process belongs to normal temperature fermentation, which requires high energy consumption

Method used

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  • High-temperature riboflavin-producing engineering bacteria and its construction method and application
  • High-temperature riboflavin-producing engineering bacteria and its construction method and application
  • High-temperature riboflavin-producing engineering bacteria and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of recombinant plasmids

[0048] The genome information of Geobacillus thermoglucosidase (NZ_CP016916.1) and Geobacillus thermodenitrification (NC_009328.1) were obtained from NCBI, the enzymes involved in riboflavin synthesis were found through annotation, the size of their gene clusters was determined, and Compared with the riboflavin cluster gene sequence of Bacillus subtilis by BLAST, it was determined that the sequence had a high similarity. Geobacillus thermoglucosidasius NCIMB 11955 and Geobacillus thermodenitrificans CGMCC 1.5331 were stored in the laboratory, and the bacterial genome extraction kit of Beijing Tiangen Company was used to extract the Genome, the riboflavin synthesis gene cluster of Geobacillus thermoglucosidase was named tgrib, and the riboflavin synthesis gene cluster of Geobacillus thermodenitrification was named dnrib. The primers designed with Primer 6.0 software are shown in Table 1.

[0049] (1) Using the plasmid pTA...

Embodiment 2

[0054] Example 2 Construction of Geobacillus and Escherichia coli engineering bacteria producing riboflavin at high temperature

[0055] The recombinant plasmid was transformed into Escherichia coli JM109, the strain containing pUCG3.8-R1 was named E.coli-R1, the strain containing pUCG3.8-R2 was named E.coli-R2, and the strain containing pUCG3.8 was named E. .coli-R0. The transformed strains were verified by colony PCR ( figure 2 ).

[0056]The recombinant plasmid was transferred to Geobacillus thermoglucosidasius (Geobacillusthermoglucosidasius) NCIMB 11955, the strain containing pUCG3.8-R1 was named GT-R1, the strain containing pUCG3.8-R2 was named GT-R2, and the strain without plasmid Designate GT as the wild type, and the strain containing pUCG3.8 as GT-R0. The transformed strains were verified by colony PCR ( image 3 ).

Embodiment 3

[0057] Example 3 Fermentation production of riboflavin by engineering bacteria

[0058] 1. Escherichia coli containing Geobacillus riboflavin synthesis gene cluster was used as the production strain

[0059] (1) Preparation of seed solution

[0060] Streak on the LB medium plate to obtain a single colony, inoculate the LB medium containing 12.5 μg / mL kanamycin, and culture in the test tube at 37°C and 220r / min for about 12h.

[0061] (2) Shake flask fermentation

[0062] Dilute the above seed solution according to the OD after inoculation 600 The inoculum with a value of 0.1 was inserted into LBG medium (LB liquid medium + 10g / L glucose + 20.9g / L MOPS) containing 12.5μg / mL kanamycin, and the volume of the shake flask was 50mL / 250mL. , riboflavin production was carried out under the fermentation condition of the shaker rotating speed of 250r / min.

[0063] When CaCO was not added to LBG medium 3 When kanamycin was 12.5μg / mL, after 48 hours of fermentation at 45°C, the OD of...

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Abstract

The invention relates to high-temperature riboflavin-producing engineering bacteria and its construction method and application. The engineering bacterium is Escherichia coli or Geobacillus, and carries an expression vector expressing Geobacillus riboflavin synthesis gene cluster. Riboflavin can be produced by high-temperature riboflavin-producing Escherichia coli engineering bacteria, and the riboflavin production in the fermentation broth can reach up to about 276mg / L. Up to about 50mg / L. The engineering bacterium provided by the invention has a shorter production cycle than the existing riboflavin engineering bacterium, saves energy, can be used for riboflavin production at low cost, and provides valuable strain resources for producing riboflavin under high temperature conditions.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and fermentation engineering, in particular to an engineering bacterium producing riboflavin at high temperature and its construction method and application. Background technique [0002] Riboflavin, namely vitamin B2 (Riboflavin), is one of the essential vitamins for bacteria and animals. It usually exists in the form of FMN and FAD in organisms and participates in intracellular redox reactions. It is mainly used in pharmaceuticals, feed additives, food additives and cosmetics. At present, the production process of riboflavin is microbial fermentation. In the early stage, its production strains were fungi, such as Candida famata, Ashbya gossypii, Eremothecium ashbyii, Saccharomy cescerevisiae, etc. (Stahmann et al, 2016, Appl Microbiol Biotechnol 100:2107-2119). However, there are some problems in the production of riboflavin by fungi, such as long fermentation period (6-7 days), l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/75C12P25/00C12R1/19C12R1/10
CPCC12N15/52C12N15/70C12N15/75C12P25/00
Inventor 李子龙齐凤仙范可强王为善王俊阳杨克迁
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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