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Method for increasing yield of riboflavin produced by fermentation of bacillus subtilis

A technology of Bacillus subtilis and riboflavin, applied in the field of genetic engineering, can solve the problems of reducing pyrimidine nucleotide metabolic flux, low yield and yield of riboflavin, increasing the ratio, etc.

Active Publication Date: 2020-04-07
HENAN JULONG BIOLOGICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the low yield and output of riboflavin in the prior art, the object of the present invention is to provide a method for improving the yield of riboflavin produced by fermentation of Bacillus subtilis. Modification to remove high levels of GTP in cells pyr The activation mechanism of the operon will pyr The expression of operon is stabilized at a low level to reduce the metabolic flux of the pyrimidine nucleotide de novo synthesis pathway and increase the proportion of PRPP entering the purine nucleotide synthesis pathway, thereby increasing the production and yield of riboflavin and reducing riboflavin fermentation cost

Method used

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  • Method for increasing yield of riboflavin produced by fermentation of bacillus subtilis
  • Method for increasing yield of riboflavin produced by fermentation of bacillus subtilis
  • Method for increasing yield of riboflavin produced by fermentation of bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 - Blocking pyr expression of operon

[0048] In this example, the riboflavin-producing bacterium Bacillus subtilis RX20 was used as the starting bacterium. pyr Insert the "CR" fragment into the operon, and replace it starting from the upstream of the promoter-35 region, including all pyrR genetic coding sequences, and pyrP Gene partial coding sequence, a total of 962 bp of sequence. get pyr operon-deficient strain RX21.

[0049] a. Using the chromosome of Bacillus subtilis RX19m as a template, using primers PU1 and PU2cr, PCR amplification pyr Homologous sequences upstream of the promoter-35 region of operon, amplified with PD1cr and PD2 pyrP For the homologous sequence downstream of the gene coding region, the "CR" fragment was amplified with primers CR1 and CR2, and the above three fragments were spliced ​​by one-step overlapping PCR method.

[0050] b. Using the competent transformation method, the above spliced ​​fragment was transformed int...

Embodiment 2

[0053] Example 2 - Weakening pyr expression of operon

[0054] This example chooses pyr The promoter-35 region sequence of operon is weakened and modified, but the technical scheme of the present invention is not limited to the following scheme, any reduction pyr operon expression level, leading to a significant reduction in the metabolic flux of the de novo pyrimidine nucleotide synthesis pathway without GTP activation, all within the protection of the present invention.

[0055] a. Using the chromosome of Bacillus subtilis RX19E as a template, using primers PyrU1 and PyrU2E, PCR amplification pyr The upstream homologous sequence of the operon promoter-35 region, the downstream homologous sequence of the promoter-35 region was amplified with PyrD1E and PyrD2cr, the erythromycin resistance gene fragment was amplified with primers E1 and E2, and the one-step overlapping PCR method was used, The above three fragments are spliced ​​together.

[0056] b. Using the compete...

Embodiment 3

[0063] Example 3 - Weakening pyrE gene start codon

[0064] This example will use further weakening pyrE The method of gene initiation codon reduces the translation efficiency of orotate phosphoribosyltransferase and further reduces the technical scheme of the reaction rate. However, the technical solution of the present invention is not limited to the following solutions, but includes any method for reducing the intracellular level and enzymatic activity of orotate phosphoribosyltransferase. "ATG" is replaced with "TTG", which reduces its translation efficiency and reduces the amount of intracellular orotate phosphoribosyltransferase, thereby reducing the consumption of PRPP in the synthesis of pyrimidine nucleotides, which is a purine nucleotide synthesis pathway More PRPPs are provided. Or reduce the translation efficiency of key enzymes,

[0065] a. Using the chromosome of Bacillus subtilis RX19E as a template, using primers PyrEU1 and PyrEU2E, PCR amplification py...

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Abstract

The invention relates to a method for increasing the yield of riboflavin produced by fermentation of bacillus subtilis, which belongs to the technical field of gene engineering. The method comprises the steps of carrying out genetic modification on a riboflavin production strain; removing the activation mechanism of intracellular high-level GTP on pyr operon; and stabilizing the expression of pyroperon at a low level to reduce the metabolic flux of pyrimidine nucleotide from the first synthesis route and increase the proportion of PRPP entering the purine nucleotide synthesis route. The yieldand yield coefficient of riboflavin are improved, and the fermentation cost of riboflavin is reduced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for improving the yield of riboflavin produced by fermentation of Bacillus subtilis. Background technique [0002] Riboflavin is a condensate of ribitol and 6,7-dimethylisoallazine, namely vitamin B 2 . Riboflavin exists in cells in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), and acts as a coenzyme for oxidoreductase and acts as a hydrogen carrier in metabolic reactions. Humans and animals lack the riboflavin synthesis pathway and belong to the riboflavin heterotrophic type. Riboflavin is an essential growth factor in humans and animals, and riboflavin deficiency occurs in some cases. Therefore, riboflavin is widely used as feed additive, food additive and medicine. At present, the annual production and sales of riboflavin in the world exceeds 10,000 tons, of which 70% is used in animal husbandry and 30% in f...

Claims

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Application Information

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IPC IPC(8): C12P25/00C12N1/21C12R1/125
CPCC12P25/00C12N15/52
Inventor 曹华杰李静谢沛李闯张孟涛刘晓东魏志娟
Owner HENAN JULONG BIOLOGICAL ENG CO LTD
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