Engineering bacteria for producing erythromycin and application of the engineering bacteria

A technology of Saccharopolyspora rubrum and recombinant bacteria, which is applied in the directions of microorganism-based methods, medical preparations containing active ingredients, bacteria, etc., can solve the problems of long cycle, poor target, heavy screening work, etc., and achieve higher titer. , the effect of low production cost

Inactive Publication Date: 2012-07-25
MAIDAN BIOLOGICAL GROUP FUJIAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional breeding methods have some limitations, such

Method used

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  • Engineering bacteria for producing erythromycin and application of the engineering bacteria
  • Engineering bacteria for producing erythromycin and application of the engineering bacteria
  • Engineering bacteria for producing erythromycin and application of the engineering bacteria

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, construction of recombinant plasmid pSET152-ermE*-eryBII-eryF

[0030] 1. Construction of recombinant plasmid pSET152-ermE*

[0031] 1. Synthesize the double-stranded DNA molecule shown in sequence 1 of the sequence list (in sequence 1, the 10th to 225th nucleotides from the 5' end are the erythromycin promoter ermE*, and the 4th to 9th nucleotides are The XbaI restriction recognition sequence, the 226th to 231st nucleotides are the XbaI restriction recognition sequence).

[0032] 2. Digest the double-stranded DNA molecule synthesized in step 1 with restriction endonuclease XbaI, and recover the digested product.

[0033] 3. Digest the vector pSET152 (Changsha Yingrun Biotechnology Co., Ltd., see the structure schematic diagram) with the restriction endonuclease XbaI figure 1 , the shuttle plasmid of Escherichia coli and Streptomyces), the vector backbone (about 5700bp) was recovered.

[0034] 4. Ligate the digested product of step 2 with the vector bac...

Embodiment 2

[0055] Embodiment 2, the construction of engineering bacteria

[0056] The recombinant plasmid pSET152-ermE*-eryBII-eryF was electroporated to transform the protoplast of Saccharopolyspora rubrum 11635, and then the primer pair composed of F1 and R2 was used for PCR identification (the one showing a specific band of about 2200bp was positive for PCR identification) to obtain the recombinant bacteria.

[0057] A recombinant strain was named as Saccharopolyspora erythraea MFSE0015. Red Saccharopolyspora erythraea (Saccharopolyspora erythraea) MFSE0015, referred to as Saccharopolyspora erythraea MFSE0015, has been preserved in the China Center for Type Culture Collection (CCTCC for short; Address: Wuhan, China, Wuhan University; Zip code: 430072) on March 2, 2012 , the deposit number is CCTCC NO: M 2012052.

Embodiment 3

[0058] Embodiment 3, application Saccharopolyspora rubrum MFSE0015 produces erythromycin

[0059] 1. Production of erythromycin using Saccharopolyspora rubrum MFSE0015

[0060] 1. Inoculate Saccharopolyspora rubrum MFSE0015 on the slant medium and culture at 33.5°C for 7 days (to produce a large number of spores).

[0061] The slant medium is composed of solute and water; the solute and its concentration in the slant medium are as follows: cornstarch 1g / 100ml, corn steep liquor 1.2g / 100ml, ammonium sulfate 0.3g / 100ml, sodium chloride 0.3g / 100ml, carbonic acid Calcium 0.25g / 100ml and agar powder 2.0g / 100ml.

[0062] 2. Take 1 cm from the slant culture medium in step 1 2 The size of the square is inoculated into 50ml seed culture medium (in 500 milliliters Erlenmeyer flasks), 33.5 ℃, 240rpm vibration culture 2 days, obtains seed liquid (contains the thalli of 10-15g wet weight in every 100 milliliters seed liquids).

[0063] Seed medium (pH7) is composed of solutes and water;...

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Abstract

The invention discloses engineering bacteria for producing erythromycin and application of the engineering bacteria. The invention claims a recombinant strain obtained by introduction of a recombinant plasmid into Saccharopolyspora erythraea. The recombinant plasmid is obtained by insertion of ermE* promoter, eryBII protein encoding gene and eryF protein encoding gene into multiple cloning sites of an initial vector; the ermE* promoter can initiate expression of the eryBII protein encoding gene and the eryF protein encoding gene; and the ATCC access number of the Saccharopolyspora erythraea strain is 11635. The engineering bacteria provided by the invention can be directly fermented to obtain erythromycin with titer up to 8,000U/ml in fermentation broth, the production cost is low, and the strain is a production strain with high production and application values.

Description

technical field [0001] The invention relates to an engineering bacterium for producing erythromycin and its application. Background technique [0002] Erythromycin is a class of macrolide antibiotics widely used to inhibit Gram-positive bacterial infections. Since its discovery in 1952, erythromycin and its derivatives have been widely used clinically. At present, the annual sales of erythromycin antibiotics exceed 3.5 billion US dollars, ranking third in the sales of antibiotics. They are considered to be the next generation of antibiotics for the treatment of drug-resistant bacteria. Clarithromycin, clarithromycin, etc.), third-generation erythromycin (such as telithromycin) are listed in Japan and Europe, and the demand for erythromycin in domestic and foreign markets has greatly increased. [0003] At present, my country is the world's largest producer of erythromycin raw materials, but it mainly relies on fermentation production, and there is a common problem of low e...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/62A61K31/7048A61P31/04C12R1/01
Inventor 吴伟斌黄钦耿施巧琴翁雪清赵燕玉黄祥峰黄维锦吴松刚
Owner MAIDAN BIOLOGICAL GROUP FUJIAN
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