Bacterial for optimizing abomacetin fermentation component, construction method and use

A technology of erythromycin and saccharopolysporidium mold, applied in the field of biotechnology engineering, can solve the problems of lack of effective pertinence, low effective components and the like

Active Publication Date: 2012-05-23
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these studies mainly focused on the improvement of substrate supply or limiting factors related to erythromycin production, and did not make specific genetic modifications on the secondary metabolic pathway of erythromycin biosynthesis. Therefore, in solving erythromycin Lack of effective pertinence when problems such as low effective components are often faced in production

Method used

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  • Bacterial for optimizing abomacetin fermentation component, construction method and use
  • Bacterial for optimizing abomacetin fermentation component, construction method and use
  • Bacterial for optimizing abomacetin fermentation component, construction method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Establishment of Genetic Transfer System of Erythromycin Industrial Production Bacteria HL3168 E3

[0053] Culture E.coli ET12567 containing appropriate plasmids to OD 600 =0.5-0.6, the bacterial cells in 25mL culture medium were collected by centrifugation, washed twice with an equal volume of LB, resuspended in 2mL LB, and used as E. coli donor cells. Take 500 μL of 20% glycerol spore suspension of S. erythraea HL3168E3 frozen at -80°C, wash twice with an equal volume of TES buffer, resuspend in an equal volume of TES buffer, and heat shock at 50°C for 10 minutes to germinate the spores. Add an equal volume of TSB and incubate at 37°C for 2-5hr. Centrifuge and resuspend in 0.5-1 mL LB as Streptomyces recipient cells. Mix 100 μL of different concentrations of recipient cells with an equal volume of donor cells and directly spread in the solution containing 10 mM MgCl 2 After incubating at 30°C for 20 hrs on MS plates, gently wash the surface of the plates ...

Embodiment 2

[0054] The shaking flask fermentation of embodiment 2 erythromycin

[0055] Erythromycin industrial production strain HL3168 E3 and its recombinant strain in slant medium [ / L: 10g cornstarch, 10ml corn steep liquor, 3g sodium chloride, 3g ammonium sulfate, 5g calcium carbonate, pH7.0, 2g agar powder] (Appropriate antibiotics are added to the culture medium when the recombinant bacteria are cultured) 34 ° C for 7 days to grow spores, which are used as seeds for liquid fermentation.

[0056] For liquid fermentation, take 1 cm from the slant medium 2 Insert 50ml of fermented seed medium into large and small squares [ / L: 50g cornstarch, 18g soybean cake powder, 13ml corn steep liquor, 3g sodium chloride, 1.2g ammonium sulfate, 1.2g ammonium nitrate, 5ml soybean oil, 6g calcium carbonate, pH6. 8~7.0] at 34°C and 250rpm for 2 days. Then transfer 5ml of seed culture solution to 50ml fermentation medium [ / L: 40g cornstarch, 30g soybean meal powder, 30g dextrin, 2g ammonium sulfate, ...

Embodiment 3

[0057] Example 3 Extraction of erythromycin fermentation broth product

[0058] In a 100ml beaker, accurately measure an appropriate amount of fermentation broth and adjust the pH to 8.9 with NaOH. If the sample is frozen, it should be thawed, mixed evenly and quantitatively transferred to a 250ml volumetric flask, made to volume with water, mixed evenly, and centrifuged at 8000-9000 for 10min.

[0059] Take 25ml of the above solution in a 125ml separating funnel, add 25ml of n-hexane, shake for 5min, collect the water phase (lower layer) in a centrifuge tube, wash the hexane layer with water twice, mix the water phase with the water phase of the centrifuge tube, add 10ml Chloroform, shake vigorously for 5 minutes, and centrifuge at 2500 for 5 minutes. Depending on the sample, an emulsion may form on the interface. Use a glass rod to disperse or centrifuge again. Discard the aqueous phase, transfer the chloroform (lower layer) to a 10ml vial, and concentrate.

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Abstract

The invention discloses a strain for optimizing a fermenting composition of erythromycin, a construction method and application thereof. The strain is saccharopolyspora erythraea which is classified and named Saccharopolyspora erythraea; and the preservation number of the strain is CGMCC2286. Through a means of metabolic engineering, erythromycin is strengthened and is subjected to biosynthesis to modify enzyme; in particular, the strength of C12-hydroxylase of a macrocyclic framework and mycarose methylase; an reconstructed erythromycin promoter is introduced; and through different mixture ratio of gene dosage, the strength and proportion of the hydroxylase and the methylase are adjusted, so as to obtain the strain for only producing a main effective composition of the erythromycin, namely erythromycin A. The strain is adopted to ferment, can reduce impurity compositions, even fully remove the impurity compositions of erythromycin A, erythromycin B and the like, simplify a production process and reduce production cost.

Description

technical field [0001] The invention belongs to the field of biotechnology engineering, and more specifically, the invention relates to a metabolically engineered erythromycin component-optimized strain, a construction method of the strain, and an application of the strain in industrial production. Background technique [0002] Erythromycin is a class of broad-spectrum macrolide antibiotics widely used clinically for the treatment of Gram-positive bacterial infections. Since Lily successfully developed the first generation of erythromycin antibiotics——erythromycin A in 1952, azithromycin, clarithromycin, roxithromycin, fluerythromycin, dirithromycin and The second-generation erythromycin antibiotics represented by weimycin, and the third-generation erythromycin antibiotics represented by telithromycin and ABT-773 have the characteristics of good antibacterial activity, low toxicity, and not easy to induce drug resistance At the same time, the course of treatment is short an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12N15/11C12P19/62C12R1/645C12N15/113C12N15/52C12N15/53
Inventor 刘文张嗣良陈云邓玮
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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