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Recombination plasmid, construction method and genetic modification for erythromycin producing strain

A technology of recombinant plasmids and erythromycin, applied in the field of genetic engineering, can solve the problems of lack of effective pertinence and low effective components

Active Publication Date: 2010-12-22
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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  • Recombination plasmid, construction method and genetic modification for erythromycin producing strain
  • Recombination plasmid, construction method and genetic modification for erythromycin producing strain
  • Recombination plasmid, construction method and genetic modification for erythromycin producing strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Contains ermE * Construction of the recombinant plasmid of -eryK structure

[0056] Using the genomic DNA of S. erythraea HL3168 E3 in industrial production as a template, primers 5'AAA CTG CAG CAC CGC GGA AGT CTC GAC ACC-3' and 5'-TTT AAG CTT CGC CGG GGC AGT GCA AGT were used ACG-3' cloned the coding sequence of the eryK gene, and the results were as follows figure 1 As shown in .A, a PCR product of 1.7 kb was obtained.

[0057] The above PCR products were recovered and purified and cloned into pGEM -Teasy vector, sequenced after digestion and verification. When we compared the sequencing results with the data submitted by Stassi in Genebank, we found two inconsistencies. One is located in the promoter region, 38bp upstream of the start codon, the base is changed from TGC to TTC, and the amino acid at this position is changed from Cys of Genebank to phenylalanine in our production bacteria (Phe); the other is located in the coding region, at 988bp down...

Embodiment 2

[0060] Example 2 contains ermE ＊ Construction of the recombinant plasmid of -eryK-eryG structure

[0061] Using the genomic DNA of S.erythraea HL3168 E3 in industrial production as a template, primers 5'AAA CTG CAG CAC CGC GGA AGT CTC GAC ACC-3' and 5'TTT AAG CTT CGC CGG GGC AGT GCA AGT ACG were used as templates -3' and 5'-TTA CCA TGGGGT GCT GTT GCC GTC CCT GCG-3' and 5'-TCG ACT AGT CTA CCG CGTGCT GCG CTC CTA-3' clone the coding sequence of eryK gene and eryG gene, and use 1% agar for the product Sugar gel electrophoresis, the results are as follows figure 1 As shown in .B, it can be seen that the product has a very specific amplification.

[0062] The eryK gene PCR product was cloned into the vector pANT841, sequenced and identified, and the vector pCY3-48-K4 with the correct eryK gene sequence was obtained. The modified erythromycin constitutive promoter PermE was excised from plasmid BSVII-41-F with EcoRI / SacI ＊ The fragment was inserted into the EcoRI / SacI digested p...

Embodiment 3

[0064] Example 3 Contains ermE ＊ -eryK-eryK-eryG structure and ermE ＊ Construction of the recombinant plasmid of -eryK-eryG-eryK structure

[0065] From the plasmid pCY3-48-K4 with correct eryK gene sequencing, the 1.3kb target fragment eryK gene was excised with XhoI and HindIII double enzymes, and cloned into the vector pGEM-7zf digested with XhoI / HindIII to obtain plasmid pCY3-58-F6, and then Then use XbaI / HindIII enzymes to excise the target fragment eryK gene, and use the characteristics of XbaI and SpeI homologous enzymes to clone into the SpeI / HindIII double-cut plasmid pCY3-58-B1, thus obtaining the PermE ＊ -eryK-eryK gene tandem copy form of recombinant plasmid pCY3-62-KK8.

[0066] In addition, from the plasmid pCY3-60-A8 with correct eryG gene sequencing, the 1.5kb target fragment eryG gene was excised with XbaI / HindIII double enzymes, and then cloned into the plasmid pCY3-62-KK8 containing the PermE ＊ -eryK-eryK-eryG gene tandem copy of the recombinant plasmid p...

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Abstract

The invention discloses a recombinant plasmid for a genetic engineering construction, a construction method for the plasmid as well as an application of the plasmid in the heredity reformation of erythromycin producing strain. The recombinant plasmid consists of a vector and a segment which contains erythromycin promoters, carbon 12 hydroxylated enzymes on a large ring backbone and mycarose methylated enzymes and is added to a multiple-clone site, wherein the vector can be the Escherichia coli streptomycete shuttling plasmid, and also be similar vector like pKC1139, pOJ260, pWHM3 or pSET152. The combination is mainly characterized by comprising m erythromycin promoters (ErmE<x>), n hydroxylated enzymes (EryK) and k methylated enzymes (EryG), wherein m is from 1 to 3, n is from 1 to 5 and k is from 0 to 4. The erythromycin promoters, the hydroxylated enzymes and the methylated enzymes can be freely matched and assembled. The recombinant plasmid which is used to carry out the heredity reformation for the erythromycin producing strain is capable of improving components obviously and even obtaining a producing strain which only produces an effective single component.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically, the invention relates to a recombinant plasmid constructed by genetic engineering, a method for constructing the recombinant plasmid, and an application of the plasmid in genetic transformation of erythromycin-producing bacteria. 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, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12N15/52C12N1/15C12R1/645
Inventor 刘文张嗣良陈云邓玮
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI