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Construction method of genetically engineered strain producing tetracycline single component

A technology of genetically engineered strains and construction methods, which is applied in the field of genetically engineered strain construction, can solve the problems of undiscovered, limited synthetic research, genetic modification, and changes in the secondary metabolite components and yield of strains, so as to save production costs, The effect of avoiding industrial pollution and simplifying the process flow

Active Publication Date: 2016-05-04
SHANGHAI JIAOTONG UNIV
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  • Description
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Problems solved by technology

Due to the difficulty of genetic manipulation of Streptomyces aureus, the research on the biosynthesis of tetracycline antibiotics can only be limited to random mutant strains, which greatly limits the research on tetracycline biosynthesis and the specific genetic modification by metabolic engineering.
[0004] After searching the existing technical literature, no related technical reports were found to change the secondary metabolite components and yield of the strain by knocking out the genes related to the chlorination reaction

Method used

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  • Construction method of genetically engineered strain producing tetracycline single component
  • Construction method of genetically engineered strain producing tetracycline single component
  • Construction method of genetically engineered strain producing tetracycline single component

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1, construction of homologous exchange plasmid

[0039] Used to import the host and undergo homologous recombination with the chromosome in order to obtain the target mutation, specifically using CopyControl TM The Streptomycesaureofaciens F3 genomic library was constructed with the FosmidLibraryProduction kit, and the probe was designed according to the halogenase gene to confirm that the cosmid 11D1 contained a complete CTC synthesis gene cluster. The cosmid 11D1 was recombined with the chlorase gene through λ-Red-mediated E. coli gene recombination The ctcP was mutated to obtain plasmid pJTU4301. First, the plasmid pIJ778 was double-digested with EcoRI / HindIII, and the 1.5kb fragment containing oriT+addA was recovered. This fragment was transformed into E. coli DH10B, and no single colony could grow on the spectinomycin-resistant plate, which was considered qualified. The recovered fragment was used as a template, amplified with primers targPF / targPR, a...

Embodiment 2

[0042] Embodiment 2, introducing the plasmid used for homologous recombination with chromosome into wild-type host

[0043] The constructed plasmid was transformed into Streptomyces aureus (Streptomycesaureofaciens F3) by conjugative transfer between Escherichia coli and Streptomyces parents. The cosmid pJTU4301 with ctcP mutation must be assisted by the helper plasmid pUZ8002 to be introduced into recipient Streptomyces aureus cells by conjugative transfer. pJTU4301 was first transformed into E.coliET12567 (carrying pUZ8002), cultured overnight in the presence of chloramphenicol, spectinomycin and kanamycin, transferred to culture for 2 hours according to 1 / 10 inoculum, collected the bacteria, and used fresh LB medium Wash the bacteria 3 times for later use. Streptomyces spores used as recipients need to be heat-shocked and pre-germinated. Suspend Streptomyces spores in TES buffer (5ml0.05mol / L, pH8.0), heat shock in a water bath at 50°C for 10min, cool to room temperatur...

Embodiment 3

[0044] Embodiment 3, screening and verification of mutant strains

[0045] Pick a single conjugative transfer from the overlay plate and inoculate it on a resistant (spectinomycin) plate to further confirm the resistance, and expand the plate culture. The total DNA of the candidate strain was used as the PCR template; the primers targPCF and TargPCR were used for the screening of mutant strains, and the DNA of the original starting strain was used as the control. The complete coding sequence of the ctcP gene of the mutant strain was replaced by the spectinomycin resistance gene sequence, and the PCR product size was 1601bp, while the PCR product size of the starting strain was 1905bp, thus finally confirming the correctness of the target mutation.

[0046] targPCF,5'-ACCACTTCGCCATCGGCGTCAAGTC-3'; (SEQ ID NO.3)

[0047] targPCR, 5'-CGAGGTGCCCAGCCCGCTGATGTAC-3'; (SEQ ID NO.4)

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Abstract

The invention relates to a method for establishing a genetic engineering strain for producing a single component tetracycline in the technical field of biological medicine. The method comprises a step of performing gene disruption of the related genes involved in aureomycin synthesis and chlorination in aureomycin producing bacteria so as to obtain a genetic engineering strain only producing tetracycline. According to the method provided by the invention, a homologous exchange plasmid is established, and DNA (deoxyribonucleic acid) homologous recombination is performed in the aureomycin producing bacteria; and the chlorination enzyme gene ctcP in charge of the chloro step is replaced by a spectinomycin resistance gene in an aureomycin synthesis process so as to disrupt the chloro step and obtain the genetic engineering strain only producing tetracycline. The strain can be applied to industrial production and has the obvious effects that (I) the strain ZTO4 obtained by genetic engineering reform only produces a single component tetracycline, and the output is as high as 18.9g / L; and (II) compared with the current fermentation technology, potassium bromide is not added as an inhibitor in a fermentation process, thus the production cost is saved, the technological flow is simplified, and industrial pollution caused by potassium bromide is avoided.

Description

technical field [0001] The invention relates to a method for constructing a genetically engineered bacterial strain producing a single component of tetracycline, and belongs to the technical field of biomedicine. Background technique [0002] Tetracycline antibiotics are a large class of broad-spectrum antibiotics that inhibit bacterial protein synthesis, and are effective against a variety of Gram-positive and negative bacteria, Rickettsia, Mycoplasma, and Spirochetes. Including the first-generation tetracycline antibiotics tetracycline, chlortetracycline and oxytetracycline, which are mainly composed of chlortetracycline-producing Streptomyces aureofacines and oxytetracycline-producing Streptomyces saureofacines ( Streptomycesrimosus) produced by fermentation. In the 1970s, semi-synthetic technology was used to chemically modify tetracycline and oxytetracycline, and the second-generation tetracycline antibiotics such as 7-chloro-6-nortetracycline, doxycycline and oxytetra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/76C12N1/21C12R1/485
Inventor 由德林朱涛邓子新
Owner SHANGHAI JIAOTONG UNIV
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