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Engineering bacteria genetically modified by neomycin biosynthetic gene cluster, and applications thereof

A technology of biosynthesis and genetic engineering, applied in genetic engineering, plant genetic improvement, microbial-based methods, etc., can solve the problems of negative mutation, time-consuming and labor-intensive, and efficiency reduction of mutagenesis breeding

Active Publication Date: 2020-04-17
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional breeding of antibiotic high-yield strains is mainly achieved through mutagenesis breeding. Although this method is simple and effective, it is time-consuming and labor-intensive. Moreover, with the increase in the yield of mutagenized strains, negative mutations are likely to occur during further mutagenesis breeding, and the efficiency will gradually decrease. decline
In the past few decades, many methods or strategies for increasing the production of antibiotics have been developed. In early practice, it was found that increasing the expression of related structural genes involved in key steps in the gene cluster can effectively increase the production, but the secondary There are often more than one key step in the metabolic product synthesis pathway, and improving the conversion efficiency of only one step may cause another catalytic reaction to become a new rate-limiting step

Method used

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  • Engineering bacteria genetically modified by neomycin biosynthetic gene cluster, and applications thereof
  • Engineering bacteria genetically modified by neomycin biosynthetic gene cluster, and applications thereof
  • Engineering bacteria genetically modified by neomycin biosynthetic gene cluster, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Cloning of the Neomycin Biosynthesis Gene Cluster

[0040]The cloning strategy of neomycin biosynthesis gene cluster adopts φBT1 attP-attB-int integration system (Zhang, L., et al. (2008). Highly efficient in vitro site-specific recombination system based on Streptomyces phage φBT1 integrase. Journal of Bacteriology, 190(19), 6392; Du, D., et al. (2015). Genome engineering and direct cloning of antibioticgene clusters via phageφBT1 integrated-mediated site-specific recombination in Streptomyces. Scientific Reports, 5, 8740.), first need to pass Two homologous single exchanges will attB 6 and attP 6 The sequence and temperature-sensitive plasmid pKC1139 was introduced into both ends of the target gene cluster through the homology arm sequences on both sides of the cluster, so two plasmids for homologous integration were constructed: pIJ778::attB 6 neoUp and pKC1139::attP 6 neoDn.

[0041] Based on a section of pIJ778 containing the resistance gene and oriT...

Embodiment 2

[0045] Embodiment 2 Transformation of Neomycin Biosynthetic Gene Cluster

[0046] The negative regulatory gene neoI (SEQ ID NO.8) in the cloned neomycin biosynthesis gene cluster was blocked by PCR targeting large fragment editing method. First, pNIK-DNI (Zhuo, J., et al.( 2017). Reconstruction of a hybrid nucleoside antibiotic gene cluster based onscarless modification of large DNA fragments. Science China Life Sciences, 60(9), 1-12.) as a template, using neoItoamp-F / neoItoamp-R as primers for PCR amplification A basic element HapI-bla-HapI was obtained. The two ends of neoItoampF / neoItoampR primers respectively have 39bp homologous sequences with the upstream and downstream of neoI gene so as to facilitate λRED recombination. The above recovered product HapI-bla-HapI was introduced into Escherichia coli E. coli BW25113 / pIJ790 / pKCZ01 strain by electrotransformation (BW25113 / pIJ790 strain was used for gene editing by PCR targeting method. The pIJ790 plasmid contains λ-RED rec...

Embodiment 3

[0048] The construction of embodiment 3 neomycin high-yielding engineering strains

[0049] The specific operation process of joint transfer is as follows:

[0050] Firstly, the plasmids constructed above (pKCZ01, pKCZ02, pKCZ03) were transformed into E.coli ET12567 / pUZ8002 (see Paget, M.S., Chamberlin, L., Atrih, A., Foster, S.J. and Buttner, M.J. (1999). Evidence that the extracytoplasmic function sigma factor sigma E is required for normal cell wall structure in Streptomyces coelicolor A3(2).JBacteriol 181,204–211.), pick the clone containing the recombinant plasmid and put it in liquid LB medium (tryptone 10g / L, yeast extract 5g / L, chloride Sodium 10g / L, pH 7.0) to OD 600 = 0.4 to 0.6 (100 µg / mL kanamycin, 100 µg / mL apramycin and 25 µg / mL chloramphenicol were added during culture). The collected bacteria were washed twice with fresh LB medium without antibiotics, and resuspended in 2×YT medium (tryptone 16g / L, yeast powder 10g / L, NaCl 5g / L, pH 7.0) in equal volume for ...

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Abstract

The invention provides a method for constructing a high-yield neomycin engineering strain streptomyces fradiae. The method comprises: introducing a neomycin biosynthetic gene cluster of streptomyces fradiae into wild streptomyces fradiae. The method can also comprise: carrying out genetic engineering modification on the neomycin biosynthetic gene cluster of streptomyces fradiae to be introduced into wild streptomyces fradiae, wherein genetic engineering modification can comprise: blocking the negative regulatory gene neoI in the neomycin biosynthetic gene cluster or blocking the negative regulatory gene neoI in the neomycin biosynthetic gene cluster while driving the transcription of a key enzyme encoding gene neoE-neoD co-transcription unit by using a strong promoter. The invention also provides high-yield neomycin streptomyces fradiae obtained by the method.

Description

technical field [0001] The invention belongs to the field of microbial pharmacy, and specifically relates to cloning a complete neomycin biosynthetic gene cluster from a strain of Streptomyces fradiae, and performing genetic manipulation on the gene cluster to obtain a neomycin high-yield engineering strain, for Medicine and industrial production provide a way to increase the production of neomycin. Background technique [0002] Streptomyces (Streptomyces) is a class of Gram-positive soil filamentous bacteria with high GC content, belonging to the prokaryotic kingdom Actinomycetes Streptomycetes. Streptomyces has complex morphological differentiation and can produce abundant secondary metabolites. About two-thirds of the known antibiotics produced by microorganisms are derived from actinomycetes, and more than 80% of them are derived from Streptomyces In addition to inhibiting and killing bacteria, these antibiotics can also be used as anticancer drugs, herbicides, enzyme i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12N15/53C12P19/52C12R1/54
CPCC12N15/74C12P19/52C12N9/0006
Inventor 谭华荣李月郑家珍张集慧田宇清关晗晔
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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