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Strong promoter suitable for streptomyces and application thereof

A strong promoter, Streptomyces technology, applied in the field of genetic engineering and microbial metabolic engineering

Pending Publication Date: 2021-11-19
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there are very few promoters that can be applied to Streptomyces. As far as constitutive promoters are concerned, there are only ermEp*, SF14p and kasOp*, and inducible promoters are only tipAp and nitAp.

Method used

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  • Strong promoter suitable for streptomyces and application thereof
  • Strong promoter suitable for streptomyces and application thereof
  • Strong promoter suitable for streptomyces and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1. Strong promoter stnK4p host adaptability test

[0037] Using Streptomyces flocculus CGMCC 4.1223 as the template, a strong promoter stnK4p was obtained by amplification, and the promoter was evaluated with xylE as the reporter gene. This strong promoter can be applied to four commonly used Streptomyces type strains. like figure 1 As shown, the four commonly used Streptomyces type strains are Streptomyces albus, Streptomyces lividans, Streptomyces coelicolor, and Streptomyces venezuelae. The promoters are ermEp* and kasOp*, the strong promoter stnK4p in each of the commonly used Streptomyces strains, the expression of the reporter gene xylE is much higher than the corresponding expression levels of the control promoters ermEp* and kasOp*.

Embodiment 2

[0038] Example 2. Construction of promoter-containing plasmid vector

[0039] (1) Using the Streptomyces flocculus CGMCC 4.1223 genome as a template, the primer pairs are stnK4p-F and stnK4p-R, and the nucleotide sequence of the primer stnK4p-F is shown in the sequence table SEQ ID NO: 2; the primer stnK4p The nucleotide sequence of -R is shown in SEQ ID NO: 3 in the sequence table; PCR amplification is performed with a high-fidelity enzyme to obtain a promoter stnK4p fragment, which is verified by electrophoresis and recovered by electrophoresis gel to obtain a purified stnK4p fragment.

[0040] (2) Using Streptomyces flocculus CGMCC 4.1223 genome as a template, using primer pairs 3HAA-F and 3HAA-R, the nucleotide sequence of primer 3HAA-F is as shown in SEQ ID NO: 4 in the sequence listing; primer 3HAA- The nucleotide sequence of R is shown in SEQ ID NO: 5 in the sequence table; PCR amplification was performed with a high-fidelity enzyme to obtain stnM1 / M2 / N / M3 gene fragment...

Embodiment 3

[0044] Embodiment 3, the construction of promoter stnK4p high-yielding 3-hydroxyanthranilic acid strain

[0045] (1) The recombinant plasmid pSET152-stnK4p-stnM1 / M2 / N / M3 was transformed into Escherichia coli ET12567 / pUZ8002, and coated on 50mg / L apramycin, 50mg / L kanamycin and 25mg / L From the LB solid plate of chloramphenicol, pick a single clone and culture it in LB medium containing 50 mg / L apramycin, 50 mg / L kanamycin and 25 mg / L chloramphenicol at 37°C for 7-8 h. The bacterial liquid was centrifuged, the supernatant was removed, the bacteria were resuspended with an appropriate amount of 20% glycerol, and stored in a -80°C refrigerator for later use.

[0046] (2) Inoculate Escherichia coli ET12567 / pUZ8002 (containing the recombinant plasmid) into 4 mL of LB medium containing 50 mg / L apramycin, 50 mg / L kanamycin and 25 mg / L chloramphenicol, and culture at 37°C overnight . Then, 50 μl of the bacterial solution was drawn and added to 5 ml of LB medium containing 50 mg / L apr...

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Abstract

The invention discloses a strong promoter suitable for streptomyces and an application thereof, and relates to the field of genetic engineering and microbial metabolism engineering. The nucleotide sequence of the strong promoter comprises a sequence (a); a sequence (b) which has 75% or more consistency with the sequence (a) and has a promoter function, or a sequence (c) which is hybridized with the sequence (a) or the sequence (b) under a highly rigorous condition and has a promoter function; a plasmid vector containing the strong promoter; and a host cell containing the plasmid vector, which comprises streptomyces albus, streptomyces lividoviride, streptomyces coelicolor and streptomyces Venezuraea. The invention also discloses an application of the strong promoter, the plasmid vector and the host cell in starting expression of a target gene. The strong promoter can be applied to common streptomyces type strains, and has important significance on high yield of important proteins including enzymes and important metabolites from actinomycetes.

Description

technical field [0001] The invention relates to the fields of genetic engineering and microbial metabolic engineering, in particular to a strong promoter suitable for Streptomyces and its application. Background technique [0002] The promoter is a special DNA sequence located upstream of the gene transcription unit, which has the function of initiating gene transcription and plays an important role in regulating the level of gene transcription. Metabolic engineering often needs to express exogenous genes or regulate the expression of endogenous genes, and the choice of promoter is very important for gene expression regulation. Promoters affect the transcription level of genes, affect the coordination between genes in the synthetic pathway or the original pathway, and then affect the metabolic function of the strain. In order to improve the yield of target substances, a common method in metabolic engineering is to use strong promoters to overexpress the genes encoding key e...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/76C12N15/67C12N1/21
CPCC07K14/36C12N15/76C12N2830/008
Inventor 林双君郭文丽黄婷婷
Owner SHANGHAI JIAO TONG UNIV
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