Nosiheptide biosynthetic regulatory protein NosP specific binding sequence and related application thereof

A technology of biosynthesis and combination of sequences, which is applied in the fields of application, biochemical equipment and methods, plant gene improvement, etc., can solve the problems such as the unknown influence of DNA sequence nosipeptide biosynthesis titer, so as to improve fermentation titer and reduce production cost effect

Inactive Publication Date: 2015-07-08
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bioinformatics analysis showed that the nosP gene was located at one end of the nosipeptide biosynthetic gene cluster in S.actuosus. It was speculated that the NosP protein belonged to the SARP family and was involved in the specific regulation of the nosipeptide biosynthetic pathway. Potency effects are unknown

Method used

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  • Nosiheptide biosynthetic regulatory protein NosP specific binding sequence and related application thereof
  • Nosiheptide biosynthetic regulatory protein NosP specific binding sequence and related application thereof
  • Nosiheptide biosynthetic regulatory protein NosP specific binding sequence and related application thereof

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Experimental program
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Effect test

Embodiment 1

[0018] Using the S. actuosus ATCC 25421 genome as a template, the 5'-end truncated nosP gene lacking 207 bases was amplified by PCR, and restriction endonuclease sites were inserted in the upstream and downstream of the gene sequence, using the technology in the art The familiar enzyme digestion-ligation method is used to construct recombinant expression plasmids containing different selection markers: Scheme 1: With the help of restriction sites NdeI / HindIII and pET22b(+), insert a histidine tag at the C-terminus of NosP to obtain a recombinant plasmid Is pET22b-his-c; Scheme 2: With the help of restriction sites NdeI and HindIII, insert a histidine tag at the N-terminus of NosP to obtain the recombinant plasmid pET28a-his-n; Scheme 2: With the help of restriction sites BamHI and XhoⅠ was inserted into the GST tag at the N-end of NosP, and the recombinant plasmid was obtained as pGEX4T-gst-n. After sequencing and verification, the above recombinant plasmid was transferred into ...

Embodiment 2

[0020] The three genetic recombinant E.coli obtained in Example 1 were cultured in 50mL LB medium (containing 100μg / mL ampicillin or 50μg / mL kanamycin), shaking overnight at 37°C and 220rpm to obtain the corresponding Seed liquid. Transfer the seed solution to LB medium (containing 100μg / mL ampicillin or 50μg / mL kanamycin) according to the inoculum amount of 5%, and shake culture to the OD of the medium at 37℃ 600 When =0.8±0.1, add the inducer IPTG with a final concentration of 0.001-2mM, induce culture at 25°C for 20 hours to obtain the engineered strain E.coli fermentation broth. The fermentation cells were collected by centrifugation at 4°C and 4500 rpm for 15 min. The cells were washed twice with 40 mM Tris-HCl buffer (pH 8.0), and the cells were collected for SDS-PAGE analysis. The results showed that the three engineering strains of pHC, pHN and pHG all obtained the best soluble expression under the condition of 0.2mM IPTG. The content of NosP accounted for 8.5%, 7.8% an...

Embodiment 3

[0022] The three genetic recombinant E.Coli obtained in Example 1 were cultured in 50mL LB medium (containing 100μg / mL ampicillin or 50μg / mL kanamycin), shaking overnight at 37°C and 220rpm to obtain the corresponding Seed liquid. Transfer the seed solution to LB medium (containing 100μg / mL ampicillin or 50μg / mL kanamycin) according to the inoculum amount of 5%, and shake culture to the OD of the medium at 37℃ 600 When =0.8±0.1, add the inducer IPTG with a final concentration of 0.2mM, and induce culture at 10~37℃ for 20 hours to obtain the engineered strain E.coli fermentation broth. The fermentation cells were collected by centrifugation at 4°C and 4500 rpm for 15 min. The cells were washed twice with 40 mM Tris-HCl buffer (pH 8.0), and the cells were collected for SDS-PAGE analysis. The results showed that the soluble expression of NosP in the three engineering strains of pHC, pHN and pHG reached the maximum at 16°C, accounting for 9.3%, 8.4% and 8.1% of the total protein, r...

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Abstract

The invention discloses a nosiheptide biosynthetic regulatory protein NosP specific binding sequence and an influence thereof upon nosiheptide biosynthesis effectiveness. The invention comprises the following contents: recombinant plasmids comprising specific affinity purification tags and truncated NosP gene are established; a NosP protein pure product is prepared through Escherichia coli heterologous expression and an affinity chromatography method; and with EMSA and DNase I footprinting assay technologies, the NosP specific binding sequence in a streptomyces actuosus nosiheptide biosynthetic gene cluster is found and verified. On this basis, through nosiheptide biosynthetic pathway renovation, nosiheptide biosynthesis effectiveness is improved by 150%, such that an application prospect is good.

Description

Technical field [0001] The invention belongs to the field of regulation and control of microbial secondary metabolite biosynthetic pathways, and specifically relates to the discovery and verification of specific binding of the specific regulatory protein NosP in the thiopeptide antibiotic nosiheptide biosynthesis gene cluster, and the discovery and verification of the specific binding of nosihtine Practical application of biosynthetic potency in active Streptomyces. Background technique [0002] Nosiheptide, also known as polythiamycin, is a typical thiopeptide antibiotic produced by Streptomyces actuosus ATCC 25421. It is one of the first thiopeptide antibiotics discovered and put into use. Nosiheptide has a broad-spectrum and strong inhibitory effect on Gram-positive bacteria, especially some drug-resistant bacteria such as Staphylococcus aureus has a strong killing effect. But due to its low yield, poor water solubility and low bioavailability, it is currently only used as an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/00C12P21/02
Inventor 陈依军张红吴旭日
Owner CHINA PHARM UNIV
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