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Biosynthetic gene cluster of noveltymycin and its application

A biosynthetic and noveltymycin technology, applied in the field of microbial genetic resources and genetic engineering, can solve undiscovered problems

Active Publication Date: 2020-05-08
SUZHOU RES INST SHANDONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After searching the literature of the prior art, no reports have been found on the biosynthetic gene cluster of cvnovycin, the vector containing it and the host, and the application of the gene expression coding protein for the catalytic synthesis of the antibiotic cnovmycin or its analogues

Method used

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  • Biosynthetic gene cluster of noveltymycin and its application
  • Biosynthetic gene cluster of noveltymycin and its application
  • Biosynthetic gene cluster of noveltymycin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Extraction and sequencing of the genome DNA of Actinomycetes florensis Jinan, a noveltymycin-producing bacterium

[0050] Inoculate the Jinan Actinomycetes strain from -80°C to a Gowell solid plate, culture at 30°C for 4 days, scrape the bacteria and inoculate to 168G (glucose 22g, beef extract 4g, peptone 5g, yeast extract 0.5g, casein Add 3g of peptone, 1.5g of table salt, add 900mL of water to sterilize at high temperature, add 10g of glycine to 100mL of water to sterilize separately, add 1 / 10th volume of 10% glycine to the medium when in use) medium 50mL / 250mL Erlenmeyer flask, 30℃ , cultivated at 150rpm for 4 days, the cells formed milky white pellets, put 1.8mL into 2mL centrifuge tubes, centrifuged at 13000rpm for 5 minutes, poured off the supernatant and washed with 1.6mL of water, centrifuged at 13000rpm for 5 minutes, discarded the supernatant, Resuspend the cells with 450μL of 10mM Tris-HCl pH8.0, add 30μL of 20mg / mL proteinase K, mix well, add 40μ...

Embodiment 2

[0055] Example 2: Direct cloning of noveltymycin biosynthetic gene cluster

[0056] After Genomic DNA was extracted from Actinomycetes jinanensis, take 200 μl, about 300 μg / μl, and perform double enzyme digestion with EcoRV and AscⅠ, 400 μl enzyme digestion system, after incubating at 37°C for 3 hours, extract DNA once with phenol chloroform, then ethanol Precipitation recovery, with a small amount of ddH 2 O dissolved. The linear cloning vector p15A-cm-HA, HA is the homologous arm of the cenomycin gene cluster, obtained by amplification of the plasmid p15A-cm-tetR-ccdB-hgy. 5 μg of genomic DNA and 0.5 μg of linear cloning vector were co-electroporated into GB05dir expressed by induced recombinase, recovered at 37°C for 1 hour, spread to LB plates containing cm, cultured overnight at 37°C, and clones were picked with a medium containing cm LB was cultured overnight, the plasmid was extracted and identified by enzyme digestion, the recombinant plasmid p15A-cm-cxm containing t...

Embodiment 3

[0057] Example 3: Heterologous expression of noveltymycin biosynthetic gene cluster

[0058] Using pSET152 as a template, amplify intgrase-attP-oriT-apra carrying a homology arm, and transform the obtained PCR product with the plasmid p15A-cm-cxm directly cloned from the gene cluster into the strain GB05red that has been induced to express the recombinase Line loop recombination, picking clones for enzyme digestion and identification, obtained the combined transfer plasmid p15A-cm-apra-cxm integrated with the noveltymycin gene cluster. apra-cxm moved to Streptomyces coelicolor A3(2) through conjugation transfer and integrated into the attB site of its chromosome, cultured at 37°C for 17h-18h, then mixed with 1 ml of sterile water containing appropriate amount of antibiotics and nalidixic acid (with to inhibit the growth of Escherichia coli) to cover, upside-down culture at 30°C for 7 days to see zygote growth, pick clones for PCR verification, and inoculate the correct clones ...

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Abstract

The invention belongs to a biosynthetic gene cluster of creatmycinum. A nucleotide sequence of the gene cluster is as shown in SEQ ID No. 1 and is named as cxm; the gene cluster comprises 10 genes related to biosynthesis of coding creatmycinum, wherein 7 genes are biosynthesis-related genes; one gene is a controlling gene, one gene is a resistance gene and one gene is a transport gene; the nucleotide sequences of the genes are as shown in SEQ ID No. 2 to SEQ ID No. 11. The gene function is validated through a test of heterogeneous expression combined with same-box gene deletion. The invention further discloses the gene cluster or an application of coded protein expressed by the gene cluster in catalyzed synthesis of the creatmycinum or analogues thereof, and biosynthesis of the creatmycinum is achieved. The gene and the protein thereof can be used for searching and discovering a compound or the gene or the protein capable of being applied to medicines, industry or agriculture.

Description

technical field [0001] The invention belongs to the field of microbial genetic resources and genetic engineering, and in particular relates to a noveltymycin biosynthetic gene cluster, a carrier containing it, a host and an application thereof. Background technique [0002] Chuangxinmycin (chuangxinmycin, CXM) has structural similarity to tryptophan (see formula (I)), and is a highly efficient selective inhibitor of bacterial tryptophanyl tRNA synthetase (TrpRS). [0003] [0004] For example, the IC50 of noveltycin on TrpRS of Escherichia coli is 30 nM, but it has no inhibitory effect on sheep TrpRS at a concentration of 30 μM, showing that noveltycin is a specific inhibitor (Brown et al, 2002). Recent studies have shown that tryptophanyl tRNA synthetase inhibitors (indomycin and noveltymycin) can disrupt the biosynthesis of tryptophan-rich proteins associated with resistance to chloramphenicol, erythromycin, and vancomycin , thereby weakening the resistance of bacteria...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00C12P17/18
CPCC12N15/52C12P17/18
Inventor 卞小莹张友明许晓坤
Owner SUZHOU RES INST SHANDONG UNIV
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