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Micafungin precursor FR901379 high-producing strain and application thereof

A technology of FR901379 and micafungin, which is used in fungi, electricity/wave energy treatment of microorganisms, peptides, etc., can solve problems such as limitations, difficulty in comprehensively improving metabolic flux of synthetic pathways, and limited targets.

Active Publication Date: 2022-08-09
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High-performance fermentation strains are a key factor in production cost control, and also a technical threshold that restricts the industry's access
Metabolic engineering of core pathways of biosynthesis is a common strategy for genetic breeding. However, systematic analysis found that the targets for rational modification in the core pathways are very limited, and transcriptome data show that the relative transcription levels of key genes in the synthetic pathways are all high. , when the regulatory mechanism is unclear, it is difficult to enhance the metabolic flux of the synthesis pathway by strengthening the expression strategy of key genes
However, the comprehensive fermentation performance of the strain needs to be improved urgently, and the relevant rational transformation needs theoretical guidance

Method used

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  • Micafungin precursor FR901379 high-producing strain and application thereof
  • Micafungin precursor FR901379 high-producing strain and application thereof
  • Micafungin precursor FR901379 high-producing strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Establishment of a high-throughput screening method for the production strain of micafungin precursor FR901379

[0033] Taking ColeophomaempetriMK01 wild strain as the starting strain, first take out a frozen glycerol tube from the -80°C refrigerator, spread it on a PDA plate after dilution, and invert at 25°C for 3 to 5 days. After the size of the single colony is suitable, use a sterile toothpick to pick the point. 15cm PDA plate, invert at 25°C for 4 days, then take the Candida albicans solution in logarithmic growth phase for 8 to 14 hours, and dilute it to OD with sterile water 600 =0.6~1, take 500 μL of soft agar and mix it in a soft agar that is not hot to the hands and cover the plate. After 24 hours, the diameter of the inhibition zone can be measured, and the diameter of the inhibition zone will not change with the extension of time ( figure 1 ). Among them, direct spraying of Candida albicans after single colony picking plate growth instead of coa...

Embodiment 2

[0034] Example 2. Mutagenesis of ColeophomaempetriMK01 by heavy ion irradiation

[0035] Take out a frozen glycerol tube from the -80°C refrigerator, spread it on a PDAS plate after dilution, and invert at 25°C for 5-8 d, so that the spores of the strain are in a mature state. Take 2-3 mL of sterile physiological saline in the PDA plate, brush the spores with a small sterile brush, filter the washed spores with a 300-500 mesh filter cloth, collect the spore suspension and wash it with sterile physiological saline 2-3 times , count with a spore counter and dilute it to 104 ~10 6 CFU / mL of spore suspension.

[0036] Pipette 1mL of spore suspension and spread it evenly in a sterile 35mm irradiated petri dish, and irradiate the flat area with the irradiation doses of 0Gy, 40Gy, 80Gy, 100Gy, 120Gy, 140Gy, 160Gy, 200Gy, 500Gy, and 800Gy, respectively. Each dose is done in three parallels.

[0037] The spore suspension after irradiation and mutagenesis was stored in a 20%-50% glyc...

Embodiment 3

[0038] Example 3. The initial screening of the FR901379 inhibition zone diameter of the strain after mutagenesis

[0039] Since the germination rate of the ColeophomaempetriMK01 strain itself is relatively low, we diluted the strains under different mutagenic doses to 1 × 10 -3 After the gradient of CFU / mL, 100 μL to 200 μL was uniformly spread on the surface of PDA medium, and cultured at 25°C for 4 to 6 days. Mutant strains with different shapes were obtained under different mutagenic doses. These strains mainly appeared after spotting. Three colony forms were obtained, one was the same as the control strain, and the other two were small black and firm, small black and shriveled ( image 3 ).

[0040] Use a sterile toothpick to select a PDA plate with a single colony point of 15cm, culture at 25°C for 3-4 days, and dilute the Candida albicans in logarithmic growth phase to OD 600 =0.6~1, take OD 600 A suitable amount of rosary albicans of = 0.6-1 is mixed in a soft agar m...

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Abstract

The invention provides a high-yield strain of FR901379, the strain is Coleophoma empetris H40-23, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.40075, the preservation date is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, the telephone number is 010-64807355, and the preservation number is CGMCC No.40075. The invention also provides a method for preparing the high-yield strain of FR901379. The invention also provides an application of the strain in production of the FR901379.

Description

technical field [0001] The invention relates to the technical field of microbial breeding, in particular to a high-yielding strain of micafungin precursor FR901379 and its application. Background technique [0002] Echinocandins are a new type of antifungal drugs, which interfere with the synthesis of fungal cell walls by inhibiting the activity of β-1,3-glucan synthase. Mammalian cells do not have cell walls, so the toxic and side effects of these drugs are small. It has high safety and has antibacterial activity against Candida, Aspergillus and some fungi that are resistant to azoles. The echinocandins that have been approved for marketing include caspofungin (launched in the United States in 2004), micafungin (launched in Japan in 2005), and anidulafungin (launched in the United States in 2006). They all have similar non-ribosomal six-membered cyclic peptide core structures, and the only difference lies in the side chain group, amino acid connection sequence and post-mod...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P21/04C12N13/00C07K7/56C07K1/107C07K1/14C07K1/36C12R1/645
CPCC12N1/145C12P21/02C12N13/00C07K7/56C12R2001/645Y02W30/40
Inventor 吕雪峰黄雪年刘永娟王贝贝门萍周宇谷猛
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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