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Strain and application thereof in fermentation production of ergot alkaloid

A technology of ergot alkaloids and bacterial strains, which is applied in the field of microbial engineering and can solve problems such as low production capacity

Active Publication Date: 2021-11-16
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As another example, opioids such as morphine are clinically important analgesics, and their production mainly comes from the extraction of opium poppy plants, and the production capacity is low

Method used

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  • Strain and application thereof in fermentation production of ergot alkaloid
  • Strain and application thereof in fermentation production of ergot alkaloid
  • Strain and application thereof in fermentation production of ergot alkaloid

Examples

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Embodiment 1

[0101] (1) Extraction of fungal genomic DNA

[0102] The fungal strains were taken, cultured on PDA plates for 3 days, the surface hyphae were scraped, washed twice with distilled water, freeze-dried and stored at -20°C. Grind the lyophilized mycelium into a fine powder using a liquid nitrogen precooled mortar, and resuspend in 500 μl of lysis buffer (40 μmol / l Tris-acetate, 20 μmol / l sodium acetate, 1 μmol / L EDTA, 1 % w / v SDS, pH 7.8), pipette until the viscosity of the suspension decreases significantly and foam forms. Add RNase A and incubate at 37°C for 5 minutes, then add 165 μl of 5 μmol / L NaCl solution and mix. After centrifugation at 13 000 rpm for 20 min, the supernatant was immediately transferred to a new test tube, and 400 μl of chloroform and 400 μl of phenol were added. Gently invert the tube until the solution becomes milky. Centrifuge for 20 minutes to remove the aqueous phase and extract with an equal volume of chloroform. Precipitate the DNA in the supern...

Embodiment 2

[0184] Example 2 Heterologous production of N-Me-DMAT and prechanoclavine (PCC) in Aspergillus nidulans

[0185] Primers were designed according to the sequences of the genes in the Aspergillus fumigatus ergot alkaloid biosynthesis gene cluster, and dmaW, easF, and easE (A.fum) gene fragments were amplified by PCR, which were used for the construction of pEA01, pEA02, and pEA03 plasmids, respectively.

[0186] The digested pYTU, pYTR, and pYTP vectors were mixed with gene fragments easF, dmaW, and easE (A.fum) respectively, and pEA01, pEA02, and pEA03 plasmids were obtained by yeast assembly. Then sequencing verified that the correct plasmids were mixed and transformed into Aspergillus nidulans to obtain the transformed strain An01, and the product was detected and quantified by fermentation.

[0187] On the basis of strain An01, additional copies of easF, dmaW, and easE (A.fum) genes were introduced to obtain strain An02; on the basis of strain An02, endogenous genes thmgR, s...

Embodiment 3

[0189] Example 3 Heterologous production of chanoclavine (CC) in Aspergillus nidulans

[0190] Design primers according to the sequence of each gene on the Aspergillus fumigatus ergot alkaloid biosynthetic gene cluster, and PCR amplify to obtain dmaW, easF, easE (A.fum), easC (A.fum) gene fragments, which are required for the expression of channoclavine Plasmid construction. Construct dmaW and easF to pYTU to obtain plasmid pEA10; construct easC(A.fum) to pYTR to obtain plasmid pEA11; construct dmaW, easF, easE(A.fum), easC(A.fum) to pYTU to obtain plasmid pEA12; co-construct easF, easE(A.fum), easC(A.fum) into pYTP to obtain plasmid pEA13; co-construct dmaW, easE(A.fum), easC(A.fum) into pYTR to obtain plasmid pEA14.

[0191] The correct plasmids verified by sequencing were mixed and transferred into Aspergillus nidulans, among which: pEA10, pEA03, pEA11 were co-transformed to obtain strain An07; pEA12, pEA13 were co-transformed to obtain strain An08; pEA12, pEA13, pEA09 wer...

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Abstract

The invention relates to the technical field of microbial engineering, in particular to a strain and application thereof in fermentation production of ergot alkaloid. According to the strain, Aspergillus nidulans is taken as a chassis cell, and 9 kinds of ergot alkaloid compounds with different structures and two kinds of path intermediates are obtained through a biosynthesis path of heterologous reconstructed ergot alkaloid. A plurality of ergot alkaloid high-yield strains are obtained through path optimization, precursor supply and P450 electron transport chain optimization. On the basis, the heterologous high yield of the ergot alkaloid compound in the aspergillus nidulans is realized.

Description

technical field [0001] The invention relates to the technical field of microbial engineering, in particular to the application of bacterial strains and their fermented production of ergot alkaloids. Background technique [0002] Parkinson's disease (PD) is a progressive, neurodegenerative movement disorder caused by the loss of brain cells that can produce dopamine, which seriously affects the daily life and work of patients. According to statistics, there are nearly 3 million PD patients in China, accounting for half of the global PD patients, and it is the country with the largest number of PD patients in the world. In my country, there are about 100,000 new patients each year, with an incidence rate of 1.7% over the age of 65 and 3%-5% at the age of 70. It is the "third killer" of middle-aged and elderly people after tumors and cardiovascular and cerebrovascular diseases. . The treatment of PD is currently mainly based on levodopa drugs, but long-term use can cause motor...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/52C12N1/15C12P17/18C12R1/66
CPCC12N15/80C12N15/52C12N9/00C12P17/182C12P17/188
Inventor 高书山姚永鹏王伟安春艳张军颜芮
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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