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Monascus industrial strain for traceless deletion of citrinin synthetic genes

A technology of citrinin and Monascus, which is applied in the field of preparation of Monascus strains, and can solve the problems of inability to completely eliminate citrinin, genetic instability and the like

Active Publication Date: 2020-08-18
GUANGDONG KELONG BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since Monascus is a multikaryotic fungus, traditional gene editing tools cannot delete all multiple copies of genes in the cell at one time, and the citrinin biosynthesis gene in the cell is not completely deleted, so the engineered strain is a heterokaryon, and heredity is extreme. Unstable, citrinin formation still detected
In addition, due to the existence of multiple spare genes or isoenzymes in Monascus, knocking out some citrinin biosynthesis genes will only lead to a reduction, but not a complete elimination of citrinin production

Method used

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  • Monascus industrial strain for traceless deletion of citrinin synthetic genes
  • Monascus industrial strain for traceless deletion of citrinin synthetic genes
  • Monascus industrial strain for traceless deletion of citrinin synthetic genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Construction of CRISPR / Cas9 plasmid pKL-03 and donor plasmid pKL-05 that seamlessly deleted the Monascus citrin biosynthetic gene cluster

[0063] (1) Extraction of Monascus Genomic DNA

[0064] Take the PDA plate cultured by Monascus strain KL-001 for 7 days, place it in -80°C and freeze it for 20 minutes, scrape the surface hyphae after taking it out, wash it twice with sterile water, freeze-dry it and store it at -20°C. Grind the lyophilized mycelia 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 / LEDTA, 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 min, then add 165 µL of 5 µmol / L NaCl solution and mix. Centrifuge at 13 000 rpm for 20 min, immediately transfer the supernatant to a new test tube, and add 400 μL of chloroform and 400 μL of phenol. ...

Embodiment 2

[0109] Embodiment 2, the complete deletion of Monascus citrin biosynthesis gene cluster

[0110] (1) Transformation of Monascus with pKL-03 and pKL-05 plasmids

[0111] The preparation method of Monascus protoplast is as follows:

[0112] a. Inoculate Monascus KL-001 onto a PDA plate and culture it in a 30°C incubator for 7 days.

[0113] b. Cut off the mycelium block with a sterile knife and inoculate into 200mL YEME liquid medium (yeast powder 3g / L, peptone 5g / L, malt extract 3g / L, glucose 10g / L, sucrose 130g / L and distilled water 1L ; Add MgCl after autoclaving 2 ·6H 2 O (2.5M) 2mL / L and glycine (10%) 100mL / L), cultured at 30°C for 30h.

[0114] c. Collect the hyphae in the culture medium by centrifugation at 10000×g, add 15 mL Osmotic Medium and wash 3 times.

[0115] d. Add 10mL of mixed enzymatic hydrolysis solution (100mg Lysing Enzyme, 30mg Yatalase, 30mg cellulase and 40mg helicase), culture in a shaker at 30°C and 80rpm, observe the enzymatic hydrolysis of mycel...

Embodiment 3

[0128] Embodiment 3, Monascus engineering strain MP-20 detects citrinin and pigment

[0129] (1) Detection of engineering strain MP-20 citrinin

[0130] The verified engineering strain MP-20 and the wild-type strain KL-001 were inoculated into PDB medium for fermentation, cultured on a shaker at 30°C and 200rpm for 7 days, and samples were taken.

[0131] Take 30 mL of fermentation broth, adjust the pH of the fermentation broth to 4.0 with HCl, extract the mycelium and supernatant with ethyl acetate for three times, and combine the organic phases. Rotary evaporate at 30°C until completely dry, add 300 μL methanol to dissolve. Filter through a 0.22 μm filter membrane, and detect citrinin by liquid chromatography-mass spectrometry (LC-MS).

[0132] Citrinin LC-MS detection condition: carry out LC-MS analysis on Agilent-1200HPLC / 6520QTOFMS (USA) system, mobile phase is acetonitrile (v / v, 0.1% formic acid) and water (v / v, 0.1% formic acid), Flow rate 0.8mL / min. 0-15min, 5%-95%...

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Abstract

The invention relates to a monascus industrial strain capable of not producing citrinin. The citrinin biosynthetic gene cluster on the monascus genome is completely deleted through a CRISPR / Cas9 mediated traceless knockout technology, and the method does not introduce any exogenous gene into the genome, so that the obtained new strain MP-20 completely loses the citrinin production capacity. Ten continuous fermentation results of the new strain MP-20 show that citrinin cannot be completely detected in the product, and the pigment yield is increased by 2.33%.

Description

technical field [0001] The invention belongs to the field of food biotechnology, and in particular relates to a genetically modified monascus strain not producing citrinin, and relates to a preparation method and application of the monascus strain. Background technique [0002] Monascus is an industrially important multinucleated filamentous fungus capable of producing natural and organic monascus pigments, and their mixtures are widely used as food colorants in Asian countries. However, Monascus also produces citrinin, a mycotoxin that is nephrotoxic due to inhibition of respiratory complex III, which greatly limits the application of Monascus pigment. Many countries have enacted regulations to strictly limit the citrinin content in Monascus and related foods. At present, the content of citrinin in China's industrial red yeast rice pigment generally exceeds the standard, which seriously affects the export of products. Moreover, as people pay more and more attention to foo...

Claims

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Application Information

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IPC IPC(8): C12P1/02C12N1/15C12N15/80C12N15/90C12N1/14C12R1/645
CPCC12P1/02C12N15/52C12N15/80C12N15/902C12N1/14C12R2001/645C12N1/145
Inventor 李绮雯容艳筠肖伟俊高书山高健信李英明
Owner GUANGDONG KELONG BIOLOGICAL SCI & TECH
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