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Method for constructing genetic engineering fungus of monascus with no citrinin

A technology of genetically engineered bacteria and a construction method, which is applied in the field of constructing and breeding high-yield engineered bacteria that do not produce citrinin and Monascus pigment, can solve problems such as pollution and concomitant occurrence, and achieve safety assurance, quality improvement, and solution to citrinin Effects of toxin concomitant and pollution problems

Inactive Publication Date: 2005-10-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Utilizing the achievement of the present invention, it can be used to block the metabolic pathway of citrinin in Monascus, and then fundamentally solve the problem of citrinin accompanying and pollution in the pigment production process, ensure the safety of Monascus products, and achieve the improvement of Monascus products Purpose of quality and depth development

Method used

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  • Method for constructing genetic engineering fungus of monascus with no citrinin
  • Method for constructing genetic engineering fungus of monascus with no citrinin
  • Method for constructing genetic engineering fungus of monascus with no citrinin

Examples

Experimental program
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Effect test

Embodiment 1

[0225] Embodiment 1: the cloning of CICC 5006 citrinin coding gene

[0226] Primer design: Two pairs of primers were designed based on the gene sequence of M. purpureus citrinin released by GeneBank, pksCT-F: 5′-GGCCGCGGCCGCGCTTCTTACCAACTTCCCTT-3′, pksCT-R: 5′-GGCCGTTAACACCTTCAGTCCGCTATCTATC-3′.

[0227] CICC5006 Chromosomal DNA Extraction Method

[0228] Take 1g of CICC5006 fresh mycelium and place it in a pre-cooled mortar, add appropriate amount of quartz sand, add 1mL CTAB extraction buffer, add proteinase K to a final concentration of 0.1mg / mL, grind on ice to form a uniform turbid solution, transfer to 5mL Centrifuge tube. Add 1 / 10 volume of 10% SDS, bathe in 65°C water bath for 25min-30min, then cool to room temperature. Extract twice with an equal volume of mixed solvent phenol: chloroform: isoamyl alcohol (25:24:1), transfer the upper aqueous phase to a new centrifuge tube, add RNase I (RNase I) without DNase (DNase) activity To a final concentration of 20ug / mL, in...

Embodiment 2

[0233] Embodiment 2: Construction of knockout vector

[0234] The Hpa I restriction fragment from the vector pBC1003 1.4kb (this fragment contains the promoter from the Aspergillusnidulans tupC gene and the hygromycin B resistance gene hph sequence) is inserted into the Sma I site of the vector pBpksCT (the Sma I site is located in the pksCT gene sequence 2253bp inside) was constructed to obtain the monascus citrinin knockout vector pBpksCTHyg. The vector pBpksCTHyg knocked out the CICC5006 citrinin gene through REMI (restrictedenzyme-mediated integration) to construct a citrinin-free high-yield engineered strain.

Embodiment 3

[0235] Embodiment 3: REMI-mediated gene knockout method

[0236] Preparation of protoplasts

[0237] Strain CICC5006 was cultured on spore-forming medium at 30°C for 7d to 10d, and the spores were collected and prepared into a uniform spore suspension (1×10 8 pieces / ml). Take 200ul potato agarose solid plate coated with cellophane, incubate at 30°C for 30h, collect mycelium, 1mol / L MgSO 4 Solution washed once. Add 30ml of lysing enzyme solution (0.3% lysing enzyme, 0.1% cellulase and 1% snailase) to the harvested mycelia on each plate, and hydrolyze at 30°C and 60r / min. Filter the protoplast body fluid, centrifuge the filtrate at 3200r / min for 10min, discard the supernatant, resuspend the precipitate in a pre-cooled 1.0mol / L sorbitol solution, and centrifuge. The protoplast pellet was resuspended in 1.0mol / L sorbitol, and placed in an ice bath for later use.

[0238] REMI-mediated gene knockout method

[0239] Method 1. The above-mentioned protoplast body fluid is centri...

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Abstract

The invention relates to a non-citrinin monascus gene engineering fungus construct method, belonging to animalcule gene engineering field. The invention offers (1) citrinin coding gene pksCT sequence origin from CICC5006;(2) construct CICC5006 citrinin gene clone carrier pBpksCTHyg;(3)REMI induces CICC5006 citrinin gene remove method. Using this invention, it can interdict the metabolizing approach of citrinin in monascus, solve associated citrinin and pollution during pigment producing and make sure of the security of red bend, fulfilling the purpose of ameliorating the equality and depth development of red bend product.

Description

technical field [0001] A method for constructing citrinin-free Monascus genetically engineered bacteria, the invention belongs to the field of microbial genetic engineering (or molecular breeding), and relates to the construction and selection of citrinin-free Monascus pigment-producing engineering bacteria through genetic engineering methods . technical background [0002] Monascus (Monascus) is a unique oriental strain and an important microbial resource in my country. It can produce a wide range of biologically active natural products and has high commercial value. The application of red yeast rice has a history of thousands of years, and its medical efficacy has long been recorded in ancient Chinese books. Previous studies have shown that red yeast rice has multiple effects such as lowering blood fat, lowering blood pressure, anti-oxidation, anti-cancer, anti-bacterial, anti-fatigue, and preventing Alzheimer's disease. Metabolites of monascus enzymes such as cholestero...

Claims

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Application Information

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IPC IPC(8): C12N1/15
Inventor 诸葛健周礼红方慧英
Owner JIANGNAN UNIV
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