Recombined monascus purpureus with characteristics of low citrinin expression and high haematochrome expression

A technology of monascus and citrinin, applied in the field of microorganisms, can solve problems such as food safety and ecological safety concerns, affecting synthesis, etc.

Inactive Publication Date: 2010-06-23
东莞市天益生物工程有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the problems with these methods are: resistance genes such as hygromycin are integrated in the genetic manipulation, which will cause food safety and ecological safety concerns if used in production; and / or knockout of pksCT will affect red pigment or lovastatin Synthesis of useful products such as lovastatin, because the synthetic pathway of citrinin is closely related to the synthetic pathway of red pigment and lovastatin

Method used

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  • Recombined monascus purpureus with characteristics of low citrinin expression and high haematochrome expression
  • Recombined monascus purpureus with characteristics of low citrinin expression and high haematochrome expression
  • Recombined monascus purpureus with characteristics of low citrinin expression and high haematochrome expression

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Knockout of embodiment one Monascus ctnR gene

[0023] 1. Monascus Genomic DNA Preparation

[0024] The common Monascus strain TY0701 used in the fermentation industry was used as the starting Monascus strain, which was preserved in the inventor's laboratory.

[0025] The Monascus genomic DNA was prepared by the following steps: 1) Scrape off the mycelium from the PDA plate cultivated for 10 days, dry it with filter paper, and weigh 200 mg. 2) Wrap the weighed mycelium in tin foil and freeze it in liquid nitrogen for 10 minutes; 3) Take 1ml CTAB (2×) and add 20 μl of β-mercaptoethanol (final concentration 2%) in a fume hood, and set aside in a 65°C water bath ;4) Put the mortar pre-frozen at -20°C on ice, dry it, add a small amount of quartz sand, and then add liquid nitrogen twice to pre-cool the mortar; 5) Add the frozen mycelium to the mortar , grind into powder, about 10min; 6) Transfer the ground powder to a 1.5ml EP tube, add CTAB preheated at 65°C, gently pipet...

Embodiment 2

[0043] Example 2 Screening and detection of recombinants with low citrinin content

[0044] 1. Preparation of Monascus protoplasts

[0045] 1) Elute the mycelium with a bacterial age of 40-50h on the cellophane, soak it in 5mmol DTT solution for 20-30min; wash it with sterile water, dry it with sterile filter paper, and transfer it to a sterile Erlenmeyer flask;

[0046] 2) Add a certain amount (10ml for every three dishes of mycelium) to the Erlenmeyer flask with 1M MgSO 4 The solution is an enzymolysis solution prepared by a solvent (helicase: lysozyme: cellulase=0.6%: 0.4%: 0.8%) and a small amount of sterilized quartz sand; under sterile conditions, 30°C, 100rpm, enzymolysis;

[0047] 3) Filtrate, centrifuge the filtrate at 3,200rpm for 10min; discard the supernatant, and resuspend the precipitate with pre-cooled 1M MgSO4 solution;

[0048] 4) Repeat 3), and equally divide into two small tubes of equal volume.

[0049] 5) Repeat step 3) for two small tubes of the same v...

Embodiment 3

[0061] Pigment and citrinin detection of embodiment three Monascus recombinant ZD19 fermented mycelia

[0062] Monascus recombinant ZD19 slant strain inoculates liquid rice flour medium (rice flour 6%, soybean flour 3%, NaNO 3 0.3%, KH 2 PO 4 0.3%, MgSO 4 ·7H 2 O 0.3%, adjust the initial pH value of the fermentation broth to 3), cultivate at 30°C and 150rpm for 7 days, extract citrinin and analyze the content of citrinin by high performance liquid chromatography. By HPLC analysis, it can be seen that the peak time of citrinin benchmark is 18.195min, the citrinin content of the starting strain fermentation liquid is 0.56 μg / ml, and the citrinin content of the recombinant fermented liquid is 0.25 μg / ml; A drop of 55%. The measurement results are shown in Table 1.

[0063] Table 1: Comparison of Citrinin and Red Pigment Produced by Fermentation of Starting Strain and Recombinant Strain ZD19

[0064] strain

[0065] Results: The citrinin produced by the recombin...

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Abstract

The invention relates to recombined monascus purpureus with the characteristics of low citrinin expression and high haematochrome expression. A bacterial strain is named purple monascus ZD19 and is preserved in CCTCC (China Center for Type Culture Collection), and the CCTCC NO. is M 208199. Citrinin on a genome synthesizes a relevant gene ctnR which is removed through knocking, monascus polyketone synthesizes a pksl gene sequence of an enzyme gene so as to replace a ctnR gene sequence, and the genome of the monascus purpureus does not contain an external source resistance gene. The invention further provides a preparation method for the monascus purpureus. The preparation method includes the following steps: the monascus polyketone synthesizes the pksl gene sequence of the enzyme gene, and the citrinin in the genome of the monascus purpureus is replaced by the homologous recombination method so as to synthesize the relevant gene ctnR, and then screening is carried out on a target recombinant by utilizing the restraining characteristic of the citrinin against bacillus subtilis as a genetic mark. The invention further provides a purpose of using the monascus purpureus to prepare monascus color.

Description

technical field [0001] The invention belongs to the field of microorganisms and genetic engineering, and relates to a new genetically modified Monascus, especially a Monascus with the characteristics of low citrinin expression and high red pigment expression, and relates to the production of the Monascus Preparation method, and the application of the monascus. Background technique [0002] Monascus purpureus is an important producer of food coloring and blood lipid-lowering drugs, and its fermented red yeast rice is often eaten directly. The bacterium has been used in the production of food additive Monascus pigment, anti-lipidemia drug lovastatin, etc. [0003] Since Blanc et al. (1995, Int.J.Food Microbiol.27(2-3): 201-213) determined that the antibacterial substance monascidin A in the pigment of Monascus was citrinin by methods such as mass spectrometry analysis, the consumption of red yeast rice The safety has been questioned, which has affected the sales of red yeast...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/09C12P1/02C12R1/645
Inventor 周世宁徐民俊梁志洲杨晓暾王民俊胡文林
Owner 东莞市天益生物工程有限公司
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