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Genetically engineered bacterium for production of beta-ionone and construction method and application thereof

A technology of genetically engineered bacteria and ionone, applied in the direction of microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problem of less bio-heterogeneous synthesis of β-ionone, and achieve good industrial application prospects , good application prospects, and the effect of shortening the construction cycle

Inactive Publication Date: 2018-12-07
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few reports on the heterogeneous biosynthesis of β-ionone

Method used

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  • Genetically engineered bacterium for production of beta-ionone and construction method and application thereof
  • Genetically engineered bacterium for production of beta-ionone and construction method and application thereof
  • Genetically engineered bacterium for production of beta-ionone and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 experimental scheme and primer design

[0049] The experimental scheme design of this embodiment is as follows:

[0050] In Yarrowia lipolytica po1f (purchased from YEASTERN BIOTECH company, strain information: MatA, leu2-270, ura3-302, xpr2-322, axp1-2, Leu - , Ura - ΔAEP, ΔAXP, Suc + ) to express CarB and CarRP from Mucor circinosa and CCD1 gene from Petunia to construct the synthesis pathway of β-ionone, and at the same time use the Ura3 gene as a screening marker to select the yeast chromosome rDNA site as the integration site , the assembly sequence of each gene module is as follows figure 2 shown. The CRISPR / cas9 operation vector, using pCAS1yl (ADDGENE No. 73226) as the starting vector, was constructed as image 3 shown.

[0051] The following gene expression modules and CRISPR / cas9 operation vectors were constructed using the method of gibson assembly:

[0052] rDNAup-hisG-Ura3-hisG, TEF1p-CarB-XPR2t, EXP1p-CarRP-LIP2t, GPD2p-CCD1-MIG1t-rDNA...

Embodiment 2

[0056] The construction of embodiment 2 genetically engineered bacteria

[0057] 2.1 Acquisition of β-ionone gene:

[0058] The genes were selected from CarB, CarRP from Mucor volvulus and CCD1 from Petunia. After codon optimization, they were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; using the primers in the sequence list, they were respectively amplified and obtained CarB, CarRP, CCD1 gene fragments.

[0059] 2.2 Extraction of yeast genomic DNA

[0060] The extraction method of the Yarrowia lipolytica po1f genome is as follows: Pick a single clone colony from a freshly recovered plate and inoculate it in a 5mL YPD liquid medium test tube, culture it at 28°C and 250rpm for 24 hours, collect 1mL of the bacterial liquid, and use the yeast genome to extract Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the genome.

[0061] The extraction method of the plasmid is as follows: inoculate the DH5α bacteria containing the corres...

Embodiment 3

[0077] Fermentation of embodiment 3 genetically engineered bacteria and the detection of β-ionone

[0078] 3.1 Fermentation

[0079] Seed liquid cultivation method is as follows:

[0080] The single colonies of the positive clones screened from the culture plate Tianqu Example 2 were connected to 10mL YPD culture solution (50mL centrifuge tube), cultivated to OD at 28°C and 250rpm 600 2 to 3.

[0081] The fermentation culture methods at different temperatures are as follows:

[0082] Inoculate the seed liquid into 20mL YPD medium (50mL shake flask), initial OD 600 The value is 0.1, the organic phase is n-dodecane (10% V / V), placed in shakers at different temperatures (15-30° C.) at 250 rpm, and cultivated for 12 days.

[0083] The fermentation and cultivation methods of different carbon sources are as follows:

[0084] Inoculate the seed liquid into 20mL YP medium (50mL shake flask) containing various carbon sources (20g / L), initial OD 600 The value is 0.1, the organic p...

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Abstract

The invention discloses genetically engineered bacterium for production of beta-ionone and a construction method and application thereof. According to the invention, by using Yarrowia lipolytica as achassis cell, the beta-ionone expression module (about 14.5 kb) is firstly linearized and then transformed into Yarrowia lipolytica. Then the expression module is integrated into a designed chromosomal location mediated by a CRISPR / cas9 technology. The construction method is simple, fast and efficient. A large fragment integrated engineering strain can be obtained within 2-3 weeks, and the construction period of the engineering bacterium is obviously shortened. The beta-ionone initial output of shaking-flask fermentation of the genetically engineering bacterium obtained according to the methodof the invention can reach about 6.3 mg / L. According to the invention, a simple medium can be used for fermentation production of the perfume beta-ionone, one-time high-efficiency transformation andintegration of multiple genes can be realized, and the construction time of the engineering bacterium can be obviously shortened. The obtained engineering bacterium can be used to perform fermentationto produce the perfume beta-ionone by the use of simple carbon sources such as glucose, glycerin, etc., and has a good industrial application prospect.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and in particular relates to a genetically engineered bacterium producing β-ionone and its construction method and application. Background technique [0002] Ionone is a high-grade spice and an indispensable and important original spice in perfumery. It has a wide range of uses and is in great demand. The molecular formula of ionone is C 13 h 20 O, according to the position of the double bond, ionone can be divided into three isomers of α-body, β-body and γ-body, and the mixed form of α-body and β-body exists in nature , where β-ionone is used as flavoring agent and flavoring agent for high-grade fine daily cosmetics, and is also an important raw material for the synthesis of vitamin A in the pharmaceutical industry. In the food industry, ionone is often used as a flavor enhancer for high-end food and beverages. It is an edible flavor allowed by China's GB2760-2011 regulations. It is main...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P7/26C12R1/645
CPCC07K14/37C07K14/415C12N15/815C12P7/26
Inventor 林章凛王婷婷杨晓锋卢彦坪张兴杨晴玉
Owner SOUTH CHINA UNIV OF TECH
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