Genetic engineering bacterium producing beta-ionone with high yield and construction method and application of genetic engineering bacterium

A technology of genetically engineered bacteria and ionones, applied in genetic engineering, microorganism-based methods, and other methods of inserting foreign genetic materials, etc., can solve the problem of less β-ionone biological heterologous synthesis and so on

Active Publication Date: 2019-12-31
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Genetic engineering bacterium producing beta-ionone with high yield and construction method and application of genetic engineering bacterium
  • Genetic engineering bacterium producing beta-ionone with high yield and construction method and application of genetic engineering bacterium
  • Genetic engineering bacterium producing beta-ionone with high yield and construction method and application of genetic engineering bacterium

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Experimental program
Comparison scheme
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Embodiment 1

[0086] Embodiment 1 experimental scheme and primer design

[0087] The experimental scheme design of this embodiment is as follows:

[0088] Based on the β-ionone-producing genetically engineered bacteria disclosed in the patent "201810695855.9, a β-ionone-producing genetically engineered bacterium and its construction method and application", the Ura3 gene was used as a screening marker to select non-essential genes for yeast chromosomes As the integration site, through the integration of overexpression of 9 genes related to the mevalonate (MVA) pathway and the integration of a β-ionone expression cassette, and the introduction of fructose in the cytoplasm through a two-step reaction -6-phosphate conversion to phosphoketolase pathway of acetyl-CoA to increase the production of β-ionone in the original engineered bacteria. The assembly sequence of each gene module is as follows: Figure 2-4 shown. The CRISPR / cas9 operation vector, using pCAS1yl (ADDGENE No. 73226) as the st...

Embodiment 2

[0103] The construction of embodiment 2 genetically engineered bacteria

[0104] 2.1 Gene acquisition:

[0105] The β-ionone synthesis genes were selected from CarB and CarRP derived from Mucor circinolus and CCD1 derived from Petunia, which were codon-optimized and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; refer to Example 2 in 201810695855.9 In step 2.1, CarB, CarRP, and CCD1 gene fragments were respectively amplified; their sequences are respectively shown in SEQ ID NO: 7, 10, and 13 of the sequence listing in 201810695855.9.

[0106] The genes related to the MVA pathway were selected from the endogenous genes of Yarrowia lipolytica. Using the primers in Table 2 and using the yeast genome as a template, GGS1 (CP028451.1: 2084792-2085775) and tHMG1 (CP028452.1: 571078-572577)[When using this gene, the start codon ATG needs to be added to the 5′ end of the sequence in the annotation], ERG8(CP028452.1:746796-748052), ERG10(CP028449.1:1141697-1143297), ERG12 ...

Embodiment 3

[0135] Fermentation of embodiment 3 genetically engineered bacteria and the detection of β-ionone

[0136] 3.1 Fermentation

[0137] The seed liquid cultivation method is as follows: select the high-yield β-ionone genetically engineered bacteria finally constructed in Example 2, select a single clone, inoculate into YPD culture solution (50mL / 250mL Erlenmeyer flask), 28 ℃, 250rpm, cultivate to OD 600 3 to 5.

[0138]Shake flask fermentation culture method is as follows: inoculate seed solution in 20mL YPD medium (50mL shake flask), initial OD 600 The value is 0.1, the organic phase is n-dodecane (10% V / V), 20° C., 250 rpm, and cultured for 12 days.

[0139] The carbon source is glucose (D-Glucose), sucrose (Sucrose), starch (Starch), glycerol (Glycerol), oleic acid (Oil Acid), the concentration is 20g / L.

[0140] Nitrogen source selection tryptone (tryptone) and peptone (peptone).

[0141] When tryptone is used as the nitrogen source, the medium is named YPDm, and the conc...

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Abstract

The invention discloses a genetic engineering bacterium producing beta-ionone with high yield and a construction method and application of the genetic engineering bacterium, and belongs to the field of microbial fermentation. In the method, by strengthening MVA pathway and introducing phosphotransketase pathway, the supply of acetyl-CoA in cytoplasm and the metabolic flux of terpenoid synthesis pathway are increased, so that the genetic engineering bacterium producing beta-ionone with high yield is successfully constructed and obtained. The genetic engineering bacteria obtained according to the method of the invention can produce beta-ionone under conventional fermentation conditions, the fermentation yield in a shake flask can reach about 358 mg/L, and the fermentation yield in a 3L fermentor can reach about 0.98 g/L, which is close to an industrialization level. According to the present invention, the perfume beta-ionone can be produced by utilizing a simple medium for fermentation,one-time and highly efficient transformation and integration of multiple genes can be realized, the construction time of the engineering bacteria can be significantly shortened, the perfume beta-ionone can be produced by the obtained engineering bacteria with simple carbon sources such as glucose and glycerin for fermentation, and the engineering bacteria has good industrial application prospects.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and in particular relates to a high-yield β-ionone genetically engineered bacterium 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 mai...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/65C12N15/90C12N9/22C12N1/19C12R1/645
CPCC07K14/37C07K14/39C12N9/0069C12N9/1029C12N9/1085C12N9/22C12N9/88C12N15/52C12N15/65C12N15/815C12N15/905C12P7/26C12Y113/11051C12Y203/01008C12Y203/01009C12Y205/0101C12Y205/01029C12Y401/02009
Inventor 杨晓锋林章凛卢彦坪杨晴玉
Owner SOUTH CHINA UNIV OF TECH
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