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Genetically engineered bacterium for synthesizing D-limonene, and construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and limonene, which is applied in the field of genetic engineering and fermentation engineering, can solve the problems of low D-limonene biosynthesis level and restrictions on the industrial production of D-limonene

Pending Publication Date: 2020-01-10
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The low level of D-limonene biosynthesis reported above restricts the industrial production of D-limonene

Method used

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  • Genetically engineered bacterium for synthesizing D-limonene, and construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for synthesizing D-limonene, and construction method and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1. The construction of the genetically engineered bacteria that synthesizes D-limonene.

[0043] The genetically engineered bacteria for synthesizing D-limonene described in this example express HMG-CoA synthetase gene mvaS, acetyl CoA acetyltransferase mvaE, geranyl pyrophosphate synthase GPPS gene, D-limonene synthase gene ClLS, MVA kinase ERG12 gene, MVAP kinase ERG8 gene, mevalonate decarboxylase ERG19 gene, isopentyl pyrophosphate isomerase IDI gene, and the host bacteria is Escherichia coli. The HMG-CoA synthase gene mvaS, GeneBank ID is AAG02439, derived from E. Phosphate synthase GPPS gene, Genbank No. AF5131121, from Abies grandis; D-limonene synthase ClLS gene, Genbank No.AF514287.1, from Citruslimon; MVA kinase ERG12 gene, GeneBank ID: 855248, from Saccharomyces cerevisiae; MVAP kinase ERG8 gene, GeneBank ID: 855260, source Saccharomyces cerevisiae, mevalonate decarboxylase ERG19 gene, GeneBank ID: 855779, source Saccharomyces cerevisiae, isopenty...

Embodiment 2

[0057] Example 2. Fermentative production of D-limonene by genetically engineered strains.

[0058] Shake flask fermentation experiment:

[0059] 1) Cultivation of the primary seed liquid: Inoculate the Example 1 on the solid LB plate in the LB seed liquid medium to obtain a single colony of the recombinant strain, and add a final concentration of 50 μg / mL kanamycin and 100 μg / ml ampicillin , grow at 37°C for 12h, and obtain the primary seed solution.

[0060] 2) Transfer the primary seed solution obtained in step 1) to a 250mL saline bottle with 2% (wt) inoculation amount, containing 50mL fermentation medium, and add 200g / L of MgSO when transferring the seed solution 4 ·7H 2 O 100μL, 500g / L glucose 2.4mL, 1000×trace elements 50μL ((NH 4 ) 6 MoO 24 4H 2 O 0.37g; ZnSO 4 ·7H 2 O 0.29g; H 3 BO 4 2.47g; CuSO 4 ·5H 2 O 0.25g; MuCl 2 4H 2O 1.58g; distilled water to 100mL), final concentration of 50μg / mL kanamycin and 100μg / ml ampicillin, culture at 37°C and 180rpm.

...

Embodiment 3

[0074] Example 3. Fermentative production of D-limonene by genetically engineered strains.

[0075] Shake flask fermentation experiment:

[0076] 1) Cultivation of the primary seed liquid: Inoculate the Example 3 on the solid LB plate in the LB seed liquid medium to obtain a single colony of the recombinant strain, and add a final concentration of 50 μg / mL kanamycin and 100 μg / ml ampicillin , grow at 37°C for 12h, and obtain the primary seed solution.

[0077] 2) Transfer the primary seed solution obtained in step 1) to a 250mL saline bottle with 2% (wt) inoculation amount, containing 50mL fermentation medium, and add 200g / L of MgSO when transferring the seed solution 4 ·7H 2 O 100μL, 500g / L glucose 2.4mL, 1000×trace elements 50μL ((NH 4 ) 6 MoO 24 4H 2 O 0.37g; ZnSO 4 ·7H 2 O 0.29g; H 3 BO 4 2.47g; CuSO 4 ·5H 2 O 0.25g; MuCl 2 4H 2 O 1.58g; distilled water to 100mL), final concentration of 50μg / mL kanamycin and 100μg / ml ampicillin, culture at 37°C and 180rpm. ...

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Abstract

The invention discloses a genetically engineered bacterium for synthesizing D-limonene, and a construction method and application of the genetically engineered bacterium, and relates to the field of genetic engineering and fermentation engineering. In the genetically engineered bacterium, an HMG-CoA synthetase gene, an acetyl CoA acetylase mvaE, a GPPS (geranyl pyrophosphate synthase) gene, a D-limonene synthetase gene C1L2, an MVA kinase ERG12 gene, an MVAP kinase ERG8 gene, a mevalonic acid decarboxylase ERG19 gene and an isopentenyl pyrophosphate isomerase IDI gene are subjected to overexpression. In an LB culture medium, after flask shaking fermentation is carried out, the D-limonene can be subjected to heterologous synthesis, and the yield of the D-limonene is 27.3mg / L of fermentationliquor. The genetically engineered bacterium can be used for the industrial production of the D-limonene.

Description

technical field [0001] The invention relates to the fields of genetic engineering and fermentation engineering, in particular to a genetically engineered bacterium for synthesizing D-limonene and its construction method and application. Background technique [0002] Limonene, also known as limonene and limonene, belongs to monocyclic terpenoids, chemical formula C 10 h 16 , is a natural compound of plant origin. It mainly exists in the peel and pulp of citrus (such as lemon, orange, citrus, etc.), and has been identified by the American Flavor and Extract Manufacturers Association (FEMA) as a GRAS level of toxicity (generally recognized as safe), and has been Approved by the FDA for consumption (Liu Shuwen. Technical Handbook of Synthetic Spices [M]. 2009.) [0003] In agriculture, D-limonene is also used as a biopesticide for crops. In the field of health care, D-limonene has antibacterial and antibacterial activities, and can be used as a good natural antibacterial act...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P5/00C12R1/19
CPCC12N15/52C12N9/0006C12N9/1029C12N9/1085C12N9/88C12N9/1205C12N9/1229C12N9/90C12N15/70C12P5/002C12Y101/01034C12Y203/01009C12Y205/01001C12Y402/03C12Y207/01036C12Y207/04002C12Y401/01C12Y503/03002
Inventor 孙超张汝兵咸漠董先娟
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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