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Engineering bacterium for producing beta-caryophyllene, and construction method and application thereof

A construction method and caryophyllene technology, applied in the field of bioengineering, can solve problems such as low synthesis efficiency, affecting downstream applications of products, and complex conditions.

Active Publication Date: 2020-04-14
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, chemical synthesis needs to be carried out under strong acid and strong alkali conditions. At the same time, there are disadvantages such as low synthesis efficiency and complicated conditions, and the chemical synthesis products are "unnatural". As food additives, consumers' acceptance is low, which affects the downstream application of the product.

Method used

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  • Engineering bacterium for producing beta-caryophyllene, and construction method and application thereof
  • Engineering bacterium for producing beta-caryophyllene, and construction method and application thereof
  • Engineering bacterium for producing beta-caryophyllene, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Cloning of tobacco β-caryophyllene synthase gene NtCAS.

[0062] Extract the RNA from the tobacco leaf tissue of the flue-cured tobacco variety Honghua Dajinyuan (purchased from the China Tobacco Germplasm Bank), and use reverse transcriptase to convert the RNA into a cDNA template. The cDNA obtained by the reverse transcription reaction is used as a template, and the forward primer is : 5'-ATGGATTTGAGCAAAGGCTTGCCGGT-3', the reverse primer is: 5'-TTATGGAACAGGATCAACCAATATTG-3', use TakaRa PrimeSTAR Max DNA polymerase for PCR amplification; PCR conditions: 95°C for 5min; 95°C for 30s, 55°C for 30s , 72°C for 30s, 35 cycles; 72°C for 5min. The PCR product was detected by 1% agarose gel electrophoresis, and the results were as follows: figure 1 As shown, lane M is the DNA marker DL10000, lanes 1 and 2 are the target gene NtCAS, and the fragment size of the gene NtCAS is about 1700bp, which is in line with the expected size.

[0063] Use the method of agarose ge...

Embodiment 2

[0064] Example 2. Biosynthesis of β-caryophyllene using β-caryophyllene synthase NtCAS.

[0065] By co-expressing the acetyl-CoA acyltransferase gene / hydroxymethylglutaryl-CoA reductase gene (mvaE) from Enterococcus faecalis in Escherichia coli, 3-hydroxy-3-methylglutaryl-CoA synthesis Enzyme gene (mvaS); mevalonate kinase gene (ERG12), mevalonate-5-phosphate kinase gene (ERG8), mevalonate-5-diphosphate decarboxylase gene (ERG19) from Saccharomyces cerevisiae , isopentenyl pyrophosphate isomerase gene (IDI1); geranyl pyrophosphate synthase gene (GPPS2) from North American fir; farnesyl pyrophosphate synthase gene (ispA) from Escherichia coli and source Based on the β-caryophyllene synthase gene (NtCAS) of tobacco, caryophyllene is biosynthesized using glucose as a raw material. The specific method is as follows:

[0066] (1) Cloning the tobacco β-caryophyllene synthase gene NtCAS, the method refers to Example 1;

[0067] (2) Construction of expression vector:

[0068] Usin...

Embodiment 3

[0072] Example 3. Fermentative production of β-caryophyllene by engineering bacteria prepared in Example 2.

[0073] The white monoclonal engineered Escherichia coli strain obtained in Example 2 was picked and placed in 20 ml of LB liquid medium (containing Cm and Amp resistance), and cultured and activated on a shaker at 37° C. as seeds. When the concentration of the seed bacteria is about 1.0, take the expanded bacteria solution and insert it into 50ml of M9 medium according to the inoculum amount of 2% (wt), and cultivate it at 37°C. When the bacterial concentration grows to about 0.6, add IPTG with a final concentration of 0.2mM , continue culturing at 30°C.

[0074] (1) Product detection

[0075] After 24 hours of fermentation, the final fermentation liquid was taken to detect the yield, and the GC and GC-MS figures of the product were as follows: Figure 4 and 5 Shown, by GC and GC-MS figure with β-caryophyllene standard ( figure 2 and 3 ) for comparison, it was sh...

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Abstract

The invention discloses an engineering bacterium for producing beta-caryophyllene, and a construction method and an application thereof, and belongs to the technical field of bioengineering. The invention provides the engineering bacterium for producing beta-caryophyllene, and the construction method and the application thereof in order to improve the biosynthesis efficiency of beta-caryophyllene. The construction method comprises the following steps: introducing an encoding gene of beta-caryophyllene synthetase cloned from tobacco into a receptor strain capable of generating farnesyl pyrophosphate to obtain a recombinant bacterium; and obtaining the engineering bacterium for producing the beta-caryophyllene from the recombinant bacterium. Heterologous reconstruction of a beta-caryophyllene biosynthetic pathway and coproduction of products are successfully realized by using a bioengineering technology, and the yield of beta-caryophyllene reaches 25.06 mg / L by using a shake flask fermentation method; and the yield of beta-caryophyllene is increased by about 85 times through a high-density fermentation technology of a recombinant strain in a fermentation tank.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to an engineering bacterium for producing β-caryophyllene, a construction method and application thereof. [0002] technical background [0003] β-Caryophyllene (also known as β-syringene, scientific name is trans-(1R,9S)-8-methylene-4,11,11-tetramethylbicyclo[7.2.0]undec-4-ene , β-caryophyllen) is a bicyclic sesquiterpenoid, naturally occurring in lemon, pomelo, nutmeg, pepper, raspberry, black currant, cinnamon leaf oil, clove leaf oil, is cinnamon oil, clove oil, etc. one of the main ingredients. Caryophyllene has spicy, woody, citrus, camphor, and mild clove aroma, so it can be used to prepare food flavors, and can also be used to synthesize other more valuable spices such as acetyl caryophyllene. At the same time, caryophyllene also has certain pharmacological value. Previous studies have shown that caryophyllene has biological activities such as anti-cancer, anti-inflamm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/54C12N15/60C12N15/61C12N15/70C12P5/02C12R1/19
CPCC12N9/80C12N9/0006C12N9/1025C12N9/1205C12N9/1229C12N9/90C12N9/1085C12N15/70C12P5/026C12Y402/03057C12Y101/01088C12Y203/0301C12Y207/01036C12Y207/04002C12Y401/01033C12Y503/03002C12Y205/0101C12Y205/01029
Inventor 张汝兵李依婷咸漠张凯杨爱国王元英
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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