A method of synthesizing beta-caryophyllene by microbial catalysis and a reconstituted cell capable of synthesizing the beta-caryophyllene

A technology for recombining cells and caryophyllene, which is applied in the direction of recombinant DNA technology, bacteria, and the use of vectors to introduce foreign genetic material, etc.

Inactive Publication Date: 2014-10-29
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Previously, there was no report on the use of m

Method used

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  • A method of synthesizing beta-caryophyllene by microbial catalysis and a reconstituted cell capable of synthesizing the beta-caryophyllene
  • A method of synthesizing beta-caryophyllene by microbial catalysis and a reconstituted cell capable of synthesizing the beta-caryophyllene
  • A method of synthesizing beta-caryophyllene by microbial catalysis and a reconstituted cell capable of synthesizing the beta-caryophyllene

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Experimental program
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Embodiment 1

[0035] By co-expressing the acetyl-CoA acyltransferase gene / hydroxymethylglutaryl-CoA reductase gene (mvaE) from Enterococcus faecalis in Escherichia coli, 3-hydroxy-3-methylglutaryl Acyl-CoA synthase gene (mvaS); mevalonate kinase gene (ERG12) from Saccharomyces cerevisiae, mevalonate-5-phosphate kinase gene (ERG8), mevalonate-5- Diphosphate decarboxylase gene (ERG19), isopentenyl pyrophosphate isomerase gene (IDI1); geranyl diphosphate synthase gene (GPPS2) derived from North American fir (Abies grandis); derived from Neurospora crassa ( The farnesyl pyrophosphate synthase gene (FPPS) from Neurospora crassa and the β-caryophyllene synthase gene (QHS1) from Artemisia annua can biosynthesize isoprene from glucose degradation intermediate acetyl-CoA Derivatives - β-caryophyllene.

[0036] 1.1 Cloning of foreign genes and construction of expression vectors

[0037] 1.1.1 Cloning of foreign genes

[0038] 1.1.1.1 Cloning of genes in the upper metabolic pathway of Enterococcus ...

Embodiment 2

[0066] The constructed plasmid was transformed into competent cells, and the recombinant bacteria were fermented and cultured by shaking flask fermentation and fermenter fermentation, and the fermentation products were detected qualitatively and quantitatively by gas chromatography.

[0067] 2.1 Construction of E.coli recombinant strain

[0068] Transform E.coli BL21 (DE3) competent cells with pYJM51 (pACY-mvaE-mvaS-GPPS2-QHS1-ispA) and pYJM14 (pTrc-low) recombinant plasmids together by heat shock, and apply to the cells added with chloramphenicol and ampicillin The LB solid plate of prime antibiotics was used to obtain positive clones by PCR screening, thereby obtaining engineering Escherichia coli containing pYJM51 and pYJM14.

[0069] 2.2 Cultivation of engineering Escherichia coli

[0070] Inoculate the activated engineering Escherichia coli into the LB liquid culture solution containing chloramphenicol and ampicillin at a ratio of 1:100, and culture it with shaking at 37...

Embodiment 3

[0079]Different fermentation conditions, such as induction temperature, rotation speed, inducer concentration, nitrogen source, substrate concentration, medium pH value and composition ratio, etc., will affect the yield of the fermentation product pinene. This patent detects the effects of different induction temperatures, inducer concentrations and nitrogen sources on the production of β-caryophyllene.

[0080] 3.1 Construction of E.coli recombinant strain

[0081] Transform E.coli BL21 (DE3) competent cells with pYJM51 (pACY-mvaE-mvaS-GPPS2-QHS1-ispA) and pYJM14 (pTrc-low) recombinant plasmids together by heat shock, and apply to the cells added with chloramphenicol and ampicillin The LB solid plate of antibiotic antibiotics was used to obtain positive clones by PCR screening, thereby obtaining engineering Escherichia coli containing pYJM27 and pYJM14.

[0082] 3.2 Study the effect of different induction temperatures on the yield of β-caryophyllene

[0083] Pick a single c...

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Abstract

A method of synthesizing beta-caryophyllene by microbial catalysis and a reconstituted cell capable of synthesizing the beta-caryophyllene are disclosed. Acetyl coenzyme A used in the method is obtained from simple initial materials such as glucose. The method includes: A) a step of constructing the reconstituted cell capable of synthesizing the beta-caryophyllene from the acetyl coenzyme A, wherein the reconstituted cell comprises gene segments comprising acetyl-CoA acyltransferase, 3-hydroxy-3-methylglutaryl coenzyme A synthetase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl diphosphate isomerase, geranyl pyrophosphate synthase, farnesyl pyrophosphate synthase, and beta-caryophyllene synthase; and B) a step of culturing the reconstituted cell in a culture medium containing glucose, inducing with a proper inducer, separating and purifying to obtain the beta-caryophyllene.

Description

technical field [0001] The present invention relates to a method for preparing terpene compounds by a biological method, in particular to a method for microorganism-catalyzed synthesis of β-caryophyllene and a recombinant cell capable of synthesizing β-caryophyllene, wherein the acetyl-CoA used is obtained from obtained from simple starting materials such as glucose. Background technique [0002] Terpene compound - β-caryophyllene, is an important platform compound of bicyclic sesquiterpene type, and is an important raw material for medicine, food, cosmetics and high-density fuel industry. As a spice, β-caryophyllene has been used in cosmetics and food additives; in the pharmaceutical industry, β-caryophyllene has local anesthetic, anti-inflammatory, mosquito repellent, anti-anxiety, anti-depressant effects, and its derivative β-caryophyllene Alcohol can also be used in antitussive and expectorant drugs, β-caryophyllene oxide has analgesic and anti-inflammatory, anti-fungal...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P5/00
Inventor 杨建明王晓璐易晓华聂庆娟
Owner QINGDAO AGRI UNIV
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