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Genetically engineered bacterium with gamma-terpinene synthesis capacity and constructing method and application thereof

A technology of genetically engineered bacteria and terpinene, applied in the field of genetic engineering, can solve problems such as low content, high energy consumption, and restrictions on mass application

Active Publication Date: 2017-11-17
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

[0003] At present, the source of γ-terpinene is mainly separated from Lantana camara essential oil, sweet orange oil and turpentine, or obtained through Birch reaction, but the energy consumption is high, the process is complicated and the content is low
Shortage of sources of γ-terpinene limits its bulk application as a precursor in the performance fuel industry
At present, there is no report on the co-expression of MVA pathway, geranyl pyrophosphate synthase (GPPS2) and γ-terpinene synthase (TPS2) using glycerol and glucose as carbon sources to synthesize γ-terpinene

Method used

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  • Genetically engineered bacterium with gamma-terpinene synthesis capacity and constructing method and application thereof
  • Genetically engineered bacterium with gamma-terpinene synthesis capacity and constructing method and application thereof
  • Genetically engineered bacterium with gamma-terpinene synthesis capacity and constructing method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Construction of recombinant plasmid pHW2 carrying genes encoding hydroxymethylglutaryl-CoA synthetase, hydroxymethylglutaryl-CoA reductase, geranyl pyrophosphate synthetase and γ-terpinene synthase

[0050] The recombinant plasmid pHW2 was constructed through molecular biology-related experiments, which carried the hydroxymethylglutaryl-CoA synthetase gene mvaS (sequence registration number: GenBank: AAG02439.1), hydroxymethylglutaryl-CoA synthetase gene Enzyme gene mvaE (sequence accession number is GenBank: AAG02439.1), geranyl pyrophosphate synthase gene GPPS2 (sequence accession number is GenBank: AAN01134.1) and γ-terpinene synthase gene TPS2 (sequence accession number is GenBank: ID: KR920616), through the heterologous expression of the recombinant vector, the above-mentioned foreign gene was overexpressed in Escherichia coli (E.coli BL21(DE3)), and the biosynthesis of γ-terpinene was realized.

[0051] The MVA pathway first converts glucose to mevalonat...

Embodiment 2

[0055] Example 2 Construction of recombinant plasmid pTrc-low carrying mevalonate kinase gene, mevalonate phosphokinase gene, mevalonate pyrophosphate decarboxylase gene and isopentenyl pyrophosphate isomerase coding gene

[0056] The pTrc-low vector was constructed according to the Lego DNA assembly method established in the laboratory, and contains the mevalonate kinase gene (ERG12) and mevalonate phosphate derived from Saccharomyces cerevisiae (S. Kinase gene (ERG8), mevalonate pyrophosphate decarboxylase gene (ERG19) and isopentenyl pyrophosphate isomerase gene (IDI1). The DNA fragment thermal denaturation assembly method has been published in relevant international journals by our laboratory, which is a mature molecular operation method (PloS one 2012doi:10.1371 / journal.pone.0030267.g001).

[0057] The pTrc-low vector construction process is as follows:

[0058]Using the commercialized plasmid pTrcHis2B plasmid (purchased from Invitrogen) and the Saccharomyces cerevisiae...

Embodiment 3

[0068] Embodiment 3 uses glycerol as raw material synthesis gamma-terpinene

[0069] 1. Fermentation of γ-terpinene

[0070] Plasmid transformation: pipette 2 μL of plasmids pHW2 and pTrc-low into Escherichia coli competent (E. coli BL21(DE3)) that is still in a frozen state, and place in an ice bath for 10-30 minutes; then heat shock in a water bath at 42°C for 60- After 90 seconds, put it in an ice bath and let it rest for 1-3 minutes; add 400 μL of LB medium, and activate it on a shaker at 37 °C and 180 rpm for 1 hour; draw 100 μL of bacterial liquid and spread it evenly on the LB solid plate containing chloramphenicol and ampicillin, Cultivate at 37°C for 12 hours, pick a single clone and inoculate it into a culture flask for culture.

[0071] Medium: 20g / L glycerol, 5g / L yeast extract, 1.5g / L MgSO 4 , 100 μL of trace elements, 100 μL of antibiotics, and 1 mL of bacterial solution were placed in a shaker at 37°C and 180 rpm for shaking culture.

[0072] The above-mentio...

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Abstract

The invention discloses a genetically engineered bacterium with gamma-terpinene synthesis capacity and a constructing method and application thereof, and belongs to the technical field of genetic engineering. According to engineered escherichia coli, a path for synthesizing MVA from mevalonic acid is reconstructed by heterogeneous expression of hydroxymethylglutaryl coenzyme A synthetase mvaS, hydroxymethylglutaryl coenzyme A reductase mvaE, mevalonate kinase Erg12, phosphomevalonate kinase Erg8, mevalonate pyrophosphate decarboxylase Erg19 and Isopentenyl diphosphate isomerase Idi1, meanwhile, geranyl pyrophosphate synthetase GPPS2 and gamma-terpinene TPS2 are led into a bacterium body, and by utilizing the biotransformation capacity of the genetically engineered bacterium, gamma-terpinene can be efficiently produced in an environment-friendly mode. Through fermentation with the genetically engineered bacterium, the output of gamma-terpinene can reach 80 mg / L. The genetically engineered bacterium and the gamma-terpinene synthesis method are suitable for practical industrial production.

Description

technical field [0001] The invention relates to a genetically engineered bacterium capable of synthesizing gamma-terpinene and its construction method and application, belonging to the technical field of genetic engineering. Background technique [0002] γ-Terpinene, or 1-methyl-4-(1-methylethyl)-1,4-cyclohexadiene, is a monoterpenoid compound that can be used as a spice and food additive. γ-Terpinene is insoluble in water, soluble in most organic solvents and non-volatile oils, with an energy density of 0.85g / cm 3 , with a boiling point of 183°C and a flash point of 53°C under standard atmospheric pressure, it has the advantages of high energy density, low freezing point, and high flash point. After catalytic hydrogenation or polymerization, it can be used as a high-density fuel in aerospace and other fields. Effective replacement of high-density fuel precursors such as adamantane and norbornene. [0003] At present, the source of γ-terpinene is mainly separated from Lant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P5/00C12R1/19
CPCC12N9/0006C12N9/1025C12N9/1085C12N9/1205C12N9/1229C12N9/88C12N9/90C12N15/70C12P5/002C12Y101/01088C12Y203/0301C12Y207/01036C12Y207/04002C12Y401/01033C12Y503/03C12Y402/03114
Inventor 张海波赵宏伟咸漠齐畅
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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