A genetically engineered bacterium with γ-terpinene synthesis ability and its construction method and application

A technology of genetically engineered bacteria and terpinene, applied in the field of genetic engineering, can solve the problems of low content, high energy consumption, complex process flow, etc.

Active Publication Date: 2020-12-18
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
The shortage of sources of γ-terpinene limits its bulk application as a precursor in the high-performance fuel industry
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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  • A genetically engineered bacterium with γ-terpinene synthesis ability and its construction method and application
  • A genetically engineered bacterium with γ-terpinene synthesis ability and its construction method and application
  • A genetically engineered bacterium with γ-terpinene synthesis ability and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0051]The MVA pathway first converts glucose into mevalonate (MVA...

Embodiment 2

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

[0056]The pTrc-low vector is constructed according to the Lego DNA assembling method established in the laboratory, and contains the mevalonate kinase gene (ERG12) and mevalonate phosphate derived from S. cerevisiae ATCC 4040002. Kinase gene (ERG8), mevalonate pyrophosphate decarboxylase gene (ERG19) and isopentenyl pyrophosphate isomerase gene (IDI1). The DNA fragment thermal denaturation assembly method has been published by our laboratory in relevant international journals, which is a mature molecular manipulation method (PloS one 2012doi:10.1371 / journal.pone.0030267.g001).

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

[0058] Using commercial plasmid pTrcHis2B (purchased from Invitrogen) and Saccharomyces cerevisiae (S.cerevisiaeATCC4040002) ...

Embodiment 3

[0068]Example 3 Synthesis of γ-terpinene with glycerin as raw material

[0069]1. Fermentation of γ-terpinene

[0070]Plasmid transformation: Take 2μL of plasmid pHW2 and pTrc-low respectively into E.coli BL21(DE3) which is still frozen, and bathe in ice for 10~30min; then heat shock in 42℃ water bath for 60~ 90s, immediately put it in an ice bath and stand still for 1 to 3 minutes; add 400 μL of LB medium and activate it on a shaker at 37°C and 180 rpm for 1 h; draw 100 μL of bacterial solution and spread it evenly on the LB solid plate containing chloramphenicol and ampicillin. Incubate at 37°C for 12 hours, pick a single clone and inoculate it in a culture flask.

[0071]Medium: 20g / L glycerol, 5g / L yeast extract, 1.5g / L MgSO4, 100μL of trace elements, 100μL of antibiotics, 1mL of bacterial solution, placed in a shaker at 37°C and 180rpm for shaking culture.

[0072]The above-mentioned trace elements are 1000 × trace element mother liquor, each 100mL contains the following inorganic salts: a...

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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 with γ-terpinene synthesis ability and a construction method and application thereof, belonging to the technical field of genetic engineering.Background technique[0002]Gamma-terpinene, namely 1-methyl-4-(1-methylethyl)-1,4-cyclohexadiene, is a monoterpene compound that can be used as a fragrance and food additive. γ-terpinene is insoluble in water, soluble in most organic solvents and non-volatile oils, and its energy density is 0.85g / cm3It has 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. Effectively replace high-density fuel precursors such as adamantane and norbornene.[0003]At present, the source of γ-terpinene is mainly separated from Lantana camara essenti...

Claims

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Application Information

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Patent Type & Authority Patents(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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