Genetic engineering stain for improving yield of isoprene and construction method and application thereof

A technology of genetically engineered bacteria and isoprene, applied in the field of genetic engineering, can solve the problems of insignificant improvement in catalytic efficiency, low catalytic efficiency, and bacterial toxicity.

Active Publication Date: 2020-07-03
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

However, there are still two problems to be solved in the biosynthesis of isoprene: one is that the exogenous MVA pathway is relatively long, and the catalytic efficiency of the downstream pathway of MVA is low. , the yield of isoprene is only 28% of the theoretical conversion rate, and the promotion of catalytic efficiency is not obvious by screening new sources of enzymes or performing directional transformation on existing enzymes; the second is that some intermediate products such as isoprene Isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP) are toxic to the bacteria and affect the normal growth of the bacteria

Method used

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  • Genetic engineering stain for improving yield of isoprene and construction method and application thereof
  • Genetic engineering stain for improving yield of isoprene and construction method and application thereof
  • Genetic engineering stain for improving yield of isoprene and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1. The construction method of the genetically engineered bacterium that improves isoprene output.

[0051] The genetically engineered bacterium prepared in this example to increase the production of isoprene overexpresses the mevalonate kinase gene ERG12 containing the D18-wrapped signal peptide sequence, the phosphomevalonate kinase gene ERG8 containing the D18-wrapped signal peptide sequence, and the Mevalonate pyrophosphate decarboxylase gene ERG19 with D18 wrapped signal peptide sequence, isopentenyl pyrophosphate isomerase gene IDI1 with D18 wrapped signal peptide sequence, isoprene synthase gene with D18 wrapped signal peptide sequence IspS pa , and recombinant plasmids containing PduA, PduB, PduJ, PduK, PduN and PduU gene clusters, and the starting strain is Escherichia coli. The amino acid sequence of the D18 wrapped signal peptide is shown in SEQ ID NO:1. The mevalonate kinase gene ERG12 is the mevalonate kinase gene ERG12 derived from Saccharomyce...

Embodiment 2

[0121] Example 2. Application of the genetically engineered bacteria prepared in Example 1 in fermentative production of isoprene.

[0122] This example illustrates the use of Example 1 to prepare the genetically engineered bacteria to produce isoprene through an in vitro enzyme-catalyzed reaction. The genetically engineered bacteria are induced to express proteosomes, and then the proteosomes are added to the in vitro enzymes. In the catalytic reaction system, the isoprene is prepared by incubating at room temperature. The specific method is as follows:

[0123] 1. Validation of Proteosomes

[0124] (1) The recombinant Escherichia coli constructed in Example 1 was inoculated in 100 mL LB medium, and the shaker was cultivated to OD at 37° C. at 180 rpm 600 0.6-1.0, add inducer IPTG to a final concentration of 0.4mM, and incubate at 30°C for 9 hours.

[0125] (2) Centrifuge the bacterial solution at 5000rpm for 10min, wash twice with Buffer A, add 10mL buffer A and 15mL BPER...

Embodiment 3

[0140] Example 3. Application of the genetically engineered bacteria prepared in Example 1 in fermentative production of isoprene.

[0141] This example illustrates the fermentation and synthesis of isoprene by using the prepared genetically engineered bacteria in a shake flask.

[0142] Inoculate the activated engineered Escherichia coli into the LB liquid culture solution containing the corresponding antibiotics at a ratio of 1:100, culture at 37°C with shaking at 180rpm, when the OD 600 When the temperature is 0.6-0.8, add IPTG with a final concentration of 0.4mM to the bacterial solution, and then transfer to 30°C and continue culturing at 180rpm. After the engineered strains were induced and cultured for 24 hours, 1 mL of the headspace gas was taken and quantitatively detected by gas-phase GC. The detection method was referred to Yang J, Xian M, Su S, Zhao G, Nie Q, JiangX, et al. -isoprene using hybrid MVA pathway and isoprene synthase in E. coli. PLoS one. 2012;7:e3350...

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Abstract

The invention provides a genetic engineering stain for improving yield of isoprene and a construction method and application thereof and belongs to the technical field of genetic engineering. In orderto improve the yield of the isoprene synthesized by a biological method and to reduce the toxicity of an intermediate product on bacteria, the invention provides the genetic engineering stain for improving the yield of the isoprene. The genetic engineering stain over-expresses a mevalonate kinase gene ERG12 containing a D18 encapsulated signal peptide sequence, a phosphomevalonate kinase gene ERG8 containing the D18 encapsulated signal peptide sequence, a mevalonate diphosphate decarboxylase gene ERG19 containing the D18 encapsulated signal peptide sequence, an isopentenyl diphosphate isomerase gene IDI1 containing the D18 encapsulated signal peptide sequence, an isoprene synthase gene IspSpa containing the D18 encapsulated signal peptide sequence, and recombinant plasmids containing PduA, PduB, PduJ, PduK, PduN and PduU gene clusters, and a starting strain is escherichia coli. The genetic engineering stain can be used for producing the isoprene through fermentation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium for increasing the yield of isoprene and its construction method and application. Background technique [0002] Isoprene is an important chemical raw material and platform compound, mainly used in synthetic rubber. In addition, it is also used in the synthesis of other isoprene compounds and their derivatives in the fine chemical industry. Its uses cover the fields of medicine, pesticides, spices, biofuels, etc., and the annual global demand reaches millions of tons. Isoprene is currently produced primarily from petroleum-based feedstocks through chemical methods. However, with the increasing depletion of fossil resources and rising prices, raw material issues and environmental issues have become important bottlenecks for the preparation of isoprene. Therefore, finding a new production route of isoprene is an important ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/12C12N9/88C12N9/90C12P5/02C12R1/19
CPCC12N9/1205C12N9/1229C12N9/88C12N9/90C12N15/70C12P5/007C12Y207/01036C12Y207/04002C12Y401/01033C12Y503/03002C12Y402/03027Y02E50/30
Inventor 张海波咸漠刘长青门潇李美洁
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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