Isoprene synthase and coding gene thereof, expression vector, engineering bacteria and method for producing isoprene and application

A technology of isoprene synthase and gene, which is applied in the field of genetic engineering, can solve the problems of low affinity of substrate DMAPP, blockage of metabolic flow, and low activity of isoprene synthase, so as to achieve good application prospects and alleviate obstacles. The effect of stagnant flux

Active Publication Date: 2018-12-28
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to solve the problems that the existing isoprene synthase has low activity and low affinity to the substrate DMAPP and they all seriously block the metabolic flow in the metabolic pathway, the

Method used

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  • Isoprene synthase and coding gene thereof, expression vector, engineering bacteria and method for producing isoprene and application
  • Isoprene synthase and coding gene thereof, expression vector, engineering bacteria and method for producing isoprene and application
  • Isoprene synthase and coding gene thereof, expression vector, engineering bacteria and method for producing isoprene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: the acquisition of gene and the construction of vector

[0035] (1) Acquisition of exogenous genes

[0036] An isoprene synthase gene IspS encoding an isoprene synthase (amino acid sequence shown in SEQ ID NO.2) as shown in SEQ ID NO.2 was obtained by optimal design ib , the gene is derived from sweet potato (Ipomoea batatas). The isoprene synthase gene (IspS) from sweet potato (Ipomoea batatas) ib ) was chemically synthesized on the pUC-57 carrier by Jinweizhi Company to obtain pUC-IspS ib carrier.

[0037] (2)pACY-IspS ib Construction of expression vector

[0038] pUC-IspS ib As a template, primer 1IspS ib -F and primer 2IspS ib -R, perform polymerase chain reaction (PCR), amplify IspS ib Fragment, its PCR amplification system is as follows:

[0039]

[0040] The PCR program is: 94°C 3min; 30×(94°C 10s, 55°C 30s, 72°C 1min); 72°C 5min; 4°C∞

[0041] Its primer sequence is as follows:

[0042] IspS ib -F: 5'-GGAGATATACATATGAGTAGCGCCCAGAATC...

Embodiment 2

[0060] Embodiment 2: Construction of recombinant bacterial strain

[0061] (1) Construction of LMJ-ib recombinant strain

[0062] pACY-IspS ib The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, spread on the LB solid plate of 34 mg / mL chloramphenicol, and obtained positive clones by PCR screening, thereby obtaining the pACY-IspS containing ib Engineering of Escherichia coli LMJ-ib.

[0063] (2) Construction of LMJ-pa recombinant strain

[0064] pACY-IspS pa The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, spread on the LB solid plate of 34 mg / mL chloramphenicol, and obtained positive clones by PCR screening, thereby obtaining the pACY-IspS containing ib Engineering of Escherichia coli LMJ-pa.

[0065] (3) Construction of LMJ11 recombinant strain

[0066] pACY-MvaE-MvaS-IspS ib Recombinant plasmid and downstream pathway plasmid pTrc-low (cited from Yang J, Xian M, Su S, Zhao G, Nie Q, Jiang X...

Embodiment 3

[0069] Example 3: Expression and purification of foreign proteins

[0070] (1) Expression of foreign proteins

[0071]Pick a single colony of LMJ-ib or LMJ-pa obtained in Example 2, place it in 3 ml of LB liquid medium containing 34 mg / mL chloramphenicol, and culture it overnight on a shaker at 37°C for activation. Transfer to 100ml LB liquid medium containing 34mg / mL chloramphenicol for expansion culture, culture until OD600 is 0.6-0.8, induce with 0.5mM isopropyl-β-D-thiogalactoside (IPTG), culture at 30°C for 6h , 5000×g, 4°C, centrifuge for 15min, discard the supernatant, wash twice with phosphate buffered solution (PBS), and finally resuspend with 10mL western / IP bacterial lysate (Beiyuntian, Cat. No. P0013), and place on ice . Take a small amount for SDS-PAGE detection, IspS ib and IspS pa The protein was successfully expressed in Escherichia coli, see the specific results Figure 4 .

[0072] (2) Purification of foreign proteins

[0073] Centrifuge the bacterial ...

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Abstract

The invention discloses an isoprene synthase and a coding gene thereof, an expression vector, engineering bacteria and a method for producing isoprene and application, and belongs to the technical field of genetic engineering. The nucleotide sequence of the isoprene synthase gene IspSib is as shown in SEQ ID NO 2, and the amino acid sequence of the isoprene synthase is as shown in SEQ ID NO. 1. The invention also provides a prokaryotic expression vector containing the isoprene synthase gene and co-transformation of the prokaryotic expression vector and a downstream expression vector of mevalonate pathway to obtain an isoprene-producing fungus The present invention also provides a method for fermentative production of isoprene and the above-mentioned isoprene synthase gene, isoprene synthase, enzyme having isoprene synthase activity, prokaryotic expression vector thereof and The application of engineering bacteria in the production of isoprene. The isoprene synthase enzyme activity provided by the present invention is high and has high affinity for the substrate DMAPP.

Description

technical field [0001] The invention relates to an isoprene synthase, its coding gene, an expression vector, an engineering bacterium, a method and application for producing isoprene, and belongs to the technical field of genetic engineering. Background technique [0002] Isoprene is an important chemical platform compound, 95% of which is used in synthetic rubber; it is also the second monomer of butyl rubber. In addition, isoprene is also widely used in the fields of pesticides, medicines, spices and adhesives. At present, the source of isoprene is mainly through petroleum-based raw material isopentane, isopentene dehydrogenation method, chemical synthesis method (including isobutylene-formaldehyde method, acetylene-acetone method, propylene dimerization method) and cracking C5 fraction extraction Distillation. However, with the increasing depletion of fossil resources, the source of raw materials is an important bottleneck issue for the preparation of isoprene from petr...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21C12P5/00C12R1/19
CPCC12N9/0006C12N9/1025C12N9/1029C12N9/1205C12N9/1229C12N9/88C12N9/90C12N15/70C12P5/007C12Y101/01088C12Y203/01016C12Y203/0301C12Y207/01036C12Y207/04002C12Y401/01033C12Y402/03027C12Y503/03002
Inventor 咸漠李美洁张海波吴桐
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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