Reconstituted cell for isoprene and preparation method of reconstituted cell

A technology of isoprene and recombinant cells, applied in microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve problems such as low yield of target products, disorder of cell self-metabolism, affecting normal cell growth and metabolism, etc.

Active Publication Date: 2012-07-11
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

When obtaining the exogenous MVA pathway in the engineered bacteria, up to 8 heterologous genes need to be transferred. The expression of too many heterologous ...

Method used

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  • Reconstituted cell for isoprene and preparation method of reconstituted cell
  • Reconstituted cell for isoprene and preparation method of reconstituted cell
  • Reconstituted cell for isoprene and preparation method of reconstituted cell

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Experimental program
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Embodiment 1

[0030] Prenyl pyrophosphate isomerization by co-overexpressing the deoxyxylulose 5-phosphate synthase gene (dxs) and deoxyxylulose 5-phosphate reductase gene (dxr) from E. coli in E. coli The enzyme gene (idi) and the isoprene synthase gene (IspS) biosynthesize isoprene using glucose degradation intermediates pyruvate and glyceraldehyde-3-phosphate.

[0031] 1.1 Cloning of foreign genes and construction of expression vectors

[0032] 1.1.1 Cloning of foreign genes

[0033] 1.1.1.1 Cloning of E.coli deoxyxylulose 5-phosphate synthase gene

[0034] Genomic DNA of E. coli (ATCC No.10798) was extracted, primers were designed according to GenBank sequence, and deoxyxylulose 5-phosphate synthase gene (dxs) was amplified by PCR, GenBank accession number: 6059543. Then use the gel recovery kit to recover the target gene fragment.

[0035] The amplification primer sequences are:

[0036] Ds-L: 5'-CGC GGATCC GAGTTTTGATATTGCCAAATACCCGA-3'

[0037] DS-R: 5'-CATG GAGCTC TTATGCCAGC...

Embodiment 2

[0086] Escherichia coli (E. coli (E. coli) or Saccharomyces cerevisiae isopentenyl pyrophosphate isomerase gene (idi), plant-derived isoprene synthase gene (IspS) (Populus alba x Populustremula; Populus nigra; Populus alba), which use glucose to degrade the intermediate acetone Acid and glyceraldehyde-3-phosphate biosynthesis of isoprene.

[0087] 2.1 Cloning of foreign genes and construction of expression vectors

[0088] 2.1.1 Cloning of foreign genes

[0089] 2.1.1.1 Cloning of the deoxyxylulose 5-phosphate synthase gene gene of Bacillus subtilis

[0090] Genomic DNA of Bacillus subtilis (ATCC NO.23857) was extracted, primers were designed according to GenBank sequence, and deoxyxylulose 5-phosphate synthase gene (dxs) was amplified by PCR, GenBank accession number: 938609. Then use the gel recovery kit to recover the target gene fragment.

[0091] The amplification primer sequence is

[0092] Ds2-L: 5′CGC GGATCC GGATCTTTTATCAATACAGGACCCG-3′

[0093] Ds2-R: 5'GC G...

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Abstract

The invention provides a method for synthesizing isoprene from pyruvic acid and 3-phosphoglyceraldehyde, and a reconstituted cell. The pyruvic acid and the 3-phosphoglyceraldehyde are finally obtained from simple starting materials such as glucose.

Description

technical field [0001] The invention relates to the field of preparation of isoprene compounds, in particular to the field of preparing isoprene by recombinant cell biology method. 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. [0003] 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 petroleum-based raw materials. [0004] There are mainly...

Claims

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Application Information

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IPC IPC(8): C12P5/02C12N15/70C12N15/75C12N15/81C12N1/21C12N1/19C08F36/08C08F210/12C08F297/04C09J147/00C10M127/02C11B9/00C12R1/19C12R1/125C12R1/865
Inventor 咸漠杨路杨建明张英伟张海波高毅
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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