Recombinant cell and production method for isoprene

一种重组细胞、异戊二烯的技术，应用在生物化学设备和方法、重组DNA技术、细菌等方向，能够解决不生产异戊二烯等问题

Inactive Publication Date: 2015-09-16
SEKISUI CHEM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology does not produce isoprene

Method used

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  • Recombinant cell and production method for isoprene
  • Recombinant cell and production method for isoprene
  • Recombinant cell and production method for isoprene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] (1) Isolation of poplar-derived isoprene synthase gene and construction of expression vector

[0135] Using the total RNA derived from the leaves of poplar (Populus nigra) as a template, by RT-PCR using the primers represented by SEQ ID NO: 3 and SEQ ID NO: 4, the coding poplar-derived isoprene synthase ( IspS) nucleic acid (IspS gene from poplar, sequence number 1, gene bank accession number: AM410988.1) was amplified. The obtained amplified DNA fragment was cloned into pT7-Blue T vector (TAKARA BIO INC.) to construct pT7IS.

[0136] On the other hand, SEQ ID NO: 5 and SEQ ID NO: 6 were introduced into the BamHI / EcoRI site of the Clostridium / Escherichia coli (E. coli) shuttle vector pIMP1 (Mermelstein LD et al., Bio / technology 1992, 10, 190-195) The indicated synthetic DNA was modified to construct pIM1A by modifying the cloning site. Furthermore, synthetic DNAs represented by SEQ ID NO: 7 and SEQ ID NO: 8 were introduced into the PstI / BamHI site of pIM1A to construc...

Embodiment 2

[0146] (1) Construction of expression vector incorporating mevalonate pathway enzyme gene and isoprene synthase gene

[0147] Using the genomic DNA of Streptomyces griseolosporeus (Kitasatospora griseola) as a template, PCR using the primers represented by SEQ ID NO: 10 and SEQ ID NO: 11 was used to detect the methylol gene encoding S. griseolosporeus. Nucleic acid (SEQ ID NO: 9) of pentanoate pathway enzyme was amplified. The nucleic acid contains codes for mevalonate kinase, mevalonate diphosphate decarboxylase, phosphomevalonate kinase, IPP isomerase, HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) Gene cluster for reductase (HMGR), and HMG-CoA synthetase. The obtained amplified DNA fragment was cloned into pT7-Blue T vector to construct pT7SMV.

[0148] On the other hand, using the pT7IS prepared in Example 1 as a template, a DNA fragment containing the poplar-derived IspS gene was amplified using the primers represented by SEQ ID NO: 3 and SEQ ID NO: 12. This DNA fragm...

Embodiment 3

[0160] (1) Construction of an expression vector incorporating the codon-changed isopentenyl diphosphate isomerase (IDI) gene and isoprene synthase (IspS) gene

[0161] In this example, it was attempted to use Yondal's Escherichia coli-derived isopentenyl diphosphate isomerase (IDI) gene and poplar-derived isoprene synthase (IspS) gene introduced with codon changes. Clostridium to produce isoprene. For codon changes, refer to the codon usage table of Clostridium kluyveri (DSM 555) (http: / / www.kazusa.or.jp / codon / cgi-bin / spsearch.cgi?species=clostridium&c= i).

[0162] A codon-changed IDI-IspS operon synthetic gene (SEQ ID NO. 15, represented by the sense strand) was introduced into the PstI / BamHI site of pIM1A produced in Example 1 to construct the expression vector pIMAIS1. Using the same method, the expression vector pIMAIS2, which introduced the IDI-IspS operon synthetic gene without codon change, was also constructed.

[0163] It should be noted that in SEQ ID NO: 15, the...

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Abstract

To provide a series of techniques capable of producing isoprene from syngas or the like. Provided is a recombinant cell prepared by introducing a nucleic acid encoding isoprene synthase into a host cell having an isopentenyl diphosphate synthesis ability by a non-mevalonate pathway, wherein the nucleic acid is expressed in the host cell, and the recombinant cell is capable of producing isoprene from at least one C1 compound selected from the group consisting of carbon monoxide, carbon dioxide, formic acid, and methanol. As the host cell, a Clostridium bacterium or a Moorella bacterium is exemplified. Also provided is a method for producing isoprene using the recombinant cell.

Description

technical field [0001] The present invention relates to a recombinant cell capable of producing isoprene from a specific C1 compound such as carbon monoxide, and a method for producing isoprene using the recombinant cell. Background technique [0002] Isoprene is a monomer raw material for the synthesis of polyisoprene, especially in the tire industry. In recent years, the development and practical application of the conversion technology from the production process of basic chemicals dependent on petroleum to the production process derived from renewable resources such as plant resources is progressing steadily. Regarding isoprene, for example, production techniques using recombinant Escherichia coli using sugar as a raw material are known (Patent Documents 1 and 2). [0003] Regarding the production process from renewable resources, including the above-mentioned isoprene production technology, the prior art basically relies on the production method using microorganisms fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/09C12P5/00
CPCC12N9/88C12P5/00C12P5/007C12Y302/01C12N9/0008C12Y102/07004C12Y102/99002C12Y402/03027Y02E50/30
Inventor 古谷昌弘上西章太岩佐航一郎S.詹尼温R.费希尔
Owner SEKISUI CHEM CO LTD
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