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Recombinant cells and method for producing isoprene or terpene

A technology for recombining cells and isoprene, which is applied in the field of isoprene or terpene production, and can solve problems such as inappropriate production of isoprene, inability to obtain transformants, and limited supply of raw materials

Inactive Publication Date: 2019-04-26
SEKISUI CHEM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the process using glucose as a raw material, there is a disadvantage that the raw material supply is limited.
Furthermore, the low carbon conversion (below 15%) and batch rather than continuous process for material production using an inducible expression system makes it commercially unsuitable for the production of isoprene
[0005] In addition, it was reported that when constitutively expressed by Methonobacterium extroquence as a methylotroph, an operon gene comprising a heterologous mevalonate pathway gene and an α-humulene (C15 cyclic terpene) synthase gene was constitutively expressed , transformants cannot be obtained due to the toxicity caused by gene expression in this pathway, and α-humulene can be efficiently produced only by an inducible expression method with tightly controlled activity (Non-Patent Document 2)

Method used

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  • Recombinant cells and method for producing isoprene or terpene
  • Recombinant cells and method for producing isoprene or terpene
  • Recombinant cells and method for producing isoprene or terpene

Examples

Experimental program
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Effect test

Embodiment 1

[0213] Preparation of Isoprene Synthesis Gene Transfer Vectors for Isoprene Production in Archaea

[0214] In this example, prepare as Figure 5 The vector pAC:Pmcr-IDI-IspS for isoprene synthesis in archaea shown in . The vector is a shuttle vector having the replication region contained in the endogenous plasmid of Methanosarcina acetivorans and the replication region p15A in Escherichia coli, and can be derived from Methanococcus voltae The promoter PmcrB (methyl coenzyme M reductase) is used to make the IDI (isopentenyl diphosphate isomerase) gene derived from Saccharomyces cerevisiae and the IspS (isoprene synthase) gene derived from Poplus canescens be constitutively expressed.

[0215] Plasmid pACYC184 (Nippon Gene Co., Ltd.) was digested with HindIII and BclI to obtain a fragment containing the chloramphenicol resistance gene (Cat) and the replication region p15A, and a linker sequence comprising sequence number 5 and sequence number 6 was introduced to obtain Plasmi...

Embodiment 2

[0219] Transformation of Methanosarcina barkeri and production of isoprene from various carbon sources

[0220] According to the method of Metcalf WW et al. (Metcalf WW et al., PNAS 1997, 94, 1997), single-cell culture of Methanosarcina barkeri (DSM800) and preparation of competent cells were carried out. That is, by ATCC 2825 modified medium (wherein 1L contains 10 grams of magnesium chloride hexahydrate, 0.76 grams of potassium chloride, 24 grams of sodium chloride) (pH 6.8), under stringent anaerobic conditions, cultured to Several growth phase (OD600=0.3-0.6). Bacteria were recovered by centrifugation in an anaerobic chamber (Coy). The recovered thalline was suspended in 0.85M sucrose aqueous solution to obtain competent cell suspension (about 10 9 cells / mL).

[0221] 15 μL of DOTAP liposomes (Boehringer Mannheim), 100 μL of 20 mM HEPES-KOH (pH 7.4), and 2 μg of pAC:Pmcr-IDI-IspS (in 50 μL of HEPES-KOH (pH 7.4)) were mixed, A 15 minute incubation was performed. Add 1 ...

Embodiment 3

[0232] Preparation of vectors for farnesene production in archaea

[0233] In this example, the carrier pAC:Pmcr-IDI-FnS ( Figure 6 ) and pAC:Pmcr-IDI-FnS-FPS ( Figure 7 ). pAC:Pmcr-IDI-FnS contains IDI from Saccharomyces cerevisiae and Farnesene synthase (FnS) genes from Artemisia annua. pAC:Pmcr-IDI-FnS-FPS contains the above-mentioned IDI, Saccharomyces cerevisiae-derived farnesene synthase (FPS), and the above-mentioned FnS gene.

[0234] Use MluI-NotI to the pAC prepared in embodiment 1:Pmcr-IDI-IspS ( Figure 5 ) to remove the IDI-IspS region and obtain a linear fragment. The linear fragment was fused with a gene fragment containing IDI-FnS (SEQ ID NO: 11) to construct pAC:Pmcr-IDI-FnS (SEQ ID NO: 13, Figure 6 ). In addition, the linear fragment was fused with a gene fragment containing the IDI-FnS-FPS gene (SEQ ID NO: 12) (Genewiz Company) to construct pAC:Pmcr-IDI-FnS-FPS gene (SEQ ID NO: 14, Figure 7 ). exist Figure 6 and 7Among them, scIDI represents I...

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Abstract

Provided are recombinant cells that are anaerobic archaea, wherein: the recombinant cells have a gene that encodes isoprene synthase, a gene that encodes monoterpene synthase, a gene that encodes sesquiterpene synthase, a gene that encodes diterpene synthase, a gene that encodes squalene synthase, or a gene that encodes phytoene synthase as a first foreign gene; and the recombinant cells are capable of expressing the first foreign gene and producing isoprene or terpenes having 10, 15, 20, 30, or 40 carbon atoms.

Description

technical field [0001] The present invention relates to recombinant cells, and methods for producing isoprene or terpene, more particularly, the present invention relates to recombinant cells capable of producing isoprene or terpene as archaea, and the use of the recombinant cell in the production of isoprene or terpene approach. Background technique [0002] In recent years, the use of microorganisms to produce combustion gases such as waste, that is, synthesis gas (CO, CO 2 、H 2 Mixed gas) technology development for the manufacture of petrochemical alternative chemicals. Among them, construction of a petrochemical alternative process for isoprene as a tire raw material is quite important, and a production technology of isoprene and terpene using a recombinant synthetic gas-utilizing bacterium belonging to the genus Clostridium has been studied ( For example, Patent Documents 1 to 3). However, in the prior art, only low productivity could be obtained due to the negative...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P5/00C12N15/09
CPCC12N15/09C12P5/007C12N15/52C12N9/88C12N9/90C12N9/0008C12Y402/03102C12Y207/04026C12N1/20C12Y101/01088C12Y102/07004C12Y203/01009C12Y203/0301C12Y205/01021C12Y205/01032C12Y207/01036C12Y401/01033C12Y402/03027C12P5/00C12N1/205C12R2001/26
Inventor 古谷昌弘松岛加奈川端一史
Owner SEKISUI CHEM CO LTD
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