Recombinant cells, method for producing recombinant cells, and method for producing 1,4-butanediol

A technology of recombinant cells and butanediol, applied in the field of recombinant cells, can solve the problems of unreported production

Inactive Publication Date: 2018-07-17
SEKISUI CHEM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, no technology has been reported to efficiently produce 1,4-butanediol from cellulose

Method used

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  • Recombinant cells, method for producing recombinant cells, and method for producing 1,4-butanediol
  • Recombinant cells, method for producing recombinant cells, and method for producing 1,4-butanediol
  • Recombinant cells, method for producing recombinant cells, and method for producing 1,4-butanediol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0189] Production of 1,4-butanediol (succinate pathway) by the synthetic gas-assimilating bacterium Clostridium listeri (C.ljungdahlii)

[0190] Clostridium / Escherichia coli (Clostridium / E.coli) shuttle vector pGn15_SUC (op) (sequence number 1, image 3 ) was introduced into Clostridium listeri (C.ljungdahlii) by electroporation method (BioRad Micropulser (BioRad, Hercules, CA, USA), 0.63kV). The obtained transformant was recovered from YTF agar medium (per 1 L, tryptone 16 g, yeast extract 10 g, sodium chloride 4 g, L-cysteine ​​2 mM, xylose 1%, agar 1.5%).

[0191]In the electroporation method, the following method is used. All operations except centrifugation and cryopreservation were carried out in a Whitley Anaerobic Workstation A55 (Don Whitley Scientific Limited, United Kingdom) under gas conditions of 10% CO 2 , 5%H 2 , 85%N 2 under obligate anaerobic conditions. A strain of Clostridium ljungdahlii (DSM No. 13528) was obtained from DSMZ (Deutsche Sammlung von Mikr...

Embodiment 2

[0208] Production of 1,4-butanediol from syngas using Clostridium ljungadhlii (alpha-ketoglutarate pathway)

[0209] Clostridium / Escherichia coli (Clostridium / E.coli) shuttle vector pGn15AKG (op) (sequence number 2, Figure 4 ) was introduced into Clostridium listeri (C.ljungdahlii) by electroporation method (BioRad Micropulser (BioRad, Hercules, CA, USA), 0.63kV). The obtained transformant was recovered from YTF agar medium (per 1 L, tryptone 16 g, yeast extract 10 g, sodium chloride 4 g, L-cysteine ​​2 mM, xylose 1%, agar 1.5%). The pGn15AKG(op) vector contains encoding α-ketoglutarate decarboxylase (sucA derived from Mycobacterium bovis), 4-hydroxybutyrate dehydrogenase (source 4hbd) from Porphyromonas gingivalis, 4-hydroxybutyrylCoA transferase (cat2 from Porphyromonas gingivalis), and difunctional 1,4-BDO synthesis gene cluster of bifunctional 4-hydroxybutyryl CoA reductase and alcohol dehydrogenase (adhE2 from Clostridium acetobutylicum) ( Figure 4 ). The resulting ...

Embodiment 3

[0215] Production of 1,4-butanediol by the cellulose-assimilating bacterium Clostridium cellulolyticum (succinate pathway)

[0216] Clostridium / Escherichia coli (Clostridium / E.coli) shuttle vector pM9_SUC (op) (sequence number 3, Figure 5 ) was introduced into Clostridium cellulolyticum (C.cellulolyticum) by electroporation method (1.6kV). By CM3 (1.3gL -1 (NH 4 ) 2 SO 4 , 1.5gL -1 KH 2 PO 4 , 2.9gL -1 K 2 HPO 4 ×3H 2 O, 0.2gL -1 MgCl 2 ×6H 2 O, 75.0mgL -1 CaCl 2 ×2H 2 O, 1.25mgL -1 FeSO 4 ×7H 2 O, 1.0 mL L -1 Trace element solution SL-10, 1.0mgL -1 Resazurin, 2.0gL -1 Yeast Extract, 2.5gL -1 Na 2 CO 3 , 0.5gL -1 L-cysteine-HCL×H 2 O, 6.0gL -1 D-cellobiose and 15.0gL -1 agar) agar plates to obtain transformants. The pM9_SUC(op) vector contains codes for succinyl CoA synthetase (sucCD from Escherichia coli), CoA-dependent succinate semialdehyde dehydrogenase (sucD from Clostridium kluyveri), 4hbd from Porphyromonas gingivalis, cat2 from Porphyrom...

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Abstract

The present invention addresses the problem of providing a technique for producing 1,4-butanediol by recombinant cells. Recombinant cells having obligate anaerobicity and acetic acid production capacity, wherein the recombinant cells have a gene that encodes at least one enzyme selected from the group comprising succinate semialdehyde dehydrogenase, succinyl CoA synthetase, CoA-dependent succinatesemialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate CoA transferase, 4-hydroxybutyrate CoA reductase, 4-hydroxybutyraldehyde dehydrogenase, and alcohol dehydrogenase, express the gene, and are capable of producing 1,4-butanediol.

Description

technical field [0001] The invention relates to a recombinant cell, a method for manufacturing the recombinant cell and a method for producing 1,4-butanediol. Background technique [0002] 1,4-Butanediol (1,4-Butanediol) is an organic compound that can serve as an important butadiene raw material as a monomer of synthetic rubber, and is an important raw material especially in the tire industry. In recent years, the development and practical use of technologies for switching from the production process of basic chemicals that depend on petroleum to the production process from renewable resources such as plant resources has been steadily progressing. Regarding 1,4-butanediol, for example, a production technique using recombinant Escherichia coli using sugar as a raw material is known (Patent Document 1). [0003] figure 1 An example of a biosynthetic pathway for 1,4-butanediol is shown. That is, 1,4-butanediol can be biosynthesized using, for example, succinate or α-ketoglu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/18C12P7/54C12N15/09
CPCC12N15/09C12P7/54C12P7/18C12N15/00Y02E50/10C12N15/74C12Y101/01002C12Y101/01001C12Y101/01061C12Y102/0101C12Y102/01076C12N1/20
Inventor 古谷昌弘S.詹内怀恩R.费希尔C.麦克埃尔罗伊S.盖达
Owner SEKISUI CHEM CO LTD
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