Method for the preparation of diols

A technology of aliphatic diols and microorganisms, which is applied in the directions of botany equipment and methods, biochemical equipment and methods, applications, etc., and can solve problems such as expensive production processes

Active Publication Date: 2012-02-01
METABOLIC EXPLORER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Specifically, the production of 1,3-propanediol has been described, which involves vitamin B12-dependent enzymes, making the production process very expensive

Method used

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  • Method for the preparation of diols
  • Method for the preparation of diols
  • Method for the preparation of diols

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Experimental program
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specific Embodiment approach

[0064] Other embodiments of the present invention will be described below. The microorganisms are modified to favor the production of hydroxy-2-keto-aliphatic acid metabolites and to convert the products obtained from the decarboxylation step of the same hydroxy-2-keto-aliphatic acid metabolites to the corresponding aliphatic diols.

[0065] The following description is for E. coli, a microorganism lacking endogenous 2-ketoacid decarboxylase activity. Thus, a heterologous gene encoding this activity is introduced into the microorganism.

[0066]The microorganism is modified to optimize the pathway for producing a hydroxy-2-ketoaliphatic acid metabolite and to convert the product obtained from the decarboxylation step of the same hydroxy-2-ketoaliphatic acid metabolite to an aliphatic diol or Based on the known metabolic pathways and endogenous genes of E. coli. However, those skilled in the art can use similar strategies to introduce or delete corresponding genes in other mi...

Embodiment 1

[0106] Construction of a strain expressing the gene encoding 2-ketoacid decarboxylase and the gene encoding hydroxyaldehyde reductase: MG1655 (pME101-kivDll-yqhD-TT07)

[0107] 1.1 Construction of plasmid pM-Ptrc01-kivD11-TT07 for overexpressing kivD of Lactococcus lactis encoding α-ketoisovalerate decarboxylase:

[0108]The synthetic gene of Lactococcus lactis kivD encoding α-ketoisovalerate decarboxylase was prepared by Geneart (Germany). The codon usage and GC content of this gene were adapted to E. coli according to the supplier matrix. Expression of this synthetic gene is driven by the constitutive Ptrc promoter. A transcription terminator was added downstream of the gene. Constructs were cloned into the supplier's pM vector and verified by sequencing. If necessary, the synthetic gene was cloned into pME101 vector (this vector was derived from plasmid pCL1920 (Lerner & Inouye, 1990, NAR 18, 15p 4631)), which was then used to transform E. coli strains.

[0109] Ptrc0...

Embodiment 2

[0152] Construction of strains with increased flux in the ethylene glycol pathway: MG1655ΔsdaAΔsdaB ΔpykFPtrc 18-gpmA Ptrc 18-gpmB(pME 101-kivDll-yqhD-TT07)

[0153] 2.1 Construction of MG1655ΔsdaAΔsdaB strain

[0154] To delete the sdaA gene, the homologous recombination strategy described by Datsenko & Wanner (2000) was used. This strategy allows the insertion of chloramphenicol or kanamycin resistance cassettes while deleting most of the genes involved. For this purpose the following oligonucleotides were used:

[0155] ΔsdaAF (SEQ ID NO 10)

[0156] gtcaggagtattatcgtgattagtctattcgacatgtttaaggtggggattggtccctcatcttcccataccgtagggccTGTAGGCTGGAGCTGCTTCGhas

[0157] - and the sequence of the sdaA gene (1894941-1895020) (see the website for the reference sequence http: / / genolist.pasteur.fr / Colibri / ) homologous region (lowercase),

[0158] - a region for amplification of the kanamycin resistance cassette (see Datsenko, K.A. & Wanner, B.L., 2000, PNAS, 97: 6640-6645 for refere...

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Abstract

The present invention concerns a new method for the biological preparation of a diol comprising culturing a microorganism genetically modified for the bioproduction of an aliphatic diol, wherein the microorganism comprises a metabolic pathway for the decarboxylation of a hydroxy-2-keto-aliphatic acid metabolite with an enzyme having a 2-keto acid decarboxylase activity, the product obtained from said decarboxylation step being further reduced into the corresponding aliphatic diol, and wherein the microorganism is genetically modified for the improved production of said hydroxy-2-keto-aliphatic acid metabolite. The invention also concerns a modified microorganism for the production of an aliphatic diol.

Description

[0001] The present invention relates to a new method for the bioproduction of diols, comprising culturing a microorganism genetically modified for the bioproduction of aliphatic diols, wherein the microorganisms comprise hydroxy-2-ketoaliphatic acid metabolites with two enzymes Metabolic pathways for decarboxylation: enzymes with 2-ketoacid decarboxylase activity and enzymes with hydroxyaldehyde reductase activity. The invention also relates to microorganisms modified for the production of aliphatic diols. Background technique [0002] Fermentative production of diols by culturing such diol-producing microorganisms is known in the art, including fermentative production with microorganisms genetically modified for improved diol production. The production of such diols is described in the following documents: WO1996 / 035796, WO2001 / 012833, WO2004 / 033646, US7267972 and others. In particular, the production of 1,3-propanediol has been described, which involves vitamin B12-dependen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/18C12N1/21C12N9/02C12N9/88C12N15/53C12N15/60
CPCC12P7/18C12N9/88C12N9/0006C12N9/1096C12N1/005C12N1/20C12N15/70C12P7/04
Inventor P.苏凯尔C.伯伊萨特
Owner METABOLIC EXPLORER
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