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Process for the biological production of n-butanol with high yield

一种正丁醇、微生物的技术，应用在以高产率生物产生正丁醇领域，能够解决连续培养不稳定性等问题

Inactive Publication Date: 2009-09-09
METABOLIC EXPLORER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Traditionally, commercial ABE fermentations have only been performed in batch mode due to the continuous culture instability of producing Clostridia

Method used

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  • Process for the biological production of n-butanol with high yield
  • Process for the biological production of n-butanol with high yield
  • Process for the biological production of n-butanol with high yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Construction of a butyrate-incapable strain: Clostridium acetobutylicum Δcac1515ΔuppΔbuk

[0051] To delete the buk gene, the homologous recombination strategy described by Croux & Soucaille (2006) in patent application PCT / EP2006 / 066997 was used. This strategy allows the insertion of an erythromycin resistance cassette while deleting most of the associated genes. The buk deletion cassette in pCons::upp was constructed as follows.

[0052] Table 1: Primer sequences

[0053]

[0054] Two DNA fragments surrounding Buk were PCR amplified with Pwo polymerase using total DNA from Clostridium acetobutylicum as template and two pairs of specific oligonucleotides. Two DNA fragments were obtained using primer pairs BUK 1-BUK2 and BUK 3-BUK 4, respectively. Primers BUK 1 and BUK 4 both introduce a BamHI site, while primers BUK 2 and BUK 3 have a complementary region that introduces a NruI site. The DNA fragments BUK 1-BUK 2 and BUK 3-BUK 4 were combined in a PCR fusion exp...

Embodiment 2

[0058] Construction of a strain incapable of producing butyrate and acetone: Clostridium acetobutylicum Δcac1515 Δupp ΔbukΔctfAB

[0059] To delete the ctfAB gene, the homologous recombination strategy described by Croux & Soucaille (2006) in patent application PCT / EP2006 / 066997 was used. This strategy allows the insertion of an erythromycin resistance cassette while deleting most of the associated genes. The ctfAB deletion cassette in pCons::upp was constructed as follows.

[0060] Table 2: Primer sequences

[0061]

[0062]Two DNA fragments surrounding ctfAB were PCR amplified with Pwo polymerase using total DNA from C. acetobutylicum as template and two pairs of specific oligonucleotides. Two DNA fragments were obtained using primer pairs CTF 1-CTF2 and CTF 3-CTF 4, respectively. Primers CTF 1 and CTF 4 both introduce a BamHI site, while primers CTF 2 and CTF 3 have complementary regions that introduce a StuI site. DNA fragments CTF 1-CTF 2 and CTF 3-CTF 4 were combi...

Embodiment 3

[0066] Construction of a strain incapable of producing butyrate, acetone and acetate: Clostridium acetobutylicum Δcac1515ΔuppΔbukΔctfABΔldh

[0067] To delete the ldh gene, the homologous recombination strategy described by Croux & Soucaille (2006) in patent application PCT / EP2006 / 066997 was used. This strategy allows the insertion of an erythromycin resistance cassette while deleting most of the associated genes. The ldh deletion cassette in pCons::upp was constructed as follows.

[0068] Table 3: Primer sequences

[0069]

[0070] Two DNA fragments surrounding ldh (CAC267) were PCR amplified with Pwo polymerase using total DNA from Clostridium acetobutylicum as template and two pairs of specific oligonucleotides. Using primer pairs LDH 1-LDH 2 and LDH 3-LDH 4, DNA fragments of 1135bp and 1177bp were obtained, respectively. Primers LDH 1 and LDH 4 both introduce a BamHI site, while primers LDH 2 and LDH 3 have complementary regions that introduce a StuI site. DNA fragm...

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Abstract

The present invention provides a method for the biological production of n-butanol at high yield from a fermentable carbon source. In one aspect of the present invention, a process for the conversion of glucose to n-butanol is achieved by the use of a recombinant organism comprising a host C. acetobutlicum transformed i) to eliminate the butyrate pathway ii) to eliminate the acetone pathway iii) to eliminate the lactate pathway and iv) to eliminate the acetate pathway. In another aspect of the present invention, the hydrogen flux is decreased and the reducing power redirected to n-butanol production by attenuating the expression of the hydrogenase gene. Optionally the n-butanol produced can be eliminated during the fermentation by gas striping and further purified by distillation.

Description

field of invention [0001] The present invention comprises a method for bioconverting a fermentable carbon source to n-butanol in high yield by metabolically engineered microorganisms. Background of the invention [0002] n-Butanol is a colorless, moderately volatile neutral liquid with limited miscibility in water (about 7-8%), but is compatible with all common solvents such as glycols, ketones, alcohols, aldehydes, ethers, and aromatic and Aliphatic hydrocarbons are freely miscible. n-Butanol is used i) in the preparation of other chemicals, ii) as a solvent and iii) as an ingredient in formulated products such as cosmetics. The main use of n-butanol as a raw material is in the synthesis of acrylates / methacrylates, glycol ethers, n-butyl acetate, amino resins and n-butylamine. The world currently consumes more than 9 million tons of n-butanol per year. [0003] More recently, n-butanol has been shown to be a superior biofuel to ethanol due to lower vapor pressure, higher...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/16C12N1/15
CPCC12P7/16Y02E50/10C12N1/20C12N15/52C12N15/74
Inventor 菲利普·索凯勒
Owner METABOLIC EXPLORER
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