Enhanced ethanol and butanol producing microorganisms and method for preparing ethanol and butanol using the same

A technology of microorganisms and recombinant microorganisms, applied in the direction of biofuels, transferases, fermentation, etc., can solve the problems of lack, impossible industrial application, and inability to be used as fuels

Active Publication Date: 2011-01-19
GS CALTEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

That is, the production of ethanol and butanol by petrochemistry has a problem in that during the production process, a large amount of hazardous waste, waste water, and exhaust gas (including carbon monoxide) are discharged, especially with restrictions on the use of fossil fuels as basic raw materials
However, the above results are examples of the production of butanol and ethanol and acetone as a by-product, with the disadvantage that without removing acetone, they cannot be used as fuel due to the properties of acetone
[0007] There are cases where ethanol and butanol are produced without the production of acetone using a recombinant microorganism constructed by introducing aad (alcohol / acetaldehyde dehydrogenase) into a mutant strain of Clostridium acetobutylicum that Loss of function for all adc (gene encoding acetoacetate decarboxylase), ctfA (gene encoding CoA transferase A), ctfB (gene encoding CoA transferase B), and aad (gene encoding ethanol / acetaldehyde dehydrogenase) but, this method has the problem of low yield because the final concentrations of butanol and ethanol are 84 mM and 8 mM respectively (Nair et al., J.Bacteriol., 176:5843, 1994)
Contains another case of butanol production by introducing a recombinant vector carrying the Clostridium acetobutylicum gene into an E. coli strain (Shota et al., Metab. Eng., In Press, 2007), but The maximum concentration of butanol produced is very low at 552 mg / l, making industrial application impossible

Method used

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  • Enhanced ethanol and butanol producing microorganisms and method for preparing ethanol and butanol using the same
  • Enhanced ethanol and butanol producing microorganisms and method for preparing ethanol and butanol using the same
  • Enhanced ethanol and butanol producing microorganisms and method for preparing ethanol and butanol using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Preparation of recombinant vector containing adhE1 gene encoding ethanol / acetaldehyde dehydrogenase and ctfAB gene encoding CoA transferase

[0033] The adhE1, ctfA, and ctfB genes of Bacillus acetobutylicum ATCC 824 with sequence number: 3, sequence number: 4, and sequence number: 5, respectively, were cloned together using a promoter and its transcription termination sequence. First, the chromosomal DNA of Bacillus acetobutylicum ATCC 824 was used as a template, and sequence 1 and sequence 2 were used as primers for PCR (Table 1), and then the obtained adhE1, ctfA and ctfB were cut with restriction enzyme SalI and inserted into the same restriction enzyme The cut Clostridium / E. coli shuttle vector pIMP1 (Mermelstein, LD et al., Bio / Technol., 10:190, 1992) was used to prepare the recombinant vector pIMP1::adhE1.ctfAB (Table 2). The genes encoding ethanol / acetaldehyde dehydrogenase and CoA transferase from Bacillus acetobutylicum ATCC 824 (adhE1, ctfAB) were thu...

Embodiment 2

[0037] Example 2: Construction of recombinant microorganisms

[0038] The M5 (pIMP1::adhE1.ctfAB) strain was constructed by introducing the recombinant vector pIMP1::adhE1.ctfAB constructed in Example 1 into the M5 strain of Bacillus acetobutylicum by electrotransformation. First, the recombinant vector of Example 1 was introduced into the vector pAN1 containing the expression Bacillus subtilis Phage Φ3T I methyltransferase (Mermelstein et al., Appl. Environ. Microbiol., 59: 1077, 1993). Escherichia coli TOP10 to induce its methylation, making the vector suitable for transformation into Clostridium. The methylated vector was isolated and purified from E. coli, and then introduced into a plasmid lacking a giant (carrying 176 genes, including genes encoding acetoacetate decarboxylase, genes encoding CoA transferase, and ethanol / acetaldehyde dehydrogenase Gene) in the mutant strain of Bacillus acetobutylicum M5 (Cornillot et al., J. Bacteriol., 179:5442, 1997), thereby preparing a ...

Embodiment 3

[0042] Example 3: Production of ethanol / butanol using recombinant microorganism M5 (pIMP1::adhE1.ctfAB)

[0043] The recombinant microorganism M5 (pIMP1::adhE1.ctfAB) prepared in Example 2 was cultured in order to check its performance. Sterilize a 30ml test tube containing 10ml of CGM culture medium, take it out at a temperature exceeding 80°C, fill it with nitrogen, and cool to room temperature in an anaerobic chamber. Then, 40 μg / ml erythromycin was added to the medium, and the recombinant microorganisms were inoculated, and then pre-cultured at 37°C under anaerobic conditions to an absorbance value of 1.0 at 600 nm. Sterilize a 250ml flask containing 100ml of the medium with the components, inoculate the medium with 6ml of the pre-culture solution, and conduct a second pre-culture at 37°C under anaerobic conditions until the absorption value at 600nm 1.0. Then, a 5.0L fermentor (LiFlus GX, Biotron Inc., Kyunggi-Do, Korea) containing 2.0L of medium with the components was st...

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Abstract

The present invention relates to a recombinant microorganism having an enhanced ability to produce ethanol and butanol and a method for preparing ethanol and butanol using the same, and more particularly to a recombinant microorganism having an enhanced ability to produce ethanol and butanol, into which a gene encoding CoA transferase and a gene encoding alcohol / aldehyde dehydrogenase are introduced, and to a method for preparing ethanol and butanol using the same. The recombinant microorganism according to the present invention, obtained by manipulating metabolic pathways of microorganisms, is capable of producing butanol and ethanol exclusively without producing any byproduct, and thus is useful as a microorganism producing industrial solvents and transportation fuel.

Description

Technical field [0001] The present invention relates to a recombinant microorganism having an enhanced ability to produce ethanol and butanol and a method for preparing ethanol and butanol using the microorganism. More specifically, the present invention relates to a recombinant microorganism having an enhanced ability to produce ethanol and butanol and to use the same A method for producing ethanol and butanol by a microorganism, wherein a gene encoding CoA transferase and a gene encoding ethanol / acetaldehyde dehydrogenase are introduced into the microorganism. Background technique [0002] Currently, ethanol and butanol have a huge market as industrial solvents, and the possibility of using them as fuels for transportation vehicles such as automobiles and the like is being realized. Therefore, the demand for ethanol and butanol is expected to continue to increase. [0003] Traditionally, ethanol (C 2 H 5 OH) is prepared by fermenting starch or sugar. Recently, most alcoholic beve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09
CPCY02E50/17C12N9/13C12P7/065C12P7/16C12N9/0006Y02E50/10C12N15/09C12P7/06
Inventor 李相烨张俞信李真英郑光燮金宰贤
Owner GS CALTEX CORP
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