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

A technology of microorganisms and butanol, which is applied in the direction of biochemical equipment and methods, botany equipment and methods, applications, etc., and can solve the problems of intermediate production efficiency

Inactive Publication Date: 2010-03-10
BIOFUELCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Recombinant bacteria capable of producing butanol into which a butanol synthesis pathway is introduced and butanol production using the bacteria are disclosed (US 2007 / 0259410 A1; US ​​2007 / 0259411 A1), but the production efficiency is intermediate

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Example 1: Preparation of recombinant mutant microorganisms with high butanol production capacity

[0057] 1-1: Knockout of lacI gene

[0058] In E. coli W3110 (ATTC 39936), the lacI gene encoding the lac operon repressor protein that inhibits transcription of the lac operon required for lactose metabolism was inactivated by a one-step method using primers SEQ ID NOS: 1 and 2 (Warner et al. ., PNAS, 6: 97(12): 6640, 2000) to remove antibiotic resistance from bacteria.

[0059] [SEQ ID NO: 1] lacI_1stup: 5'-gtgaaaccagtaacgttatacgat

[0060] gtcgcagagtatgccggtgtctcttagattgcagcattacacgtcttg-3′

[0061] [SEQ ID NO: 2] lacI_1stdo: 5'-tcactgcccgctttccagtcgg

[0062] gaaacctgtcgtgccagctgcattaatgcacttaacggctgacatggg-3′

[0063] 1-2: Knockout of ldhA, pta and adhE genes

[0064] In the lacI-knockout Escherichia coli W3110 of Example 1-1, ldhA (encoding lactate dehydrogenase), pta (encoding phosphotransacetylase) were further eliminated by one-step inactivation using prime...

Embodiment 2

[0134] Example 2: Introduction of recombinant mutant microorganisms from Escherichia coli and Clostridium acetobutylicum production of butanol

[0135] In this example, when the part of the gene responsible for the butanol biosynthesis pathway from Clostridium acetobutylicum was replaced by the gene from Escherichia coli, the production capacity of butanol ( Figure 10 ). In detail, when adhE, crt, hbd and thiL from Clostridium were replaced by genes from Escherichia coli, respectively, the butanol-producing ability of the resulting recombinant mutant microorganisms was determined.

[0136] 2-1: Construction of pKKmhpFpaaFGHatoB vector

[0137]PCR was performed using the chromosomal DNA of E. coli W3110 as a template using primers SEQ ID NOS: 33 to 38 to amplify the genes necessary for the butanol biosynthesis pathway, including mhpF (encoding aldehyde dehydrogenase), paaFG (encoding enoyl coenzyme) A hydratase), paaH (encodes 3-hydroxy-acetyl-CoA dehydrogenase) and atoB...

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Abstract

The present invention relates to a method for producing butanol using a bacterium capable of biosynthesizing butanol from butyryl-CoA as an intermediate. More particularly, a method for producing butanol, the method comprising generating bytyryl-CoA in a bacterium which contains a gene coding for AdhE (an enzyme responsible for the conversion of butyryl-CoA to butanol) using various methods, and converting the butyryl-CoA into butanol.

Description

technical field [0001] The present invention relates to a recombinant mutant microorganism with enhanced butanol-producing ability, wherein genes encoding enzymes responsible for lactate, ethanol and / or acetate biosynthesis are deleted, and genes encoding enzymes involved in butanol biosynthesis are introduced, the present invention It also relates to a method of producing butanol using the microorganism. Background technique [0002] Biofuels produced using microorganisms have received increasing attention in recent years with the sharp rise in oil prices and growing concerns about global warming and greenhouse gases. In particular, compared to bioethanol, biobutanol has the advantage that it is easier to mix with fossil fuels due to its lower oxygen content. The market size of biobutanol, which has emerged as an alternative fuel to gasoline in recent years, has increased rapidly. The biobutanol volume in the US market reaches 370 million gallons per year at a price of $3...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00
Inventor 李相烨朴真焕埃莱夫塞里奥斯·特里·帕保特塞克斯
Owner BIOFUELCHEM
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