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Method for preparing mutant escherichia coli capable of simultaneously utilizing glucose and xylose

一种大肠杆菌、葡萄糖的技术，应用在基于微生物的方法、使用微生物的方法、生物化学设备和方法等方向，能够解决缺少除去、消除CCR等问题

Active Publication Date: 2015-02-25
UNIST (ULSAN NAT INST OF SCI & TECH)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the absence of the glucose phosphotransferase system (PTS) in E. coli partially deprived the CCR, and deletion of the methylglyoxal synthase gene favored regulation of glucose utilization patterns
However, these methods cannot eliminate CCRs to a sufficient extent to produce cellulosic biofuels, therefore, new methods are still needed

Method used

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  • Method for preparing mutant escherichia coli capable of simultaneously utilizing glucose and xylose
  • Method for preparing mutant escherichia coli capable of simultaneously utilizing glucose and xylose
  • Method for preparing mutant escherichia coli capable of simultaneously utilizing glucose and xylose

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1: Preparation of mutant E. coli by promoter replacement and evolutionary adaptation

[0051] Mutant E. coli was prepared by replacing the inducible promoters of the araBAD operon, araFGH operon, araE gene, xylAB operon, and xylFGH operon of wild-type E. coli with the respective constitutive promoters in the following manner.

[0052] Inducible activation of araBAD operon, araFGH operon and araE gene substituting the respective constitutive promoters

[0053] The λ- Red recombination system, the inducible promoter (SEQ ID NO:1) of the araBAD on the chromosome of E.coli MG1655 is replaced by the constitutive CP25 promoter (SEQ ID NO:6) (see figure 1 ).

[0054] In short, such as literature [Datta, S., N. Costantino, et al. (2006). "A set of recombineering plasmamids for gram-negative bacteria." Gene 379 (0): 109-115; Datsenko KA et al ., Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6640-6645, 2000; and Cherepano...

Embodiment 2

[0079] Example 2: Preparation of mutant E. coli by promoter replacement and evolutionary adaptation in minimal medium of arabinose and xylose

[0080] In addition to using the bacterial strain obtained in the embodiment in the basic medium of arabinose and xylose (containing arabinose 2g / L, xylose 2g / L, disodium hydrogen phosphate 6.78g / L, dihydrogen phosphate Potassium 3.0g / L, sodium chloride 0.5g / L, ammonium chloride 1.0g / L, magnesium sulfate 2mM and calcium chloride 0.1mM M9 basal medium) cultured in 50 days to replace xylose basal medium, according to The same method as in Example 1 was used to prepare mutant E. coli strains.

[0081] Evolved in minimal media with arabinose and xylose after replacing the inducible promoters of the araBAD operon, araFGH operon, and araE and xylAB operon and xylFGH operon with constitutive promoters The adapted strain was named "AXcpAX50".

Embodiment 3

[0082] Embodiment 3: the preparation of the bacterial strain that knocks out ptsG

[0083] Preparation of wild-type E.coli with knockout of ptsG

[0084] To examine the molecular mechanism of the mutant strain (AXcpAX50) for CCR elimination, ptsG gene disruption was performed in the chromosome. As previously reported (Nichols et al., Appl. Microbiol. Biotechnol., 2001, 56:120–125), it is known that EIIBC, which encodes the glucose transporter Glc Strains mutated on ptsG utilize both glucose and xylose. To elucidate that the molecular mechanism of the mutant E. coli strain is due to the new mutant factor, wild-type E. coli MG1655 with ptsG deletion was prepared. By replacing the ptsG gene coding sequence with the kanamycin resistance gene in the same manner as described for the promoter replacement in Example 1, and then finally deleting the kanamycin resistance in the same manner as in Example sex genes for ptsG disruption. Thus, E. coli MG1655 in which ptsG was knocke...

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Abstract

The present invention relates to a method for preparing a mutant E. coli strain, capable of simultaneously using glucose and xylose, by genetic engineering and evolutionary adaptation; the mutant E. coli prepared using the same; and a method for producing biofuels, biologically active ingredients, medicinal materials or base chemicals for the chemical industry using the mutant E. coli. Being capable of simultaneously using glucose and xylose, in contrast to wild-type E. coli, the mutant E. coli can be effectively applied to the enzymatic saccharification process of producing biofuels from a biomass.

Description

technical field [0001] The present invention relates to a method for preparing mutant E. coli capable of utilizing both glucose and xylose. More specifically, the present invention relates to a method for preparing a mutant E.coli strain capable of simultaneously utilizing glucose and xylose through genetic engineering and evolutionary adaptation; a mutant E.coli prepared using the method; and using the mutant E.coli to produce biological Methods for fuels, bioactive ingredients, pharmaceutical materials, or chemicals for chemical use. Background of the invention [0002] With the depletion of fossil fuels, there has been intense global focus on alternative energy sources. Fuel ethanol, one of the alternative energy sources, can be obtained from cellulosic biomass. Every year, a large amount of cellulose is produced due to cellulose being fixed by photosynthesis. Additionally, cellulose is renewable. Cellulose is functionally and economically very advantageous when its r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12R1/19
CPCC12P7/18C12N15/70C12N1/22C12N15/01C12N15/52C12P7/10C12P2203/00Y02E50/10C12N1/20
Inventor 李成国金久熙丁圣勋金奭玟崔倍荣
Owner UNIST (ULSAN NAT INST OF SCI & TECH)
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