Method for preparing mutant
View Patent ↗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.
1. A method for preparing a mutant E. coli capable of simultaneously utilizing glucose and xylose from a wild-type E. coli , comprising:
(1) replacing inducible promoters of araBAD operon, araFGH operon, araE gene, xylAB operon, and xylFGH operon of the wild-type E. coli with respective constitutive promoters; and
(2) growing the promoter-replaced E. coli in a xylose minimal medium or an arabinose and xylose minimal medium for 10 days or longer.
2. The method of claim 1 , wherein the inducible promoters of araBAD operon, araFGH operon, araE gene, xylAB operon, and xylFGH operon have nucleotide sequences of SEQ ID NOS: 1 to 5, respectively.
3. The method of claim 1 , wherein the constitutive promoters have the nucleotide sequence of SEQ ID NO: 6 or 7.
4. The method of claim 1 , wherein the xylose minimal medium is an M9-minimal medium containing xylose 4 g/L, disodium hydrogen phosphate 6.78 g/L, potassium phosphate monobasic 3.0 g/L, sodium chloride 0.5 g/L, ammonium chloride 1.0 g/L, magnesium sulfate 2 mM, and calcium chloride 0.1 mM.
5. The method of claim 1 , wherein the arabinose and xylose minimal medium is an M9-minimal medium containing arabinose 2 g/L, xylose 2 g/L, disodium hydrogen phosphate 6.78 g/L, potassium phosphate monobasic 3.0 g/L, sodium chloride 0.5 g/L, ammonium chloride 1.0 g/L, magnesium sulfate 2 mM, and calcium chloride 0.1 mM.
6. A mutant E. coli , capable of simultaneously utilizing glucose and xylose, prepared by replacing inducible promoters of araBAD operon, araFGH operon, araE gene, xylAB operon, and xylFGH operon of wild-type E. coli with respective constitutive promoters, and growing the promoter-replaced E. coli in a xylose minimal medium or an arabinose and xylose minimal medium for 10 days or longer.
7. The mutant E. coli of claim 6 , wherein the inducible promoters of araBAD operon, araFGH operon, araE gene, xylAB operon and xylFGH operon have nucleotide sequences of SEQ ID NOS: 1 to 5, respectively.
8. The mutant E. coli of claim 6 , wherein the constitutive promoters have the nucleotide sequence of SEQ ID NO: 6 or 7.
9. The mutant E. coli of claim 6 , which comprises mutations represented by the nucleotide sequences of SEQ ID NOS: 55, 56, 58, and 60.
10. The mutant E. coli of claim 6 , which comprises mutations represented by the amino acid sequences of SEQ ID NOS: 51 and 53.
11. The mutant E. coli of claim 6 , which comprises mutations represented by the nucleotide sequences of SEQ ID NOS: 50, 52, 54, and 55.
12. The mutant E. coli of claim 6 , which comprises mutations represented by the amino acid sequences of SEQ ID NOS: 57 and 59.
13. A method for producing a biofuel, a biologically active ingredient, a medicinal material, or a chemical substance for the chemical industry from a biomass by using a mutant E. coli capable of simultaneously utilizing glucose and xylose, said mutant E. coli being prepared by replacing inducible promoters of araBAD operon, araFGH operon, araE gene, xylAB operon, and xylFGH operon of a wild-type E. coli with respective constitutive promoters, and growing the promoter-replaced E. coli in a xylose minimal medium or an arabinose and xylose minimal medium for 10 days or longer.