Genetically modified microorganism for production of aspartic acid and downstream metabolites from aspartic acid as target substance, and method for producing target substance using same

A method for producing a target substance, the method comprising:

• (p) culturing a genetically modified microorganism in a reaction medium (X) comprising a sugar serving as a substrate, to produce a target substance;
• and
• (q) recovering the target substance from the reaction medium (X), wherein
• the genetically modified microorganism belongs to the genus Escherichia or coryneform bacteria, and is genetically modified:
• (I) to have an inactivated or disrupted chromosomal gene, the gene encoding a fumarate reductase catalyzing a conversion reaction from fumarate to succinate in a counterclockwise TCA cycle under reducing conditions or anaerobic conditions;
• (II) to have an inactivated or disrupted chromosomal lactate dehydrogenase gene;
• and
• (III) to have an operable gene that encodes a mutant-type phosphoenolpyruvate carboxylase showing increased resistance to feedback inhibition by aspartic acid as compared to a wild-type phosphoenolpyruvate carboxylase,
• in step (p), the genetically modified microorganism is cultured so as to produce aspartic acid or a downstream metabolite from aspartic acid as the target substance via the counterclockwise TCA cycle under reducing conditions or anaerobic conditions, and
• the mutant-type phosphoenolpyruvate carboxylase comprises SEQ ID NO:
• 2, with mutations including at least one amino acid substitution selected from the group consisting of:
• (a) an amino acid substitution at the position 299 with asparagine, (b) an amino acid substitution at the position 653 with glycine, (c) an amino acid substitution at the position 813 with serine or glycine, (d) an amino acid substitution at the position 869 with glycine, (e) an amino acid substitution at the position 873 with glycine or serine, and (f) an amino acid substitution at the position 917 with glycine, and
• wherein the genetically modified microorganism produces more aspartic acid or a downstream metabolite from aspartic acid as the target substance as compared to a wild-type unmodified microorganism, wherein the wild-type unmodified microorganism is a wild type of the genetically modified microorganism.

A mutant-type phosphoenolpyruvate carboxylase comprising an amino acid mutation with respect to an amino acid sequence of a wild-type phosphoenolpyruvate carboxylase of a microorganism belonging to coryneform bacteria, the amino acid mutation being capable of reducing feedback inhibition by aspartic acid in the wild-type phosphoenolpyruvate carboxylase activity,

• wherein the amino acid mutation at least comprises:
• based on the amino acid sequence set forth in SEQ ID NO:
• 2,
• (g) an amino acid substitution of an amino acid corresponding to the 299th aspartic acid with asparagine;
• (i) an amino acid substitution of an amino acid corresponding to the 813th lysine with a predetermined amino acid, wherein the substituted amino acid is not lysine;
• or
• (l) an amino acid substitution of an amino acid corresponding to the 917th asparagine with a predetermined amino acid, wherein the substituted amino acid is not asparagine, wherein
• the mutant-type phosphoenolpyruvate carboxylase has higher resistance to feedback inhibition by aspartic acid than that of a protein having only the amino acid substitution defined in (g), (i), or (1) above with respect to the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase.