Method for L-threonine production
View Patent ↗A method for producing L-threonine using a microorganism is provided. In the method, additional one or more copies of each of the phosphoenolpyruvate carboxylase (ppc) gene and the threonine operon are integrated into a particular site of the chromosomal DNA of a microorganism, while its inherent ppc gene and threonine operon remain. Accordingly, two or more ppc genes and threonine operons are included in the chromosomal DNA of the microorganism to thereby enhance the expression of the ppc gene encoding an enzyme to convert phosphoenolpyruvate to a threonine biosynthesis precursor, oxaloacetete, and the genes encoding enzymes involved in the synthetic pathway of threonine from oxaloacetate, including thrA (aspartokinasel-homoserine dehydrogenase), thrB (homoserine kinase), and thrC (threonine synthase), thereby markedly increasing L-threonine productivity.
1. A method of producing L-threonine, comprising:
constructing a recombinant chromosomal DNA integration system comprising one or more copies of each of a nucleic acid encoding phosphoenolpyruvate carboxylase which is obtained using PCR primers of SEQ ID NO: 1 and SEQ ID NO: 2 from Escherichia coli strain TF 4076 Accession No. KFCC 10718 and a threonine operon from a microorganism belonging to the Eseherichia or Corynebacterium genus resistant to threonine analogs, lysine analogs, isoleucine analogs, or methionine analogs;
introducing the recombinant chromosomal DNA integration system into a microorganism belonging to the Escherichia genus to integrate the one or more additional copies into the lacZ gene site of the Eseherichia chromosomal DNA while the microorganism's inherent phosphoenolpyruvate carboxylase gene and threonine operon remain;
culturing the transformed Escherichia with substrates to produce L-threonine; and
isolating the produced L-threonine.
2. The method of claim 1 , wherein the nucleic acid encoding phosphoenolpyruvate carboxylase of the recombinant chromosomal DNA integration vector is obtained from the chromosome of a threonine producing E.coli strain, TF4076, of Accession No. KFCC 10718 by polymerase chain reaction (PCR), and the threonine operon of the recombinant chromosomal DNA integration vector is cloned from said E.coli strain TF4076, wherein the obtained nucleic acid encoding phosphoenolpyruvate carboxylase and the cloned threonine operon are incorporated into the chromosome of the host strain E.coli TF4076.
3. The method of claim 1 , wherein the microorganism is constructed with recombinant plasmid pGmTN-PPC of FIG. 2 .