Recombinant Microorganisms Having Enhanced Propanol and Method for Preparing Propanol Using the Same

Construction of Microbial Strain Having High Ability to Produce L-threonine

[0060]1-1: Construction of pSacHR06

[0061]In order to disrupt feedback inhibition of thrA, a pSacHR06 vector was constructed for the purpose of using homologous recombination of sacB originated from Bacillus subtilis (Wohlleben et. al., J. Bacteriol., 174:5462, 1992) to substitute a specific base or bases of chromosomal DNA (see FIG. 2).

[0062]First, in order to substitute the ampicillin resistance gene of a pUC19 vector (New England Biolab, USA) with kanamycin resistance, a 1.5 kb fragment obtained by digesting the pUC19 vector with NdeI and AhdI was ligated with a 1.3 kb fragment obtained by digesting a pACYC177 vector (New England Biolabs, USA) with StuI, thus obtaining a pUC19KM vector.

[0063]Then, a 2.5 kb fragment obtained by digesting the pUC19KM vector with PvuII was ligated with a 400 bp fragment obtained by digesting a pBluescriptIIKS(+) vector with PvuII, thus obtaining a pUC19KKS vector. In order to ...

Construction of Strain Deficient in ilvIH Gene

[0099]From the strain constructed in Example 1, the ilvIH operon was disrupted by one-step inactivation (Warner et al., PNAS, 6:6640, 2000) using primers of SEQ ID NO: 51 and SEQ ID NO: 52.

ilvIHk / o F:(SEQ ID NO: 51)5′-atggagatgttgtctggagccgagatggtcgtccgatcgcttatcgatcaTAGGTGACACTATAGAACGCG-3′ilvIHk / o R::(SEQ ID NO: 52)5′-tcaacgcattattttatcgccgcgcgaaagtccgaccacaccagagcgagTAGTGGATCTGATGGGTACC-3′

Construction of Strain Deficient in ilvBN Gene

[0100]From the strain constructed in Example 2, the ilvBN operon was disrupted by one-step inactivation (Warner et al., PNAS, 6:6640, 2000) using primers of SEQ ID NO: 53 and SEQ ID NO: 54.

ilvBNk / o F:(SEQ ID NO: 53)5′-atggcaagttcgggcacaacatcgacgcgtaagcgctttaccggcgcagaTAGGTGACACTATAGAACGCG-3′ilvBNk / o R::(SEQ ID NO: 54)5′-tttactgaaaaaacaccgcgatcttgttaaacatcgtcggatcggactgatTAGTGGATCTGATGGGTACC-3′