Microorganism producing l-methionine precursor and the method of producing l-methionine precursor using the microorganism
A technology of methionine and microorganisms, which is applied in the field of microorganisms for preparing L-methionine precursor O-succinylhomoserine, and can solve the problems of small methionine yield and the like
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Embodiment 1
[0067] Embodiment 1: Construction of methionine precursor production strain
[0068] Deletion of the metB gene
[0069] To delete the metB gene encoding cystathionine synthase in E. coli, a FRT-one-step PCR deletion was performed (PNAS (2000) vol 97: p6640-6645). Primers of SEQ ID NO: 1 and NO: 2 were used for PCR using pKD3 vector (PNAS (2000) vol 97: p6640-6645) as a template to obtain the construction of the deletion gene cassette. PCR was performed as follows: 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute.
[0070]The PCR product was subjected to 1.0% agarose gel electrophoresis, followed by purification of the DNA obtained from the 1.2 kbp band. The recovered DNA fragment was electroporated into Escherichia coli (K12) W3110 transformed with pKD46 vector (PNAS (2000) vol 97: p6640-6645). Before electroporation, W3110 transformed with pKD46 was grown to OD at 30°C in LB medium containing 100 μg / ...
Embodiment 2
[0107] Example 2: Fermentation for the production of L-methionine precursor
[0108] Glass bottle culture experiment
[0109] In order to examine the productivity of the methionine precursor of each strain constructed in Example 1, Erlenmeyer flask culture was used. Each strain was cultured overnight at 31°C in LB plate medium. Single clones were inoculated into 3 ml of LB medium, followed by culturing at 31°C for 5 hours. The culture solution was diluted 200 times into a 250ml Erlenmeyer flask containing 25ml of methionine precursor production medium, and then cultured at 31°C and 200rpm for 64 hours. HPLC was performed to compare the methionine precursor production capacity (Table 2 and Table 3). As a result, the methionine-producing ability was significantly increased in the methionine precursor-producing strain prepared from the L-threonine-producing strain having no methionine requirement.
[0110] Table 1
[0111] Glass bottle media components for methionine pr...
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