CAMP receptor protein variants and methods for producing l-amino acids using the same
A receptor protein and amino acid technology, applied in the field of cAMP receptor protein variants, can solve the problem that the mechanism has not been clearly revealed, and achieve the effects of reduced production cost and convenient production
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Embodiment 1
[0064] Example 1. Preparation of recombinant vector pCC1BAC-crp
[0065] 1-1. Preparation of crp gene fragment
[0066] In order to obtain a DNA fragment of about 0.96kb including the SEQ ID NO: 5 of the crp gene and the expression regulatory region, the Genomic-tip system of Qiagen (company) was used to extract the genomic DNA (gDNA) of wild-type Escherichia coli W3110, using gDNA as a template PCR (polymerase chain reaction) was performed with PCR HL Premix Kit (manufactured by BIONEER, hereinafter the same applies). PCR for amplification of the crp gene fragment was performed using primers of SEQ ID NOS: 6 and 7 for 27 cycles consisting of denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds and extension at 72°C for 2 minutes.
[0067] The PCR product was digested with EcoR I, electrophoresed on 0.8% agarose gel and eluted to obtain a 0.96 Kb DNA fragment (hereinafter referred to as "crp fragment").
[0068] [Table 1]
[0069] SEQ ID NO. Pr...
Embodiment 2
[0072] Example 2. Preparation of recombinant vector pCC1BAC-crp variant library
[0073] 2-1. Preparation of mutant crp fragments by error-prone PCR
[0074] PCR was performed using genomic DNA of wild-type E. coli W3110 as a template and clonetech's Diverse PCR Random Mutagenesis Kit (Cat. No.: K1830-1, Table III, Mutagenesis Reaction 4). In detail, PCR was performed using the primers of SEQ ID NOS: 6 and 7 used in Example 1-1 for 27 cycles consisting of denaturation at 94°C for 30 seconds and extension at 68°C for 1 minute.
[0075]The PCR product was digested with EcoR I, electrophoresed and eluted on a 0.8% agarose gel to obtain a mutated crp fragment of 0.96 Kb (hereinafter referred to as "crp m Fragment").
[0076] 2-2. Preparation of recombinant vector pCC1BAC-crp variant library
[0077] Vector pCC1BAC was treated with restriction enzyme EcoR I, followed by alkaline phosphatase (NEB). The prepared carrier and the crp obtained in Example 2-1 m The fragments were ...
Embodiment 3
[0078] Example 3. Introduction of a library of crp variants into threonine-producing strains and selection of growth-improved strains
[0079] 3-1. Add pCC1BAC-crp m Library introduction of threonine-producing strains
[0080] The pCC1BAC-crp obtained in Example 2 was electroporated m The library was transformed into electrocompetent cells of KCCM10541, a threonine-producing microorganism. Escherichia coli KCCM10541 (Korean Patent No. 10-0576342) used in this example was prepared by inactivating the galR gene in L-threonine-producing Escherichia coli KFCC10718 (Korean Patent No. 10-0058286).
[0081] Introduced as pCC1BAC-crp m As a control group of microorganisms of the library, pCC1BAC-crp was transformed into KCCM10541 in the same manner as above to prepare KCCM10541 / pCC1BAC-crp (WT).
[0082] 3-2. Comparison of growth rate of recombinant microorganisms
[0083] M9 basal medium containing 1% glucose and 0.2 g / L yeast extract was dispensed into deep-well microplates, ...
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