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Site‐directed mutagenesis of the aspartokinase gene lys C and its characterization in Brevibacterium flavum
Author(s) -
Lu J.H.,
Liao C.C.
Publication year - 1997
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1046/j.1472-765x.1997.00383.x
Subject(s) - brevibacterium , mutagenesis , biology , gene , site directed mutagenesis , genetics , microbiology and biotechnology , mutation , bacteria , mutant , microorganism
Using overlap extension polymerase chain reaction (PCR), five transformants of Escherichia coli containing site‐directed mutagenized lys Cβ gene were generated and analysed. Exchange of C to A and C to T at nucleotide 1118 of the mutated lys Cβ gene causes a substitution of serine 301 in the wild‐type enzyme for tyrosine 301 and phenylalanine 301 in the mutant enzymes, respectively. Enzyme assays showed that Brevibacterium flavum cells harbouring pSUMN18 with mutated lys Cβ genes exhibited 16–20 fold lower specific activities of aspartokinase as compared to that of host containing wild‐type lys C gene. The mutation introduced into lys Cβ of B. flavum CCRC18271 resulted in partial feedback‐resistant aspartokinase activity.