Classical whole-cell mutagenesis based on iterative random mutation and analogue specific screening has achieved great success in development of many industrial fermentation strains. During each iterative random mutation and selection, a mutant of improved or desired property is identified and the single best performer is taken forwards. However, in proportion to recursive mutagenesis, it has the serious disadvantage of accumulation of uncharacterized secondary mutations that are detrimental to their performance. For reconstruction of optimal amino acid over-producing strain which has desired property without detrimental mutations, we developed a simple and generally applicable method for the isolation of threonine and lysine producing mutant harboring positive relevant gene fragment on the indicator solid plate screening system without analogue oriented selection. Based on the metabolic pathway analysis, we reconstructed the metabolic map for L-threonine and L-lysine biosynthesis, and selected the target genes which might be crucial for construction of an L-threonine and L-lysine overproducing strain, respectively. Of the tested target genes, the four genes, aspC, ppc, lysC, and thrABC, exhibited the positive effect on the production of amino acid. In controlled flask fermentations, introduction of mutated ppc, lysC, and thrABC resulted in an increase of L-threonien and L-lysine, respectively. It is anticipated that the approach developed here can be used for development of an amino acid-overproducing strain.