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A comparative genomic analysis was performed to study the genetic variations between the l-serine-producing strain Corynebacterium glutamicum SYPS-062 and the mutant strain SYPS-062-33a, which was derived from SYPS-062 by random mutagenesis with enhanced l-serine production. Some variant genes between the two strains were reversely mutated or deleted in the genome of SYPS-062-33a to verify the influences of the gene mutations introduced by random mutagenesis. It was found that a His-594 → Tyr mutation in aceE was responsible for the more accumulation of by-products, such as l-alanine and l-valine, in SYPS-062-33a. Furthermore, the influence of this point mutation on the l-serine production was investigated, and the results suggested that this point mutation led to a better growth profile and a higher l-serine production in the high-yield strain 33a∆SSAAI, which was derived from SYPS-062-33a by metabolic engineering with the highest l-serine production to date. [ABSTRACT FROM AUTHOR]