Control of the Lysine Biosynthesis Sequence in Corynebacterium glutamicum as Analyzed by Overexpression of the Individual Corresponding Genes

Abstract

The gene cluster that codes for feedback-resistant aspartate kinase ( lysC α and lysC β) and aspartate semialdehyde dehydrogenase ( asd ) was cloned from a mutant strain of Corynebacterium glutamicum. Its functional analysis by subcloning, enzyme assays, and type of aspartate kinase regulation enabled the isolation of a fragment for separate expression of the feedback-resistant kinase without aspartate semialdehyde dehydrogenase expression. This was used together with other clones constructed (J. Cremer, L. Eggeling, and H. Sahm, Mol. Gen. Genet. 220:478-480, 1990) to overexpress individually each of the six genes that convert aspartate to lysine. Analysis of lysine formation revealed that overexpression of the feedback-resistant kinase alone suffices to achieve lysine formation (38 mM). Also, sole overexpression of wild-type dihydrodipicolinate synthase resulted in lysine formation but in a lower amount (11 mM). The other four enzymes had no effect on lysine secretion. With a plasmid overexpressing both relevant enzymes together, a further increase in lysine yield was obtained. This shows that of the six enzymes that convert aspartate to lysine the kinase and the synthase are responsible for flow control in the wild-type background and can be useful for construction of lysine-producing strains.