초록
<P><I>Corynebacterium glutamicum</I> shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a <I>C. glutamicum</I> strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of <I>E. coli</I> ornithine decarboxylase gene <I>speC</I> was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene <I>snaA</I> and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting <I>C. glutamicum</I> strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, <I>i.e</I>., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g<SUP>−1</SUP>·h<SUP>−1</SUP> and a yield on glucose of 0.26 g·g<SUP>−1</SUP>.</P>