STABLE EXPRESSION OF HOM-1-THRB IN CORYNEBACTERIUM-GLUTAMICUM AND ITSEFFECT ON THE CARBON FLUX TO THREONINE AND RELATED AMINO-ACIDS

The hom-1-thrB operon encodes homoserine dehydrogenase resistant to fe edback inhibition by L-threonine and homoserine kinase. Stable express ion of this operon has not yet been attained in different Corynebacter ium glutamicum strains. We studied the use of chromosomal integration and of a low-copy-number vector for moderate expression of the hom-1-t hrB operon to enable an analysis of the physiological consequences of its expression in C. glutamicum. Strains tarrying one, two, or three c opies of hom-1-thrB were obtained. They showed proportionally increase d enzyme activity of feedback-resistant homoserine dehydrogenase and o f homoserine kinase. This phenotype was stably maintained in all recom binants for more than 70 generations. In a lysine-producing C. glutami cum strain which does not produce any threonine, expression of one cop y of hom-1-thrB resulted in the secretion of 39 mM threonine. Addition al copies resulted in a higher, although not proportional, accumulatio n of threonine (up to 69 mM). This indicates further limitations of th reonine production. As the copy number of hom-1-thrB increased, increa sing amounts of homoserine (up to 23 mM) and isoleucine (up to 34 mM) were secreted. Determination of the cytosolic concentration of the res pective amino acids revealed an increase of intracellular threonine fr om 9 to 100 mM and of intracellular homoserine from 4 to 74 mM as the copy number of hom-1-thrB increased. These results suggest that threon ine production with C. glutamicum is limited by the efflux system for this amino acid. Furthermore, the results show the successful use of m oderate and stable hom-1-thrB expression for directing the carbon flux from aspartate to threonine.