MECHANISM OF ALANINE EXCRETION IN RECOMBINANT STRAINS OF ZYMOMONAS-MOBILIS

A thiamine-auxotrophic strain of Zymomonas mobilis (CP4thi/pZY73), in which the alaD gene of Bacillus sphaericus coding for the alanine dehy drogenase was expressed, synthesizes and excretes alanine at high rate s after thiamine starvation and in the presence of high external ammon ium concentrations. The mechanism of alanine excretion was studied in this recombinant Zymomonas mobilis strain. Under production conditions the internal alanine concentration reached values of up to 280 mM and excretion rates of up to 140 nmol min(-1) mg dry mass(-1) were obtain ed. The membrane integrity and the energetic properties of the cells r emained intact and were comparable to growing wild-type cells. Unspeci fic leakage of solutes was not observed. We did not find any indicatio n of a carrier-mediated excretion of alanine, since typical properties of this type of mechanism, i.e., saturation at increasing internal su bstrate concentration, substrate specificity and functional inhibition were absent. Furthermore, a counterflow maximum, which would indicate the involvement of a carrier protein, was not observed either. Conseq uently, alanine excretion in recombinant Z. mobilis cells is interpret ed as mediated by simple diffusion through the intact cytoplasmic memb rane at high rates (diffusion constant 10(-8) l s(-1) mg dry mass(-1) or 0.28 min(-1)). For comparison, the diffusion constant for alanine e fflux was also measured in Corynebacterium glutamicum cells and the va lues obtained were significantly lower than those determined in Z. mob ilis. The consequences of this finding are discussed.