Mechanism of alanine excretion in recombinant strains of Zymomonas mobilis

Abstract

A thiamine-auxotrophic strain of Zymomonas mobilis (CP4thi/pZY73), in which the alaD gene of Bacillus sphaericus coding for the alanine dehydrogenase was expressed, synthesizes and excretes alanine at high rates after thiamine starvation and in the presence of high external ammonium 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 obtained. The membrane integrity and the energetic properties of the cells remained intact and were comparable to growing wild-type cells. Unspecific leakage of solutes was not observed. We did not find any indication of a carrier-mediated excretion of alanine, since typical properties of this type of mechanism, i.e., saturation at increasing internal substrate 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. Consequently, alanine excretion in recombinant Z. mobilis cells is interpreted as mediated by simple diffusion through the intact cytoplasmic membrane 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 efflux was also measured in Corynebacterium glutamicum cells and the values obtained were significantly lower than those determined in Z. mobilis. The consequences of this finding are discussed.