The gene encoding glucose-fructose oxidoreductase (gfo) The bacterium Zymomonas mobilis ferments sugars to ethanol and CO2 as major end products; however, under certain conditions, substantial amounts of sorbitol are also found in the culture supernatant (3,15,37). It has been shown that sorbitol formation requires the presence of both glucose and fructose and is maximum when the organism is grown on a mixture of these monosaccharides or on sucrose. The sorbitol originates from fructose, which is reduced simultaneously with the oxidation of glucose (21). The gluconolactone product is hydrolyzed and phosphorylated to 6-phosphogluconate (40), which is then further metabolized to ethanol by the main Entner-Doudoroff pathway enzymes in this organism. The sorbitol is unable to be further metabolized and accumulates to levels as high as 5 to 6% in the culture. Although in the context of industrial alcohol production, this was regarded as wasted substrate, lowering the ethanol yield, the sorbitol has subsequently been considered as a useful end product in itself (30).The enzyme responsible for sorbitol formation has been identified and isolated (41) and characterized kinetically extensively (17). It belongs to a enzyme category in which an oxidoreduction is carried out without any external cofactor: the enzyme contains nondissociable NADP which transfers reducing equivalents between the glucose and fructose. The enzyme is a tetramer with subunits of approximately 40 kDa. This enzyme, glucose-fructose oxidoreductase (GFOR), is present at levels of up to 1% of the soluble protein in Z. mobilis cells, although it has been noted that the level of expression is less in those conditions, i.e., the presence of both glucose and fructose, needed for it to be active. The enzyme has unusually low affinities for its substrates, requiring fructose concentrations of up to 1 M for maximum activity and glucose concentrations of 50 to 100 mM. Nevertheless, high levels of glucose and fructose accumulate inside Z. mobilis, because both sugars are thought to enter the cells by facilitated diffusion (13) glucose concentrations inhibit fructokinase (3,14), enabling fructose to accumulate to higher levels than glucose, which is metabolized first.Many of the genes coding for the enzymes of glycolysis in Z. mobilis have been cloned and sequenced (2,5,(9)(10)(11)(12)24). Features such as codon usage, consensus promoter sequences, and operon occurrence have been noted. We present here the isolation, sequencing, and structure of the gene encoding GFOR, with some observations on its transcription and translation by both Z. mobilis and Escherichia coli cells.
MATERILLS AND METHODSBacterial strains and plasmids. Z. mobilis ZM6 (ATCC 29191) and ZM6100, a Met-mutant of ZM6, were obtained from P. L. Rogers and S. Delaney, respectively, University of New South Wales, Sydney, Australia. ZM6 was the strain used in most experiments; strain ZM6100 was used to study the overexpression of GFOR in Z. mobili&. The growth conditions for Z. mobilis were as des...