Cloning, sequence analysis, and expression of the structural gene encoding glucose-fructose oxidoreductase from Zymomonas mobilis

Abstract

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...

The Substitution of a Single Amino Acid Residue (Ser-116 → Asp) Alters NADP-containing Glucose-Fructose Oxidoreductase ofZymomonas mobilis into a Glucose Dehydrogenase with Dual Coenzyme Specificity

“…During protein purification, the cofactor is not removed from the enzyme (1,6). The published DNA sequence and the amino acid sequence derived thereof reveal that GFOR has little similarity to other enzymes (5). This, together with the mode of tight cofactor binding, suggests a novel dinucleotide binding mode.…”

“…The apparent physiological role of GFOR is the production of sorbitol from the two sugar moieties of sucrose, a natural carbon source of the bacterium which dwells in sugar-rich habitats (4). The enzyme is synthesized as a precursor (pre-GFOR) with an NH 2 -terminal signal peptide of 52 amino acid residues (5) and is exported to the periplasm, at least partially, via the Sec pathway (6). The mature enzyme is located in the periplasm (7,8).…”

Crystallization and preliminary x‐ray analysis of glucose‐fructose oxidoreductase from zymomonas mobilis

“…Because the preform was enzymatically fully active and displayed fluorescence at 365 nm after cathodic gel electrophoresis (Loos et al, 1993), binding of the cofactor before protein secretion is probable. In the deduced amino acid sequence, however, no significant sequence homology to other structurally known proteins could be detected (Kanagasundaram & Scopes, 1992). This fact, and the unusual type of cofactor binding (in a tight, but not covalent manner), suggests a novel dinucleotide binding mode, thus prompting us to crystallize the enzyme with the goal of an eventual X-ray analysis.…”

“…The g fo gene encoding GFOR was isolated and its DNA sequence was determined (Kanagasundaram & Scopes, 1992). From the deduced amino acid sequence, an unusually long signal peptide of 52 amino acids was predicted.…”

“…From the deduced amino acid sequence, an unusually long signal peptide of 52 amino acids was predicted. The enzyme ap-parently undergoes processing from a preform to the mature form during export into the periplasmic space of Z. mobilis (Kanagasundaram & Scopes, 1992; Loos et al, 1993). The preform was purified from a GFOR-overproducing strain that had accumulated the preform.…”