Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism

Abstract

The Zymomonas mobilis genes that encode glucose-6-phosphate dehydrogenase (zwj), 6-phosphogluconate dehydratase (edd), and glucokinase (gik) were cloned independently by genetic complementation of specific defects in Escherichia coli metabolism. The identity of these cloned genes was confirmed by various biochemical means. Nucleotide sequence analysis established that these three genes are clustered on the genome and revealed an additional open reading frame in this region that has significant amino acid identity to the E. coli xylose-proton symporter and the human glucose transporter. On the basis of this evidence and structural analysis of the deduced primary amino acid sequence, this gene is believed to encode the Z. mobilis glucose-facilitated diffusion protein, gif. The four genes in the 6-kb cluster are organized in the order gif, zwf, edd, gik. The gif and zwf genes are separated by 146 bp. The zwf and edd genes overlap by 8 bp, and their expression may be translationally coupled. The edd and gik genes are separated by 203 bp. The gik gene is followed by tandem transcriptional terminators. The four genes appear to be organized in an operon. Such an arrangement of the genes that govern glucose uptake and the first three steps of the Entner-Doudoroff glycolytic pathway provides the organism with a mechanism for carefully regulating the levels of the enzymes that control carbon flux into the pathway.Zymomonas mobilis is a gram-negative bacterium which, during evolution, has become highly specialized for growth in plant saps with a high sugar content (37,51,53). This obligately fermentative organism possesses remarkably simple carbon and energy metabolism. Z. mobilis is only able to utilize glucose, fructose, and sucrose, which are converted to the sole fermentation products ethanol and carbon dioxide. Yet, in terms of biosynthetic capabilities, this organism has only two growth factor requirements. Z. mobilis uses the Entner-Doudoroff pathway exclusively for conversion of carbohydrates to pyruvate and the decarboxyclastic mechanism for ethanol production, with the key enzyme pyruvate decarboxylase. The enzymes responsible for this fermentation compose as much as 50% of the total soluble protein. Z. mobilis is totally dependent on substrate-level phosphorylation for energy production and, due to its use of the Entner-Doudoroff pathway, obtains only a single mole of ATP per mole of glucose fermented. For this organism, rapid carbon flux is necessitated by inefficient energy production and is facilitated by high levels of the pathway enzymes.Despite enormous carbon flux, Z. mobilis must keep the levels of toxic metabolic intermediates low while providing sufficient pools of precursor metabolites for biosynthetic pathways. The physiology and biochemistry of this organism are dictated by these constraints on metabolism. The glycolytic enzymes appear to operate near their maximal capacity, and there is no substantial allosteric control of physiologically irreversible enzymes (53). It can be concluded that carbo...

Cloning, characterization, and nucleotide sequence analysis of a Zymomonas mobilis phosphoglucose isomerase gene that is subject to carbon source-dependent regulation

“…The remaining glucose' clone expressed high glucose-6-phosphate dehydrogenase activity. A study of the Z. mobilis zwfgene was published elsewhere (2).…”

“…Genomic DNA from Z. mobilis CP4 was isolated as previously described (3). A library of Z. mobilis genomic DNA containing 4-to 6-kb fragments was generated by partial digestion of genomic DNA with Sau3A and subsequent ligation into the BamHI site of pUC18 as described previously (2). Amplification in E. coli DHSa produced a 10,000-member library with a 75% insertion frequency.…”

Site‐directed mutagenesis to facilitate X‐ray structural studies of leuconostoc mesenteroides glucose 6‐phosphate dehydrogenase

“…Secondary structure prediction from the amino acid sequence (Lee et al, 1991) was made using the Leeds package of algorithms (Eliopoulos et al, 1982). Regions of sequence identity and homology were identified by comparisons of the amino acid sequence of L. mesenteroides G6PD with those of G6PDs from human (Persico et al, 1986), rat (Ho et al, 1988), Drosophila (Fouts et al, 1988), Saccharomyces cerevisiae (Nogae & Johnston, 1990;Thomas et al, 1991), E. coli (Rowley & Wolf, 1991), and Zymomonas mobilis (Barnell et al, 1990), using a multiple sequence alignment and prediction protocol (Russell et al, 1992).…”

Combined expression of glucokinase and invertase in potato tubers leads to a dramatic reduction in starch accumulation and a stimulation of glycolysis

“…The gene encoding a glucokinase from Zymomonas mobilis (Barnell et al, 1990) was cloned in the sense orientation into the plant transformation vector pBinAR Hyg between the patatin B33 promoter and the ocs terminator. Potato leaves of U-IN2-30 (Sonnewald et al, 1997) were transformed with this construct using an Agrobacteriummediated protocol (Rocha-Sosa et al, 1989), and 80 transgenic lines were transferred to the greenhouse for screening.…”