Expression of Zymomonas mobilis adhB (encoding alcohol dehydrogenase II) and adhB-lacZ operon fusions in recombinant Z. mobilis

The Zymomonas mobilis alcohol dehydrogenase II gene (adhB) was overexpressed 7- to 14-fold on a recombinant plasmid, accompanied by a small decrease in growth rate. A fragment containing the truncated gene with promoter reduced expression from the chromosomal gene as measured immunologically and enzymatically, consistent with the presence of a trans-active regulatory factor and positive regulatory control. Both the complete gene and the promoter fragment increased pyruvate decarboxylase and glucokinase activities, with no effect on alcohol dehydrogenase I or eight glycolytic enzymes. Tandem promoters from adhB expressed beta-galactosidase at higher levels than did either promoter alone in operon fusions. Addition of 50 microM zinc sulfate in minimal medium reduced the expression of adhB and of the operon fusions. Abundant but inactive alcohol dehydrogenase II was produced in iron-limited cells. This inactive enzyme did not form intracellular aggregates, and no morphological changes were apparent by transmission electron microscopy.

[1]  L. Ingram,et al.  Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis , 1987, Journal of bacteriology.

[2]  L. Enquist,et al.  Experiments With Gene Fusions , 1984 .

[3]  R. Scopes.  An iron‐activated alcohol dehydrogenase , 1983, FEBS letters.

[4]  R. Simons,et al.  Improved single and multicopy lac-based cloning vectors for protein and operon fusions. , 1987, Gene.

[5]  L. Ingram,et al.  Glycolytic flux in Zymomonas mobilis: enzyme and metabolite levels during batch fermentation , 1987, Journal of bacteriology.

[6]  L. Ingram,et al.  Modulation of alcohol dehydrogenase isoenzyme levels in Zymomonas mobilis by iron and zinc , 1989, Journal of bacteriology.

[7]  F. Richaud,et al.  New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. , 1983, Gene.

[8]  E. Lin,et al.  Ferrous-activated nicotinamide adenine dinucleotide-linked dehydrogenase from a mutant of Escherichia coli capable of growth on 1, 2-propanediol. , 1969, Journal of bacteriology.

[9]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[10]  R. Scopes,et al.  Simultaneous purification and characterization of glucokinase, fructokinase and glucose-6-phosphate dehydrogenase from Zymomonas mobilis. , 1985, The Biochemical journal.

[11]  L. Ingram,et al.  Gene expression in Zymomonas mobilis: promoter structure and identification of membrane anchor sequences forming functional lacZ' fusion proteins , 1987, Journal of bacteriology.

[12]  M. Ciriacy.  Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae: I. Isolation and genetic analysis of adh mutants , 1975 .

[13]  R. Scopes,et al.  An iron-activated alcohol dehydrogenase: metal dissociation constants and magnetic and spectroscopic properties , 1988 .

[14]  L. Ingram,et al.  Isolation and Characterization of Xylan-Degrading Strains of Butyrivibrio fibrisolvens from a Napier Grass-Fed Anaerobic Digester , 1988, Applied and environmental microbiology.

[15]  R. Wettenhall,et al.  Pyruvate decarboxylase of Zymomonas mobilis: isolation, properties, and genetic expression in Escherichia coli , 1987, Journal of bacteriology.

[16]  C. Wills,et al.  Characterization of the two alcohol dehydrogenases of Zymomonas mobilis. , 1981, Archives of biochemistry and biophysics.

[17]  J. Kelly,et al.  The two alcohol dehydrogenases of Zymomonas mobilis. Purification by differential dye ligand chromatography, molecular characterisation and physiological roles. , 1986, European journal of biochemistry.