Expression of the Escherichia coli pmi gene, encoding phosphomannose-isomerase in Zymomonas mobilis, leads to utilization of mannose as a novel growth substrate, which can be used as a selective marker

Wild-type Zymomonas mobilis can utilize only three substrates (sucrose, glucose, and fructose) as sole carbon sources, which are largely converted into ethanol and carbon dioxide. Here, we show that although D-mannose is not used as a growth substrate, it is taken up via the glucose uniport system (glucose facilitator protein) with a Vmax similar to that of glucose. Moreover, D-mannose was phosphorylated by a side activity of the resident fructokinase to mannose-6-phosphate. Fructokinase was purified to homogeneity from an frk-recombinant Z. mobilis strain showing a specific activity of 205 +/- 25 U of protein mg-1 with fructose (K(m), 0.75 +/- 0.06 mM) and 17 +/- 2 U mg-1 (relative activity, 8.5%) with mannose (K(m), 0.65 +/- 0.08 mM). However, no phosphomannoseisomerase activity could be detected for Z. mobilis, and this appeared to be the reason for the lack of growth on mannose. Therefore, we introduced the Escherichia coli gene pmi (manA) in Z. mobilis under the control of a lacIq-Ptac system on a broad-host-range plasmid (pZY507; Cmr). Subsequently, in pmi-recombinant cells of Z. mobilis, phosphomannoseisomerase was expressed in a range of from 3 U (without isopropyl-beta-D-thiogalactopyranoside [IPTG]) to 20 U mg-1 of protein in crude extracts (after IPTG induction). Recombinant cells of different Z. mobilis strains utilized mannose (4%) as the sole carbon source with a growth rate of 0.07 h-1, provided that they contained fructokinase activity. When the frk gene was additionally expressed from the same vector, fructokinase activities of as much as 9.7 U mg-1 and growth rates of as much as 0.25 h-1 were detected, compared with 0.34 h-1 on fructose for wild-type Z. mobilis. Selection for growth on mannose was used to monitor plasmid transfer of pZY507pmi from E. coli to Z. mobilis strains and could replace the previous selection for antibiotic resistance.

[1]  R. Sydiskis,et al.  Genetic and Biochemical Studies on Mannose-Negative Mutants That Are Deficient in Phosphomannose Isomerase in Escherichia coli K-12 , 1967, Journal of bacteriology.

[2]  E. Lin,et al.  Replacement of a Phosphoenolpyruvate-dependent Phosphotransferase by a Nicotinamide Adenine Dinucleotide-linked Dehydrogenase for the Utilization of Mannitol , 1967, Journal of bacteriology.

[3]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[4]  C. Asensio,et al.  Purification and properties of the mannokinase from Escherichia coli. , 1972, Archives of biochemistry and biophysics.

[5]  J. Beckwith,et al.  Genetic Characterization of Mutations Which Affect Catabolite-Sensitive Operons in Escherichia coli, Including Deletions of the Gene for Adenyl Cyclase , 1973, Journal of bacteriology.

[6]  W. Epstein,et al.  Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase , 1975, Journal of bacteriology.

[7]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[8]  B. Chassy,et al.  Partial purification and properties of a mannofructokinase from Streptococcus mutans SL-1 , 1980, Infection and immunity.

[9]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[10]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[11]  A. Pühler,et al.  A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria , 1983, Bio/Technology.

[12]  J. S. Miles,et al.  Nucleotide sequence and transcriptional start point of the phosphomannose isomerase gene (manA) of Escherichia coli. , 1984, Gene.

[13]  Antonio H. Romano,et al.  d-Glucose Transport System of Zymomonas mobilis , 1985, Applied and environmental microbiology.

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

[15]  K. Mullis,et al.  Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. , 1987, Methods in enzymology.

[16]  C. Eddy,et al.  Cosmid cloning of five Zymomonas trp genes by complementation of Escherichia coli and Pseudomonas putida trp mutants , 1988, Journal of bacteriology.

[17]  L. Viikari,et al.  CARBOHYDRATE METABOLISM IN ZYMOMONAS , 1988 .

[18]  H. Sahm,et al.  Metabolic shifts in Zymomonas mobilis in response to growth conditions , 1988 .

[19]  H Sahm,et al.  Expression of an L-alanine dehydrogenase gene in Zymomonas mobilis and excretion of L-alanine , 1991, Applied and environmental microbiology.

[20]  J. Lengeler,et al.  Molecular analysis of two fructokinases involved in sucrose metabolism of enteric bacteria , 1991, Molecular microbiology.

[21]  J. Liu,et al.  Cloning, sequencing, and expression of the Zymomonas mobilis fructokinase gene and structural comparison of the enzyme with other hexose kinases , 1992, Journal of bacteriology.

[22]  C. Drainas,et al.  Genetics and genetic engineering ofZymomonas mobilis , 1993, World journal of microbiology & biotechnology.

[23]  G. Sprenger.  Approaches to broaden the substrate and product range of the ethanologenic bacterium Zymomonas mobilis by genetic engineering , 1993 .

[24]  C. Sander,et al.  Convergent evolution of similar enzymatic function on different protein folds: The hexokinase, ribokinase, and galactokinase families of sugar kinases , 1993, Protein science : a publication of the Protein Society.

[25]  M. Walsh,et al.  Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose , 1994, Journal of bacteriology.

[26]  L. Ingram,et al.  Reconstruction of glucose uptake and phosphorylation in a glucose-negative mutant of Escherichia coli by using Zymomonas mobilis genes encoding the glucose facilitator protein and glucokinase , 1994, Journal of bacteriology.

[27]  L. Ingram,et al.  Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport , 1995, Molecular microbiology.

[28]  H. Sahm,et al.  Functional expression of the glucose transporter of Zymomonas mobilis leads to restoration of glucose and fructose uptake in Escherichia coli mutants and provides evidence for its facilitator action , 1995, Journal of bacteriology.