Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon.
We have constructed a shuttle plasmid for Bacillus megaterium and Escherichia coli that contains the promoter and repressor gene of the B. megaterium-borne operon for xylose utilization. A polylinker downstream of the promoter allows versatile cloning of genes under its transcriptional control. We have placed gdhA (encoding glucose dehydrogenase) from B. megaterium, lacZ (encoding beta-galactosidase) from E. coli, mro (encoding mutarotase) from Acinetobacter calcoaceticus, and human puk (encoding single-chain urokinase-like plasminogen activator, rscuPA) under xylose control in this vector. All four genes were between 130-fold and 350-fold inducible by 0.5% xylose in the growth medium in B. megaterium. Enzymatically active glucose dehydrogenase and mutarotase accumulated to 20% and 30% of the total soluble protein, respectively. beta-Galactosidase and rscuPA were also expressed at a high level. A gel analysis of the products demonstrated their proteolytic stability in the cytoplasm, even up to 5 h after induction. The expression properties of this new host-vector system are discussed in comparison to the ones available for B. subtilis and E. coli.
Rygus
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Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie und Biochemieder Friedrich-Alexander Universität Erlangen-Nürnberg, Federal Republic of Germany.
Hillen
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W
D000150:Acinetobacter / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000235:genetics*; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004792:Enzyme Precursors / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics; D014994:Xylose / Q000378:metabolism*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D014994:Xylose; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; D001616:beta-Galactosidase; D014568:Urokinase-Type Plasminogen Activator; D002238:Carbohydrate Epimerases; C019646:aldose 1-epimerase