Heterologous hyper-expression of a glucansucrase-type glycosyltransferase gene.
Heterologous expression of the large glucansucrase-type glycosyltransferases genes is still a challenge, and typically yields are poor. Therefore, a number of different Escherichia coli systems for the expression of such a gene, encoding the glycosyltransferase R (GtfR) from Streptococcus oralis, were constructed and evaluated. We thereby obtained a strain producing the highest molar yields described so far for this class of enzymes. Cloning of a 5'-terminally truncated version of the gene in the expression vector pET33b(+) yielded, in dissolved form, about 2 micromol (300 mg) of enzyme per liter of culture of an optical density at 600 nm of four. Problems frequently encountered in the heterologous biosynthesis of this class of enzymes, such as formation of a high fraction of insoluble aggregates and/or proteolytic degradation, were not observed in the described system. The over-produced enzyme, devoid of almost its entire variable region, retained its characteristic activities.
Swistowska
Anna Maria
AM
Division of Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Wittrock
Sabine
S
Collisi
Wera
W
Hofer
Bernd
B
D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019533:Streptococcus oralis / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
D016695:Glycosyltransferases; C033837:alternansucrase