Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium.
Leuconostoc mesenteroides dextransucrase DsrS was recombinantly produced in Bacillus megaterium and exported into the growth medium. For this purpose a plasmid-based xylose-inducible gene expression system was optimized via introduction of a multiple cloning site and an encoded optimal B. megaterium ribosome binding site. A cre mediating glucose-dependent catabolite repression was removed. Recombinant DsrS was found in the cytoplasm and exported via its native leader sequence into the growth medium. Elimination of the extracellular protease NprM increased extracellular DsrS concentrations by a factor of 4 and stabilized the recombinant protein for up to 12 h. Cultivation in a semi-defined medium resulted in a further doubling of extracellular DsrS concentration up to an activity of 65 Units/L. To develop an industrial process a high cell density cultivation of B. megaterium was established yielding cell dry weights of up to 80 g/L. After induction of dsrS expression high specific (362 Units/g) and volumetric (28,600 Units/L) activities of dextran free DsrS were measured. However, using high cell density cultivation, most DsrS was found cell-associated indicating current limitations of the production process. A protease accessibility assay identified the major limitation of DsrS production at the level of protein folding. Intracellular misfolding of DsrS hampered DsrS export via the SEC pathway at high cell densities. The subsequent use of a semi-defined mineral medium and the induction of DsrS production at lower cell densities increased protein export efficiency remarkably, but also led to extracellular DsrS aggregation. Further optimization strategies for the production of recombinant DsrS in B. megaterium are discussed.
Malten
Marco
M
Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
Hollmann
Rajan
R
Deckwer
Wolf-Dieter
WD
Jahn
Dieter
D
D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000235:genetics*; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014169:Transformation, Bacterial / Q000235:genetics
D011994:Recombinant Proteins; D005964:Glucosyltransferases; C020025:dextransucrase