Production and Secretion of Recombinant Leuconostoc mesenteroides Dextransucrase DsrS in Bacillus megaterium Marco Malten, 1, * Rajan Hollmann, 2, * Wolf-Dieter Deckwer, 2 Dieter Jahn 1 1 Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany; telephone: + 49 (0) 531-391-5801; fax: + 49 (0) 531-391-5854; e-mail: d.jahn @tu-bs.de 2 Biochemical Engineering, Technical University Braunschweig, Mascheroder Weg 1, 38124 Braunschweig, Germany Received 11 May 2004; accepted 9 September 2004 Published online 7 December 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20341 Abstract: 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 repres- sion was removed. Recombinant DsrS was found in the cytoplasm and exported via its native leader sequence into the growth medium. Elimination of the extracellular pro- tease 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 re- sulted in a further doubling of extracellular DsrS con- centration 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) ac- tivities of dextran free DsrS were measured. However, using high cell density cultivation, most DsrS was found cell-associated indicating current limitations of the pro- duction process. A protease accessibility assay identified the major limitation of DsrS production at the level of pro- tein 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 optimiza- tion strategies for the production of recombinant DsrS in B. megaterium are discussed. B 2004 Wiley Periodicals, Inc. Keywords: Bacillus megaterium; secretion; heterolo- gous gene expression; dextransucrase; high cell density cultivation INTRODUCTION The dextransucrase DsrS from L. mesenteroides NRRL B- 512F is a glucosyltransferase, which catalyzes the transfer of a D-glucopyranosyl unit from sucrose to acceptor molecules. This polymerization reaction results in dextran, an a-1,6 glycosidic linked D-glucan with 5% of a-1,3 glycosidic linked branches (Buchholz and Monsan, 2002; Remaud-Simeon et al., 2000). Due to its extensive use as a blood plasma substitute and as the basis for many chromatography support materials, e.g., SephadexR, dex- tran is of significant commercial interest. Additionally, the use of dextransucrase for the synthesis of new oligosac- charides via the employment of acceptor molecules apart from sucrose is presently investigated (Demuth et al., 2002; Richard et al., 2003). Currently, commercially available DsrS is produced and secreted using L. mesenteroides. En- zyme production is induced by the addition of sucrose to the culture medium and leads to enzyme preparations con- sisting of 99% dextran and only 1% of protein (C.R.I.T.T., Toulouse, France). These preparations are difficult to use for the investigation of enzyme structure and catalysis. To obtain purified dextran-free DsrS, recombinant production in E. coli was established. Intracellularly localized DsrS with activities of up to 200 units per liter culture broth were obtained (Monchois et al., 1997). Key amino acids involved in catalysis were identified and the role of the C-terminal domain in catalysis and dextran binding was investi- gated despite problems with considerable proteolytic degra- dation (Monchois et al., 1998). Characterization of DsrD from L. mesenteroides Lcc4 revealed 99.7% identity at the amino acid sequence level to DsrS (Neubauer et al., 2003). Expression of L. mesenteroides dsrD was carried out in Lactococcus lactis resulting in 800 Units dextransu- crase activity per liter culture broth. DsrD was found secreted, but also cell-associated. Hence, heterologous recombinant production and secretion of DsrS are currently B 2004 Wiley Periodicals, Inc. Correspondence to: Dieter Jahn * M. Malten and R. Hollmann contributed equally to this work. Contract grant sponsors: Sonderforschungsbereich 578 ‘‘From gene to product,’’ Deutsche Forschungsgemeinschaft; Fonds der Chemischen Industrie