Molecular characterization and expression analysis of the dextransucrase DsrD of Leuconostoc mesenteroides Lcc4 in homologous and heterologous Lactococcus lactis cultures.
The gene encoding the dextransucrase DsrD from the industrial strain Leuconostoc mesenteroides Lcc4 was isolated by PCR using degenerate primers recognizing conserved regions present in other dextransucrase-encoding genes from Leuconostoc spp. and Southern blot analyses on total genomic DNA. N-terminal sequence analysis of the active protein recovered in the culture showed that the secreted protein of 165 kDa is devoid of a 42 aa prepeptide which is removed post-translationally, most likely by signal peptidase cleavage. Primer extension and Northern blot analysis identified a monocistronic dsrD mRNA with two transcription initiation sites. Expression of the dextransucrase DsrD was investigated in pH-controlled fed-batch cultures via Northern blot analysis and enzyme activity measurement during the experiments. Sucrose levels of 20 g l(-1) were shown to induce the DsrD biosynthesis around 10-fold. The combination of pH-controlled fed-batch fermentation and Northern analysis clearly showed that dsrD expression was related to the growth of the bacteria. dsrD was transferred to and expressed in Lactococcus lactis MG1363. Controlled fed-batch cultures revealed that active dextransucrase was produced and secreted by the recombinant L. lactis strain. The expression was independent of sucrose levels. These results show that dextransucrase can be efficiently expressed and secreted in a non-Leuconostoc, heterologous host and is able to drive dextran synthesis.
Neubauer
Heike
H
Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, PO Box 44, CH-1000 Lausanne 26, Switzerland.
Bauché
Anne
A
Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, PO Box 44, CH-1000 Lausanne 26, Switzerland.
Mollet
Beat
B
Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, PO Box 44, CH-1000 Lausanne 26, Switzerland.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D003911:Dextrans / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013395:Sucrose / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D003470:Culture Media; D003911:Dextrans; D013395:Sucrose; D005964:Glucosyltransferases; C020025:dextransucrase