Molecular characterization and expression analysis of the dextransucrase DsrD of Leuconostoc mesenteroides Lcc4 in homologous and heterologous Lactococcus lactis cultures

Abstract

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 21 wereshown 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.

Heterologous Expression of a Position 2-Substituted (1→3)-β- d -Glucan in Lactococcus lactis

“…As far as we know, this is the first instance of the production of a ␤-glucan by L. lactis, although the production of ␣-glucan dextran in a recombinant L. lactis strain by the expression of the Leuconostoc mesenteroides dextransucrase DsrD has been reported (17).…”

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

“…In order to elucidate which enzyme activities are encoded, these genes were cloned and heterologously expressed. The distribution of glucosidic linkages has been elucidated for the glucans synthesized from sucrose by heterologously produced (mostly using Escherichia coli as the host) GS enzymes from (i) seven Streptococcus strains (13 GS enzymes, synthesizing either dextran or mutan polymers) (67,90,124), (ii) four Leuconostoc strains (seven GS enzymes, synthesizing mainly dextran polymers, but also one alternan, and a GS synthesizing large numbers of ␣-(132,6) branch points) (4,17,55,124,135), and (iii) seven Lactobacillus strains (seven GS enzymes, synthesizing mainly dextrans, but also reuteran, and a highly branched mutan) (94,95,97) (Table 1).…”

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis

“…L. lactis is thus able to produce and secrete <10-kDa proteins through a Secdependent pathway. This bacterium is also able to secrete >160-kDa proteins (3). Taken together, these facts show that protein size is not a serious bottleneck for heterologous protein secretion in L. lactis.…”

“…Consequently, an understanding of the molecular processes involved in protein secretion has become important for both basic and applied research. Secretion of highmolecular weight proteins such as dextran sucrase (165 kDa) has been reported for L. lactis (3); however, little is known about the ability of L. lactis to secrete low-molecular weight proteins, even though this feature has been found to be a limiting step for protein production in other bacteria (4).…”