Heterologous expression and characterization of the exopolysaccharide from Streptococcus thermophilus Sfi39.

The genes responsible for exopolysaccharide (EPS) synthesis in Streptococcus thermophilus Sfi39 were identified on a 20-kb genomic fragment. The two genes, epsE and epsG, were shown to be involved in EPS synthesis as their disruption lead to the loss of the ropy phenotype. Several naturally selected nonropy mutants were isolated, one acquired an insertion sequence (IS)-element (IS905) in the middle of the eps gene cluster. The eps gene cluster was cloned and transferred into a nonEPS-producing heterologous host, Lactococcus lactis MG1363. The EPS produced was shown by chemical analysis and NMR spectroscopy to be identical to the EPS produced by S. thermophilus Sfi39. This demonstrated first that all genes needed for EPS production and export were present in the S. thermophilus Sfi39 eps gene cluster, and second that the heterologous production of an EPS was possible by transfer of the complete eps gene cluster alone, provided that the heterologous host possessed all necessary genetic information for precursor synthesis.     

Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6.

We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)]-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.     

Identification and characterisation of a gene encoding aminoacylase activity from Lactococcus lactis MG1363

Analysis of the sequence of a randomly cloned chromosomal DNA fragment (3.2 kb) from Lactococcus lactis revealed the presence of part of an open reading frame, designated amd1, which specifies a protein displaying significant similarity to aminoacylases from various bacteria. The presence of an immobilised copy of an IS982 element immediately upstream of the coding region of amd1 has probably resulted in the displacement of amd1's native promoter. This genetic organisation was shown to be retained in seven other dairy strains, one of which was only slightly different. The amd1 gene was overexpressed in L. lactis NZ9800 under the control of the inducible nisA promoter and the deacetylating capacity of its gene product was measured on a number of substrates.     

Citrate permease gene expression in Lactococcus lactis subsp. lactis strains IL1403 and MG1363

Abstract Citrate permease gene expression in the plasmid-free Lactococcus lactis strains IL1403 and MG1363 was studied. The ability to transport citrate results in diacetyl and acetoin production in IL1403 but not in MG1363. Citrate lyase, α-acetolactate decarboxylase, diacetyl and acetoin reductase were detected in IL1403. These data show that L. lactis ssp. lactis strain IL1403 is a citrate permease mutant of the biovar. diacetylactis . Immunological analysis revealed the α-and β-subunits of citrate lyase not only in IL1403 but also in MG1363 where no citrate lyase activity was found.     

Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363

We have cloned usp45, a gene encoding an extracellular secretory protein of Lactococcus lactis subsp. lactis strain MG1363. Unidentified secreted 45-kDa protein (Usp45) is secreted by every mesophilic L. lactis strain we tested so far and it is chromosomally encoded. The nucleotide sequence of the usp45 gene revealed an open reading frame of 1383 bp encoding a protein of 461 amino acids (aa), composed of a 27-aa signal peptide and a mature protein initiated at Asp28. The gene contains a consensus promoter sequence and a weak ribosome-binding site; the latter is rather uncommon for Gram-positive bacteria. Expression studies in Escherichia coli showed efficient synthesis and secretion of the protein. Usp45 has an unusual aa composition and distribution, and it is predicted to be structurally homologous with P54 of Enterococcus faecium. Up to now, no biological activity could be postulated for this secreted protein.     

Identification and characterization of a cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363

This paper describes the nucleotide sequence of a gene encoding cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363, its overexpression in Escherichia coli and some functional characteristics of the purified recombinant protein.     

Cloning and sequence analysis of putative histidine protein kinases isolated from Lactococcus lactis MG1363.

Eight recombinant plasmids harboring chromosomal fragments of Lactococcus lactis MG1363 were shown to phenotypically suppress a histidine protein kinase (HPK) deficiency in either of two different E. coli strains. Sequence analysis of the plasmid inserts revealed five different complete or partial open reading frames (ORFs) specifying proteins with high similarity to HPKs. One of the plasmids also harbored an additional ORF, unrelated to HPKs, with suppressing activity.     

南极假丝酵母脂肪酶B(CALB)基因在乳酸乳球菌MG1363中的整合及表达

根据南极假丝酵母脂肪酶B (CALB)的基因序列将CALB基因进行TA克隆、酶切鉴定及测序后,亚克隆至大肠杆菌-乳酸乳球菌穿梭表达栽体pMG36e-Nisl中,构建重组表达栽体pMG36e-Nisl-CALB.设计特异性引物P3和P4,对重组质粒pMG36e-NisI-CALB进行红霉素抗性基因的敲除,以构建食品级表达载体pMG36N-CALB,后再将两种重组质粒分别电转化入乳酸乳球菌MG1363,以Nisin为选择压力,考察CALB在MG1363中的表达情况.结果显示,成功构建了表达载体pMG36e-NisI-CALB及pMG36N-CALB,两株重组菌在含有20 IU Nisir/mL的培养基中均生长情况良好,遗传性能稳定,且经水解圈鉴定,CALB能够进行活性表达.进一步研究发现,CALB基因整合到乳酸乳球菌MG1363染色体中.     

Unraveling the function of glycosyltransferases in Streptococcus thermophilus Sfi6.

Streptococcus thermophilus Sfi6 produces a texturizing exopolysaccharide (EPS) consisting of a -->3)[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->3)-alpha-D-GalpNAc-(1--> 3)-beta-D-Galp-(1--> repeating unit. We previously identified and analyzed a 14.5-kb gene cluster from S. thermophilus Sfi6 consisting of 13 genes responsible for its EPS production. Within this gene cluster, we found a central region of genes (epsE, epsF, epsG, and epsI) that showed similarity to glycosyltransferases. In this study, we investigated the sugar specificity of these enzymes. EpsE catalyzes the first step in the biosynthesis of the EPS repeating unit. It exhibits phosphogalactosyltransferase activity and transfers galactose onto the lipophilic carrier. The second step is fulfilled by EpsG, which transfers an alpha-N-acetylgalactosamine onto the first beta-galactoside. The activity of EpsF was determined by characterizing the EPS produced by an S. thermophilus epsF deletion mutant. This EPS consisted of the monosaccharides Gal, Glc, and GalNAc in an approximately equimolar ratio, thus suggesting that epsF codes for the branching galactosyltransferase. epsI probably codes for the beta-1,3-glucosyltransferase, since it is the only glycosyltransferase to which no gene has been assigned and it exhibits similarity to other beta-glycosyltransferases. EpsE shows the conserved features of phosphoglycosyltransferases, whereas EpsF and EpsG exhibit the primary structure of alpha-glycosyltransferases, belonging to glycosyltransferase family 4, whose members are conserved in all major phylogenetic lineages, including the Archaea and Eukaryota.     

Engineering of EPA/DHA omega-3 fatty acid production by Lactococcus lactis subsp. cremoris MG1363

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be of major importance in human health. Therefore, these essential polyunsaturated fatty acids have received considerable attention in both human and farm animal nutrition. Currently, fish and fish oils are the main dietary sources of EPA/DHA. To generate sustainable novel sources for EPA and DHA, the 35-kb EPA/DHA synthesis gene cluster was isolated from a marine bacterium, Shewanella baltica MAC1. To streamline the introduction of the genes into food-grade microorganisms such as lactic acid bacteria, unnecessary genes located upstream and downstream of the EPA/DHA gene cluster were deleted. Recombinant Escherichia coli harboring the 20-kb gene cluster produced 3.5- to 6.1-fold more EPA than those carrying the 35-kb DNA fragment coding for EPA/DHA synthesis. The 20-kb EPA/DHA gene cluster was cloned into a modified broad-host-range low copy number vector, pIL252m (4.7 kb, Ery) and expressed in Lactococcus lactis subsp. cremoris MG1363. Recombinant L. lactis produced DHA (1.35?±?0.5 mg g^?1 cell dry weight) and EPA (0.12?±?0.04 mg g^?1 cell dry weight). This is believed to be the first successful cloning and expression of EPA/DHA synthesis gene cluster in lactic acid bacteria. Our findings advance the future use of EPA/DHA-producing lactic acid bacteria in such applications as dairy starters, silage adjuncts, and animal feed supplements.     

Increased exopolysaccharide production in Lactococcus lactis due to increased levels of expression of the NIZO B40 eps gene cluster

Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented food products. This is the first report demonstrating that homologous overexpression of a complete eps gene cluster in Lactococcus lactis leads to increased EPS production levels. A ninefold-elevated EPS plasmid copy number led to an almost threefold increase in the eps expression level, resulting in an almost fourfold increase in the NIZO B40 EPS production level. It was previously reported that increased EPS precursor levels did not influence NIZO B40 EPS production levels. However, the present results indicate that the maximal NIZO B40 EPS production level is limited by the activity level of the expression products of the eps gene cluster rather than by the level of EPS precursors.