Exopolysaccharide biosynthesis in Lactococcus lactis : a molecular characterisation

Lactic acid bacteria are Gram-positive bacteria which are used for industrial food fermentation processes. Some have the ability to form exopolysaccharides (EPSs) and these bacteria or the produced EPSs can be used to enhance the structural properties of food products. Furthermore, these EPSs are claimed to be health beneficial. This thesis describes the results of a study on the biosynthesis of these polymers in Lactococcus lactis strains.Chapter 1 provides an overview of the current knowledge of cell-surface polysaccharide biosynthesis, the glycosyltransferases involved, and export and polymerisation processes. Special attention is paid to genetics, regulation, and EPSs produced by LAB.Chapter 2 describes the characterisation of EPS production by L. lactis NIZO B40. The strain produces an extracellular phosphopolysaccharide containing galactose, glucose, and rhamnose. The EPS production is encoded on a 40-kb plasmid, which was isolated after conjugation and subsequent plasmid curing. On this plasmid, a 12-kb region containing 14 genes with the order epsRXABCDEFGHIJKL was identified encoding putative gene products which shared sequence homologies with gene products involved in cell-surface polysaccharide biosynthesis of other bacteria. Based on these homologies, predicted functions as regulation ( epsR ), polymerisation and export ( epsA , epsB , epsI , epsK ), or biosynthesis of the repeating unit ( epsD , epsE / epsF , epsG , epsH ) could be assigned. The eps genes are co-ordinately expressed and transcribed as a single 12-kb mRNA from a promoter upstream of epsR . Heterologous expression of epsD in Escherichia coli showed that its gene product is the so-called priming glucosyltransferase, linking the first sugar of the repeating unit to the lipid carrier.Chapter 3 describes the functional analysis of the glycosyltransferase genes of the NIZO B40 eps gene cluster. The genes were cloned and expressed in E. coli and L. lactis to determine their function and the sugar-specificity of the encoded enzymes. The EPS consists of repeating units containing a trisaccharide backbone of two glucose and one galactose moieties. The epsDEFG gene products are involved in the synthesis of this trisaccharide, linking glucose to a lipid carrier in the membrane (EpsD), glucose to lipid-linked glucose (EpsE/EpsF), and galactose to lipid-linked cellobiose (EpsG), respectively. The epsJ gene product was found to be involved in the biosynthesis of EPS and is likely to act either as a galactosyl phosphotransferase or as an enzyme which releases the backbone oligosaccharide from the lipid carrier.Chapter 4 describes the variety of EPS production by L. lactis . Sixteen EPS-producing L. lactis strains were analysed and based on the chemical composition of the EPSs they formed and the genotype of their eps genes, they were grouped in three major groups and two unique strains. Representatives of the three major groups were studied in detail. Group I comprises strain NIZO B40 which was characterised in the previous chapters. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating unit synthesis.First successful attempts for genetic engineering of the EPS production were achieved by replacing the NIZO B40 priming glucosyltransferase gene, epsD , by an erythromycin resistance gene which resulted in the loss of EPS production and the complementation of the EPS-producing phenotype by controlled expression of priming glycosyltransferase genes from Gram-positive organisms with known function and substrate specificity.In Chapter 5 the regions involved in replication and mobilisation of the NIZO B40 EPS-plasmid pNZ4000 were characterised. The plasmid contains four highly conserved replication regions that belong to the lactococcal theta replicon family and all are functional and compatible in L. lactis . Plasmid pNZ4000 was shown to be a mobilisation plasmid and two regions involved in mobilisation were identified. Both regions contained a functional origin of transfer ( oriT ). One oriT sequence was followed by a mobA gene, coding for a trans -acting protein involved in conjugative transfer and likely to be the relaxase nicking the nic sites of the oriT sequences.Chapter 6 describes the complete nucleotide sequence of the EPS-plasmid pNZ4000, which amounts to 42810 bp and represents one of the largest sequenced plasmids in LAB to date. Apart from the regions involved in EPS biosynthesis, replication, and mobilisation, described in Chapters 2 and 5, two regions potentially involved in transport of divalent cations were localised on pNZ4000.In Chapter 7 the results of the previous chapters are discussed and their implications on practical applications and in particular the perspectives for polysaccharide engineering are described