Characterization of the individual glucose uptake systems of Lactococcus lactis: mannose-PTS, cellobiose-PTS and the novel GlcU permease.

According to previous reports, Lactococcus lactis imports glucose via two distinct phosphoenolpyruvate:phosphotransferase systems (mannose-PTS and cellobiose-PTS) and one or more unknown non-PTS permease(s). GlcU was identified as the sole non-PTS permease involved in the transport of glucose. Additionally, the biochemical properties of PTS(Man), PTS(Cel) and GlcU were characterized in double knockout mutants with glucose uptake restricted to a single system. Transport susceptibility to protonophores indicated that glucose uptake via GlcU is proton-motive force dependent. Competition assays revealed a high specificity of GlcU for glucose. Furthermore, the permease has low affinity for glucose and displays strong preference for the beta-anomer as shown by the profiles of consumption of the two glucose anomers studied by (13)C-NMR. Similar kinetic properties were found for PTS(Cel), while PTS(Man) is a high-affinity system recognizing equally well the two anomeric forms of glucose. Transcripts of the genes encoding the three transporters are present simultaneously in the parent strain NZ9000 as shown by reverse transcription-PCR. Investigation of the distribution of GlcU homologues among bacteria showed that these proteins are restricted to the low-GC Gram-positive Firmicutes. This work completes the identification of the glucose transport systems in L. lactis MG1363.

Towards enhanced galactose utilization by Lactococcus lactis.

Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.

Overview on sugar metabolism and its control in Lactococcus lactis - the input from in vivo NMR.

The wide application of lactic acid bacteria in the production of fermented foods depends to a great extent on the unique features of sugar metabolism in these organisms. The relative metabolic simplicity and the availability of genetic tools made Lactococcus lactis the organism of choice to gain insight into metabolic and regulatory networks. In vivo nuclear magnetic resonance has proven a very useful technique to monitor non-invasively the dynamics of intracellular metabolite and co-factor pools following a glucose pulse. Examples of the application of this methodology to identify metabolic bottlenecks and regulatory sites are presented. The use of this information to direct metabolic engineering strategies is illustrated.

Natural sweetening of food products by engineering Lactococcus lactis for glucose production.

We show that sweetening of food products by natural fermentation can be achieved by a combined metabolic engineering and transcriptome analysis approach. A Lactococcus lactis ssp. cremoris strain was constructed in which glucose metabolism was completely disrupted by deletion of the genes coding for glucokinase (glk), EII(man/glc) (ptnABCD), and the newly discovered glucose-PTS EII(cel) (ptcBAC). After introducing the lactose metabolic genes, the deletion strain could solely ferment the galactose moiety of lactose, while the glucose moiety accumulated extracellularly. Additionally, less lactose remained in the medium after fermentation. The resulting strain can be used for in situ production of glucose, circumventing the need to add sweeteners as additional ingredients to dairy products. Moreover, the enhanced removal of lactose achieved by this strain could be very useful in the manufacture of products for lactose intolerant individuals.

The alpha-phosphoglucomutase of Lactococcus lactis is unrelated to the alpha-D-phosphohexomutase superfamily and is encoded by the essential gene pgmH.

alpha-Phosphoglucomutase (alpha-PGM) plays an important role in carbohydrate metabolism by catalyzing the reversible conversion of alpha-glucose 1-phosphate to glucose 6-phosphate. Isolation of alpha-PGM activity from cell extracts of Lactococcus lactis strain MG1363 led to the conclusion that this activity is encoded by yfgH, herein renamed pgmH. Its gene product has no sequence homology to proteins in the alpha-d-phosphohexomutase superfamily and is instead related to the eukaryotic phosphomannomutases within the haloacid dehalogenase superfamily. In contrast to known bacterial alpha-PGMs, this 28-kDa enzyme is highly specific for alpha-glucose 1-phosphate and glucose 6-phosphate and showed no activity for mannose phosphate. To elucidate the function of pgmH, the metabolism of glucose and galactose was characterized in mutants overproducing or with a deficiency of alpha-PGM activity. Overproduction of alpha-PGM led to increased glycolytic flux and growth rate on galactose. Despite several attempts, we failed to obtain a deletion mutant of pgmH. The essentiality of this gene was proven by using a conditional knock-out strain in which a native copy of the gene was provided in trans under the control of the nisin promoter. Growth of this strain was severely impaired when alpha-PGM activity was below the control level. We show that the novel L. lactis alpha-PGM is the only enzyme that mediates the interconversion of alpha-glucose 1-phosphate to glucose 6-phosphate and is essential for growth.