Polyol production during heterofermentative growth of the plant isolate Lactobacillus florum 2F.

AIMS: This study examined the fermentative growth and polyol production of Lactobacillus florum and other plant-associated lactic acid bacteria (LAB). METHODS AND RESULTS: Sugar consumption and end-product production were measured for Lact. florum 2F in the presence of fructose, glucose and both sugars combined. The genome of Lact. florum was examined for genes required for mannitol and erythritol biosynthesis. The capacity for other plant-associated LAB to synthesize polyols was also assessed. CONCLUSIONS: Lactobacillus florum exhibited higher growth rates and cell yields in the presence of both fructose and glucose. Lactobacillus florum 2F produced lactate, acetate and ethanol as well as erythritol and mannitol. Lactobacillus florum 2F synthesized mannitol during growth on fructose and erythritol during growth on glucose. Gene and protein homology searches identified a mannitol dehydrogenase in the Lact. florum 2F genome but not the genes responsible for erythritol biosynthesis. Lastly, we found that numerous other heterofermentative LAB species synthesize erythritol and/or mannitol. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus florum is a recently identified, plant-associated, fructophilic LAB species. Our results show that Lact. florum growth rates and heterofermentation end-products differ depending on the sugar substrates present and growth yields can be improved when combinations of sugars are provided. Lactobacillus florum 2F produces erythritol and mannitol, two polyols that are relevant to foods and potentially also in plant environments. The capacity for polyol biosynthesis appears to be common among plant-associated, LAB species.

Functional genomics for food fermentation processes.

This review describes recent scientific and technological drivers of food fermentation research. In addition, a number of practical implications of the results of this development will be highlighted. The first part of the manuscript elaborates on the message that genome sequence information gives us an unprecedented view on the biodiversity of microbes in food fermentation. This information can be made applicable for tailoring relevant characteristics of food products through fermentation. The second part deals with the integration of genome sequence data into metabolic models and the use of these models for a number of topics that are relevant for food fermentation processes. The final part will be about metagenomics approaches to reveal the complexity and understand the functionality of undefined complex microbial consortia used in a diverse range of food fermentation processes.

Lactobacillus plantarum WCFS1 electron transport chains.

Lactobacillus plantarum WCFS1 requires both heme and menaquinone to induce respiration-like behavior under aerobic conditions. The addition of these compounds enhanced both biomass production, without progressive acidification, and the oxygen consumption rate. When both heme and menaquinone were present, L. plantarum WCFS1 was also able to reduce nitrate. The ability to reduce nitrate was severely inhibited by the glucose levels that are typically found in L. plantarum growth media (1 to 2% [vol/vol] glucose). In contrast, comparable mannitol levels did not inhibit the reduction of nitrate. L. plantarum reduced nitrate with concomitant formation of nitrite and ammonia. Genes that encode a bd-type cytochrome (cydABCD) and a nitrate reductase (narGHJI) were identified in the genome of L. plantarum. The narGHJI operon is part of a cluster of genes that includes the molybdopterin cofactor biosynthesis genes and narK. Besides a menaquinone source, isogenic mutants revealed that cydA and ndh1 are required for the aerobic-respiration-like response and narG for nitrate reduction. The ndh1 mutant was still able to reduce nitrate. The existence of a nonredundant branched electron transport chain in L. plantarum WCFS1 that is capable of using oxygen or nitrate as a terminal electron acceptor is proposed.

Generation of a membrane potential by Lactococcus lactis through aerobic electron transport.

Lactococcus lactis, a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in L. lactis under these conditions. Proton motive force generation in whole cells was measured using a fluorescent probe (3',3'-dipropylthiadicarbocyanine), which is sensitive to changes in membrane potential (Delta psi). Wild-type cells, grown aerobically in the presence of heme, generated a Delta psi even in the presence of the F(1)-F(o) ATPase inhibitor N,N'-dicyclohexylcarbodiimide, while a cytochrome bd-negative mutant strain (CydA Delta) did not. We also observed high oxygen consumption rates by membrane vesicles prepared from heme-grown cells, compared to CydA Delta cells, upon the addition of NADH. This demonstrates that NADH is an electron donor for the L. lactis ETC and demonstrates the presence of a membrane-bound NADH-dehydrogenase. Furthermore, we show that the functional respiratory chain is present throughout the exponential and late phases of growth.

Expression of Sulfolobus solfataricus alpha-glucosidase in Lactococcus lactis.

The industrial potential to use extreme thermophilic microorganisms and their enzymes lies in applications in which the temperature cannot be adjusted (cooled) at will. The production of enzymes from wild-type thermophiles is very low, therefore, for industrial applications, it is necessary to use recombinant microorganisms. In this paper, the cloning of a heat-stable alpha-glucosidase from Sulfolobus solfataricus using lactic acid bacteria as expression system is reported. The extremophilic alpha-glucosidase was cloned in Lactococcus lactis and correctly folded despite being expressed at a lower temperature. The recombinant cells were assayed for enzyme residual activity at 75 degrees C in order to analyze the direct use of whole cells as biocatalysts. Maximum activity corresponded to 40 U/l in static cultures. The protein yield was further improved by optimizing fermentation and reached 600 U/l in batch mode. Microfiltration led to an even higher enzyme production of 850 U/l as a result of increased biomass. The overall production of alpha-glucosidase using the engineered L. lactis strain in microfiltration fermentation is 1,000-fold higher than obtained using the wild-type.

Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy.

The effect of agitation speeds on the performance of producing pyruvate by a multi-vitamin auxotrophic yeast, Torulopsis glabrata, was investigated in batch fermentation. High pyruvate yield on glucose (0.797 g g(-1)) was achieved under high agitation speed (700 rpm), but the glucose consumption rate was rather low (1.14 g l(-1) h(-1)). Glucose consumption was enhanced under low agitation speed (500 rpm), but the pyruvate yield on glucose decreased to 0.483 g g(-1). Glycerol production was observed under low agitation speed and decreased with increasing agitation speed. Based on process analysis and carbon flux distribution calculation, a two-stage oxygen supply control strategy was proposed, in which the agitation speed was controlled at 700 rpm in the first 16 h and then switched to 500 rpm. This was experimentally proven to be successful. Relatively high concentration of pyruvate (69.4 g l(-1)), high pyruvate yield on glucose (0.636 g g(-1)), and high glucose consumption rate (1.95 g l(-1)h(-1)) were achieved by applying this strategy. The productivity (1.24 g l(-1) h(-1)) was improved by 36%, 23% and 31%, respectively, compared with fermentations in which agitation speeds were kept constant at 700 rpm, 600 rpm, and 500 rpm. Experimental results indicate that the difference between the performances for producing pyruvate under a favorable state of oxygen supply (dissolved oxygen concentration >50%) was caused by the different regeneration pathways of NADH generated from glycolysis.

Metabolic engineering of acetaldehyde production by Streptococcus thermophilus.

The process of acetaldehyde formation by the yogurt bacterium Streptococcus thermophilus is described in this paper. Attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the glyA gene. In S. thermophilus, SHMT also possesses threonine aldolase (TA) activity, the interconversion of threonine into glycine and acetaldehyde. In this work, several wild-type S. thermophilus strains were screened for acetaldehyde production in the presence and absence of L-threonine. Supplementation of the growth medium with L-threonine led to an increase in acetaldehyde production. Furthermore, acetaldehyde formation during fermentation could be correlated to the TA activity of SHMT. To study the physiological role of SHMT, a glyA mutant was constructed by gene disruption. Inactivation of glyA resulted in a severe reduction in TA activity and complete loss of acetaldehyde formation during fermentation. Subsequently, an S. thermophilus strain was constructed in which the glyA gene was cloned under the control of a strong promoter (P(LacA)). When this strain was used for fermentation, an increase in TA activity and in acetaldehyde and folic acid production was observed. These results show that, in S. thermophilus, SHMT, displaying TA activity, constitutes the main pathway for acetaldehyde formation under our experimental conditions. These findings can be used to control and improve acetaldehyde production in fermented (dairy) products with S. thermophilus as starter culture.

Time dependent responses of glycolytic intermediates in a detailed glycolytic model of Lactococcus lactis during glucose run-out experiments.

Glucose addition and subsequent run-out experiments were compared to simulations with a detailed glycolytic model of Lactococcus lactis. The model was constructed largely on bases of enzyme kinetic data taken from literature and not adjusted for the specific simulations shown here. Upon glucose depletion a rapid increase in PEP, inorganic phosphate and a gradual decrease in fructose 1,6-bisphosphate (FBP) were measured and predicted by simulation. The dynamic changes in these and other intermediate concentrations as measured in the experiments were well predicted by the kinetic model.

Fluorescent method for monitoring cheese starter permeabilization and lysis.

A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed.

Spontaneous formation of a mannitol-producing variant of Leuconostoc pseudomesenteroides grown in the presence of fructose.

We report the spontaneous formation of a stable mannitol-producing variant of Leuconostoc pseudomesenteroides. The mannitol-producing variant showed mannitol dehydrogenase activity which was absent in the parental strain. It was also able to use fructose and glucose simultaneously, whereas the parental strain showed diauxic growth with these sugars. A possible explanation of these observations is discussed.

Physiological function of exopolysaccharides produced by Lactococcus lactis.

The physiological function of EPS produced by Lactococcus lactis was studied by comparing the tolerance of the non-EPS producing strain L. lactis ssp. cremoris MG1614 and an EPS producing isogenic variant of this strain to several anti-microbial factors. There was no difference in the sensitivity of the strains to increased temperatures, freezing or freeze-drying and the antibiotics, penicillin and vancomycin. A model system showed that EPS production did not affect the survival of L. lactis during passage through the gastrointestinal tract although the EPS itself was not degraded during this passage. The presence of cell associated EPS and EPS in suspension resulted in an increased tolerance to copper and nisin. Furthermore, cell associated EPS also protected the bacteria against bacteriophages and the cell wall degrading enzyme lysozyme. However, it has not been possible, so far, to increase EPS production using the presence of copper, nisin, lysozyme or bacteriophages as inducing factors.

Influence of different substrate limitations on the yield, composition and molecular mass of exopolysaccharides produced by Lactococcus lactis subsp. cremoris in continuous cultures.

The type of substrate limitation had a remarkable influence on the molecular mass of exopolysaccharides (EPS) produced by Lactococcus lactis subsp. cremoris NIZO B40 and NIZO B891. Under glucose/energy limitation, the molecular mass was much smaller than under leucine or phosphate limitation, resulting in a marked decrease of the intrinsic viscosity of this EPS. The sugar composition of EPS produced by both strains, and the phosphate content of EPS produced by NIZO B40, were not affected by the type of nutrient limitation. Both strains produced comparable amounts of EPS under leucine and glucose limitation, but the efficiency of EPS production was highest under glucose limitation. The EPS yields of the phosphorylated B40 EPS as well as the unphosphorylated B891 EPS were reduced, with about 40% under conditions of phosphate limitation.

Changes in glycolytic activity of Lactococcus lactis induced by low temperature.

The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium Lactococcus lactis were studied. The maximal glycolytic activity measured at 30 degrees C increased approximately 2.5-fold following a shift from 30 to 10 degrees C for 4 h in a process that required protein synthesis. Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures. In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain. This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr [HPr(Ser-P)] probably is involved. Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures. The las operon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed. Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock. This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.

Lactococcus lactis as a cell factory for high-level diacetyl production.

We report the engineering of Lactococcus lactis for the efficient conversion of sugar into diacetyl by combining NADH-oxidase overproduction and alpha-acetolactate decarboxylase inactivation. Eighty percent of the carbon flux was found to be rerouted via alpha-acetolactate to the production of diacetyl by preloading the cells with NADH-oxidase before their use as a cell factory.

Lactic acid bacteria as a cell factory: rerouting of carbon metabolism in Lactococcus lactis by metabolic engineering.

Lactic acid bacteria display a relatively simple metabolism wherein the sugar is converted mainly to lactic acid. The extensive knowledge of metabolic pathways and the increasing information of the genes involved allows for the rerouting of natural metabolic pathways by genetic and physiological engineering. We discuss several examples of metabolic engineering of Lactococcus lactis for the production of important compounds, including diacetyl, alanine and exopolysaccharides.

Analysis of sugar metabolism in an EPS producing Lactococcus lactis by 31P NMR.

Sugar metabolism and exopolysaccharide (EPS) production was analysed in Lactococcus lactis by in vivo 31P NMR. Transient production of several sugar phosphates, transient depletion of intracellular phosphate, transient production of ATP and UTP, transient acidification of the medium and alkalinisation of the cytoplasm could be observed in a period of 20 min upon energization by the addition of glucose. EPS and non-EPS producing variants showed similar NMR spectra, the exception being two pH-dependent resonances observed in the former. They were already observed before addition of glucose and their response to glucose incubation reflected exposure to the medium. They are presumably phosphorylated poly- or oligosaccharides being loosely adhered to cell walls. By freezing and perchloric acid extraction of the cell material, different types of phosphorylated compounds could be recognised in the NMR spectra such as fructose-1-6-diphosphate, nucleotides (like ADP, ATP, UTP and TDP) and several nucleotide sugars. The ongoing work is focused on identifying the unknown peaks and quantifying the differences between wild-type cells and the EPS producing variant.

Regulation of exopolysaccharide production by Lactococcus lactis subsp. cremoris By the sugar source.

Lactococcus lactis produced more exopolysaccharide (EPS) on glucose than on fructose as the sugar substrate, although the transcription level of the eps gene cluster was independent of the sugar source. A major difference between cells grown on the two substrates was the capacity to produce sugar nucleotides, the EPS precursors. However, the activities of the enzymes required for the synthesis of nucleotide sugars were not changed upon growth on different sugars. The activity of fructosebisphosphatase (FBPase) was by far the lowest of the enzymes involved in precursor formation under all conditions. FBPase catalyzes the conversion of fructose-1, 6-diphosphate into fructose-6-phosphate, which is an essential step in the biosynthesis of sugar nucleotides from fructose but not from glucose. By overexpression of the fbp gene, which resulted in increased EPS synthesis on fructose, it was proven that the low activity of FBPase is indeed limiting not only for EPS production but also for growth on fructose as a sugar source.