scholarly journals Engineering Lactococcus lactis for Production of Mannitol: High Yields from Food-Grade Strains Deficient in Lactate Dehydrogenase and the Mannitol Transport System

2004 ◽  
Vol 70 (3) ◽  
pp. 1466-1474 ◽  
Author(s):  
Paula Gaspar ◽  
Ana Rute Neves ◽  
Ana Ramos ◽  
Michael J. Gasson ◽  
Claire A. Shearman ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Lactococcus Lactis ◽  
Selection Marker ◽  
Phosphotransferase System ◽  
Acid Fermentation ◽  
Food Grade ◽  
Mixed Acid ◽  
Glucose Depletion ◽  
Mannitol Production

ABSTRACT Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTSMtl). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (ΔldhΔmtlA and ΔldhΔmtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo 13C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTSMtl. Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.

scholarly journals High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+Cofactor Recycling

2011 ◽  
Vol 77 (19) ◽  
pp. 6826-6835 ◽  
Author(s):  
Paula Gaspar ◽  
Ana Rute Neves ◽  
Michael J. Gasson ◽  
Claire A. Shearman ◽  
Helena Santos
Keyword(s):  
Lactate Dehydrogenase ◽  
Lactococcus Lactis ◽  
Growth Parameters ◽  
Genetic Redundancy ◽  
Acid Fermentation ◽  
Content Type ◽  
Mixed Acid ◽  
Reverse Transcription Pcr ◽  
Theoretical Yield ◽  
Maximal Yield

ABSTRACTManipulation of NADH-dependent steps, and particularly disruption of thelas-located lactate dehydrogenase (ldh) gene inLactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome ofL. lactis, i.e., theldh,ldhB, andldhXgenes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, theldhBandldhXgenes were sequentially deleted inL. lactisFI10089, a strain with a deletion of theldhgene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, theadhEgene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of theldhBgene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhBis a valuable basis for engineering strategies aiming at the production of reduced compounds.

scholarly journals Regulatory Functions of Serine-46-Phosphorylated HPr in Lactococcus lactis

2001 ◽  
Vol 183 (11) ◽  
pp. 3391-3398 ◽  
Author(s):  
Vicente Monedero ◽  
Oscar P. Kuipers ◽  
Emmanuel Jamet ◽  
Josef Deutscher
Keyword(s):  
Lactococcus Lactis ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Inducer Exclusion ◽  
Gram Positive Bacteria ◽  
In Vivo Experiments ◽  
Regulatory Functions

ABSTRACT In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a β-glucoside-specific EII and a 6-P-β-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methylβ-d-thiogalactoside (TMG) and 2-deoxy-d-glucose (2-DG). In vivo experiments with theptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

scholarly journals Pyruvate Formate Lyase Is Required for Pneumococcal Fermentative Metabolism and Virulence

2009 ◽  
Vol 77 (12) ◽  
pp. 5418-5427 ◽  
Author(s):  
Hasan Yesilkaya ◽  
Francesca Spissu ◽  
Sandra M. Carvalho ◽  
Vanessa S. Terra ◽  
Karen A. Homer ◽  
...  
Keyword(s):  
Wild Type ◽  
Product Analysis ◽  
Acid Fermentation ◽  
Pyruvate Formate Lyase ◽  
Growth And Survival ◽  
Mixed Acid Fermentation ◽  
Mixed Acid ◽  
Fermentative Metabolism ◽  
In The Wild

ABSTRACT Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pfl A genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.

scholarly journals Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus lactis by Controlled Expression of NADH Oxidase

1998 ◽  
Vol 180 (15) ◽  
pp. 3804-3808 ◽  
Author(s):  
Felix Lopez de Felipe ◽  
Michiel Kleerebezem ◽  
Willem M. de Vos ◽  
Jeroen Hugenholtz
Keyword(s):  
Lactococcus Lactis ◽  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Metabolic Effects ◽  
Initial Growth ◽  
Acid Fermentation ◽  
Aerobic Conditions ◽  
Mixed Acid Fermentation ◽  
Mixed Acid ◽  
Nadh Oxidase Activity

ABSTRACT NADH oxidase-overproducing Lactococcus lactis strains were constructed by cloning the Streptococcus mutans nox-2gene, which encodes the H2O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the L. lactis nisA promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via α-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD+ratios in the cells.

Altered Superoxide Dismutase Activity by Carbohydrate Utilization in a Lactococcus lactis Strain

2014 ◽  
Vol 77 (7) ◽  
pp. 1161-1167 ◽  
Author(s):  
H. KIMOTO-NIRA ◽  
N. MORIYA ◽  
H. OHMORI ◽  
C. SUZUKI
Keyword(s):  
Superoxide Dismutase ◽  
Lactococcus Lactis ◽  
Dairy Products ◽  
Nadh Oxidase ◽  
Acid Fermentation ◽  
Sod Activity ◽  
Carbohydrate Utilization ◽  
Carbohydrate Source ◽  
Mixed Acid Fermentation ◽  
Mixed Acid

Reactive oxygen species, such as superoxide, can damage cellular components, such as proteins, lipids, and DNA. Superoxide dismutase (SOD) enzymes catalyze the conversion of superoxide anions to hydrogen peroxide and dioxygen. SOD is present in most lactococcal bacteria, which are commonly used as starters for manufacturing fermented dairy products and may have health benefits when taken orally. We assessed the effects of carbohydrate use on SOD activity in lactococci. In Lactococcus lactis ssp. lactis G50, the SOD activity of cells grown on lactose and galactose was higher than that on glucose; in Lactococcus lactis ssp. cremoris H61, SOD activity was independent of the type of carbohydrate used. We also investigated the activity of NADH oxidase, which is related to the production of superoxide in strains G50 and H61. Activity was highest in G50 cells grown on lactose, lower on galactose, and lowest on glucose, whereas activity in H61 cells did not differ with the carbohydrate source used. The SOD and NADH oxidase activities of strain G50 in three carbohydrates were linked. Strain G50 fermented lactose and galactose to lactate, acetate, formate, and ethanol (mixed-acid fermentation) and fermented glucose to mainly lactate (homolactic fermentation). Strain H61 fermented glucose, lactose, and galactose to mainly lactate (homolactic fermentation). In strain G50, when growth efficiency was reduced by adding a metabolic inhibitor to the growth medium, SOD activity was higher than in the control; however, the metabolism was homofermentative. Aerobic conditions, but not glucose-limited conditions, increased SOD activity, and mixed-acid fermentation occurred. We conclude that the effect of carbohydrate on SOD activity in lactococci is strain dependent and that the activity of commercial lactococci can be enhanced through carbohydrate selection for mixed-acid fermentation or by changing the energy distribution, thus enhancing the value of the starter and the resulting dairy products.

scholarly journals The Global Repressor SugR Controls Expression of Genes of Glycolysis and of the l-Lactate Dehydrogenase LdhA in Corynebacterium glutamicum

2008 ◽  
Vol 190 (24) ◽  
pp. 8033-8044 ◽  
Author(s):  
Verena Engels ◽  
Steffen N. Lindner ◽  
Volker F. Wendisch
Keyword(s):  
Lactate Dehydrogenase ◽  
Corynebacterium Glutamicum ◽  
Target Genes ◽  
Lactate Production ◽  
Oxygen Deprivation ◽  
Mrna Levels ◽  
Phosphotransferase System ◽  
Gel Retardation ◽  
Activity Measurements

ABSTRACT The transcriptional regulator SugR from Corynebacterium glutamicum represses genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Growth experiments revealed that the overexpression of sugR not only perturbed the growth of C. glutamicum on the PTS sugars glucose, fructose, and sucrose but also led to a significant growth inhibition on ribose, which is not taken up via the PTS. Chromatin immunoprecipitation combined with DNA microarray analysis and gel retardation experiments were performed to identify further target genes of SugR. Gel retardation analysis confirmed that SugR bound to the promoter regions of genes of the glycolytic enzymes 6-phosphofructokinase (pfkA), fructose-1,6-bisphosphate aldolase (fba), enolase (eno), pyruvate kinase (pyk), and NAD-dependent l-lactate dehydrogenase (ldhA). The deletion of sugR resulted in increased mRNA levels of eno, pyk, and ldhA in acetate medium. Enzyme activity measurements revealed that SugR-mediated repression affects the activities of PfkA, Fba, and LdhA in vivo. As the deletion of sugR led to increased LdhA activity under aerobic and under oxygen deprivation conditions, l-lactate production by C. glutamicum was determined. The overexpression of sugR reduced l-lactate production by about 25%, and sugR deletion increased l-lactate formation under oxygen deprivation conditions by threefold. Thus, SugR functions as a global repressor of genes of the PTS, glycolysis, and fermentative l-lactate dehydrogenase in C. glutamicum.

scholarly journals Expression of Genes Encoding F1-ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

2002 ◽  
Vol 68 (9) ◽  
pp. 4274-4282 ◽  
Author(s):  
Brian J. Koebmann ◽  
Christian Solem ◽  
Martin B. Pedersen ◽  
Dan Nilsson ◽  
Peter R. Jensen
Keyword(s):  
Atpase Activity ◽  
Lactococcus Lactis ◽  
Biomass Production ◽  
Energy State ◽  
Sugar Transport ◽  
Growth Conditions ◽  
Glycolytic Flux ◽  
Acid Fermentation ◽  
Mixed Acid ◽  
Genes Encoding

ABSTRACT We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F1 domain of the membrane-bound (F1F0) H+-ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F1-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F1-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.

scholarly journals Reengineering Escherichia coli for Succinate Production in Mineral Salts Medium

2009 ◽  
Vol 75 (24) ◽  
pp. 7807-7813 ◽  
Author(s):  
X. Zhang ◽  
K. Jantama ◽  
K. T. Shanmugam ◽  
L. O. Ingram
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Phosphotransferase System ◽  
Acid Fermentation ◽  
Mineral Salts ◽  
Gene Encoding ◽  
Succinate Production ◽  
Atp Production ◽  
Mixed Acid ◽  
Fermentative Metabolism ◽  
Genes Encoding

ABSTRACT The fermentative metabolism of glucose was redirected to succinate as the primary product without mutating any genes encoding the native mixed-acid fermentation pathway or redox reactions. Two changes in peripheral pathways were together found to increase succinate yield fivefold: (i) increased expression of phosphoenolpyruvate carboxykinase and (ii) inactivation of the glucose phosphoenolpyruvate-dependent phosphotransferase system. These two changes increased net ATP production, increased the pool of phosphoenolpyruvate available for carboxylation, and increased succinate production. Modest further improvements in succinate yield were made by inactivating the pflB gene, encoding pyruvate formate lyase, resulting in an E scherichia coli pathway that is functionally similar to the native pathway in Actinobacillus succinogenes and other succinate-producing rumen bacteria.

scholarly journals Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

2004 ◽  
Vol 70 (7) ◽  
pp. 4286-4292 ◽  
Author(s):  
H. Wouter Wisselink ◽  
Astrid E. Mars ◽  
Pieter van der Meer ◽  
Gerrit Eggink ◽  
Jeroen Hugenholtz
Keyword(s):  
Metabolic Engineering ◽  
Lactococcus Lactis ◽  
High Performance ◽  
Nuclear Magnetic Resonance Analysis ◽  
Resting Cells ◽  
Resonance Analysis ◽  
Glucose Depletion ◽  
Dehydrogenase Gene ◽  
Mannitol Production ◽  
Phosphofructokinase Activity

ABSTRACT To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and 13C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

scholarly journals Deciphering the regulation of the mannitol operon paves the way for efficient production of mannitol in L. lactis

2021 ◽  
Author(s):  
Hang Xiao ◽  
Claus Heiner Bang-Berthelsen ◽  
Peter Ruhdal Jensen ◽  
Christian Solem
Keyword(s):  
Stationary Phase ◽  
Lactococcus Lactis ◽  
Transcriptional Activator ◽  
High Yield ◽  
Efficient Production ◽  
Phosphotransferase System ◽  
Transcriptional Terminator ◽  
Mannitol Production ◽  
Transcriptional Units ◽  
Foreign Genes

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated, and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positives, mtlF and mtlD , encoding the Enzyme IIA component (EIIA mtl ) of the mannitol phosphotransferase system (PTS), and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA , encoding the Enzyme IIBC component (EIIBC mtl ) of the mannitol PTS, mtlR , encoding a transcriptional activator, and mtlF , and a separately expressed mtlD . The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive elements ( cre ). Presence of carbon catabolite repression was demonstrated, and was shown to be relieved in stationary phase cells. The transcriptional activator MtlR ( mtlR ), in some Gram-positives, is repressed by phosphorylation by EIIA mtl , and when we knocked-out mtlF we indeed observed enhanced expression from the two promotors, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary phase cells as biocatalysts, we attained 10.1 g/L mannitol with a 55% yield, which is the highest titer ever reported for L. lactis . Summing up, the results of our study should be useful for improving the mannitol producing capacity of this important industrial organism. Importance Lactococcus lactis is the most studied species of the Lactic Acid Bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis into producing mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis , with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated, and demonstrate how this impacts on mannitol production capability. We harness this information and manage to establish efficient mannitol production, without introducing foreign genes.

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