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 Aerobic Fermentation of d-Glucose by an Evolved Cytochrome Oxidase-Deficient Escherichia coli Strain

2008 ◽  
Vol 74 (24) ◽  
pp. 7561-7569 ◽  
Author(s):  
Vasiliy A. Portnoy ◽  
Markus J. Herrgård ◽  
Bernhard Ø. Palsson
Keyword(s):  
Escherichia Coli ◽  
Oxygen Uptake ◽  
Growth Conditions ◽  
Aerobic Growth ◽  
Acid Fermentation ◽  
Mixed Acid Fermentation ◽  
E Coli ◽  
Knockout Strain ◽  
Mixed Acid ◽  
Cytochrome Oxidases

ABSTRACT Fermentation of glucose to d-lactic acid under aerobic growth conditions by an evolved Escherichia coli mutant deficient in three terminal oxidases is reported in this work. Cytochrome oxidases (cydAB, cyoABCD, and cbdAB) were removed from the E. coli K12 MG1655 genome, resulting in the ECOM3 (E. coli cytochrome oxidase mutant) strain. Removal of cytochrome oxidases reduced the oxygen uptake rate of the knockout strain by nearly 85%. Moreover, the knockout strain was initially incapable of growing on M9 minimal medium. After the ECOM3 strain was subjected to adaptive evolution on glucose M9 medium for 60 days, a growth rate equivalent to that of anaerobic wild-type E. coli was achieved. Our findings demonstrate that three independently adaptively evolved ECOM3 populations acquired different phenotypes: one produced lactate as a sole fermentation product, while the other two strains exhibited a mixed-acid fermentation under oxic growth conditions with lactate remaining as the major product. The homofermenting strain showed a d-lactate yield of 0.8 g/g from glucose. Gene expression and in silico model-based analyses were employed to identify perturbed pathways and explain phenotypic behavior. Significant upregulation of ygiN and sodAB explains the remaining oxygen uptake that was observed in evolved ECOM3 strains. E. coli strains produced in this study showed the ability to produce lactate as a fermentation product from glucose and to undergo mixed-acid fermentation during aerobic growth.

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 Increasing Acidification of Nonreplicating Lactococcus lactis ΔthyA Mutants by Incorporating ATPase Activity

2002 ◽  
Vol 68 (11) ◽  
pp. 5249-5257 ◽  
Author(s):  
Martin B. Pedersen ◽  
Brian J. Koebmann ◽  
Peter R. Jensen ◽  
Dan Nilsson
Keyword(s):  
Lactic Acid ◽  
Dna Replication ◽  
Atpase Activity ◽  
Lactococcus Lactis ◽  
Growth Medium ◽  
Thymidylate Synthase ◽  
De Novo ◽  
Sugar Transport ◽  
Appl Environ ◽  
Atp Supply

ABSTRACT Lactococcus lactis MBP71 ΔthyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis.

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 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 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 The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis

2004 ◽  
Vol 70 (9) ◽  
pp. 5477-5484 ◽  
Author(s):  
Johan Palmfeldt ◽  
Marco Paese ◽  
Bärbel Hahn-Hägerdal ◽  
Ed W. J. van Niel
Keyword(s):  
Growth Rate ◽  
Alcohol Dehydrogenase ◽  
Lactococcus Lactis ◽  
Product Formation ◽  
Glycolytic Flux ◽  
Resting Cells ◽  
Acid Product ◽  
Mixed Acid ◽  
Intracellular Concentrations ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of Pi was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.

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