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 Analysis of the Proteome of Intracellular Shigella flexneri Reveals Pathways Important for Intracellular Growth

2013 ◽  
Vol 81 (12) ◽  
pp. 4635-4648 ◽  
Author(s):  
Rembert Pieper ◽  
C. R. Fisher ◽  
Moo-Jin Suh ◽  
S.-T. Huang ◽  
P. Parmar ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Intracellular Bacteria ◽  
Intracellular Growth ◽  
Iron Starvation ◽  
Acid Fermentation ◽  
Nadph Regeneration ◽  
Content Type ◽  
Mixed Acid Fermentation ◽  
Mixed Acid

ABSTRACTGlobal proteomic analysis was performed withShigella flexneristrain 2457T in association with three distinct growth environments:S. flexnerigrowing in broth (in vitro),S. flexnerigrowing within epithelial cell cytoplasm (intracellular), andS. flexnerithat were cultured with, but did not invade, Henle cells (extracellular). Compared toin vitroand extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread ofS. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in theS. flexneritransition from the extra- to the intracellular milieu.

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.

scholarly journals Increasing the Heme-Dependent Respiratory Efficiency of Lactococcus lactis by Inhibition of Lactate Dehydrogenase

2012 ◽  
Vol 79 (1) ◽  
pp. 376-380 ◽  
Author(s):  
Stefania Arioli ◽  
Daniele Zambelli ◽  
Simone Guglielmetti ◽  
Ivano De Noni ◽  
Martin B. Pedersen ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Lactococcus Lactis ◽  
Biomass Production ◽  
Growth Efficiency ◽  
Industrial Processes ◽  
Aerobic Respiration ◽  
Lactate Dehydrogenase Activity ◽  
Content Type ◽  
Respiratory Efficiency

ABSTRACTThe discovery of heme-induced respiration inLactococcus lactishas radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity ofL. lactisduring growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism.

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 Genomic Features of a Bumble Bee Symbiont Reflect Its Host Environment

2014 ◽  
Vol 80 (13) ◽  
pp. 3793-3803 ◽  
Author(s):  
Vincent G. Martinson ◽  
Tanja Magoc ◽  
Hauke Koch ◽  
Steven L. Salzberg ◽  
Nancy A. Moran
Keyword(s):  
Nadh Dehydrogenase ◽  
Tca Cycle ◽  
Nitrate Respiration ◽  
Bumble Bee ◽  
Metabolic Reconstruction ◽  
Low Oxygen ◽  
Acid Fermentation ◽  
Content Type ◽  
Mixed Acid ◽  
Complex Interactions

ABSTRACTHere, we report the genome of one gammaproteobacterial member of the gut microbiota, for which we propose the name “CandidatusSchmidhempelia bombi,” that was inadvertently sequenced alongside the genome of its host, the bumble bee,Bombus impatiens. This symbiont is a member of the recently described bacterial orderOrbales, which has been collected from the guts of diverse insect species; however, “Ca. Schmidhempelia” has been identified exclusively with bumble bees. Metabolic reconstruction reveals that “Ca. Schmidhempelia” lacks many genes for a functioning NADH dehydrogenase I, all genes for the high-oxygen cytochromeo, and most genes in the tricarboxylic acid (TCA) cycle. “Ca. Schmidhempelia” has retained NADH dehydrogenase II, the low-oxygen specific cytochromebd, anaerobic nitrate respiration, mixed-acid fermentation pathways, and citrate fermentation, which may be important for survival in low-oxygen or anaerobic environments found in the bee hindgut. Additionally, a type 6 secretion system, a Flp pilus, and many antibiotic/multidrug transporters suggest complex interactions with its host and other gut commensals or pathogens. This genome has signatures of reduction (2.0 megabase pairs) and rearrangement, as previously observed for genomes of host-associated bacteria. A survey of wild and laboratoryB. impatiensrevealed that “Ca. Schmidhempelia” is present in 90% of individuals and, therefore, may provide benefits to its host.

scholarly journals The Pentose Moiety of Adenosine and Inosine Is an Important Energy Source for the Fermented-Meat Starter Culture Lactobacillus sakei CTC 494

2011 ◽  
Vol 77 (18) ◽  
pp. 6539-6550 ◽  
Author(s):  
T. Rimaux ◽  
G. Vrancken ◽  
B. Vuylsteke ◽  
L. De Vuyst ◽  
F. Leroy
Keyword(s):  
Starter Culture ◽  
Energy Sources ◽  
Lactobacillus Sakei ◽  
Acid Fermentation ◽  
Content Type ◽  
Mixed Acid ◽  
Arginine Catabolism ◽  
Adenosine Deaminase Activity ◽  
End Products ◽  
Ph Range

ABSTRACTThe genome sequence ofLactobacillus sakei23K has revealed that the speciesL. sakeiharbors several genes involved in the catabolism of energy sources other than glucose in meat, such as glycerol, arginine, and nucleosides. In this study, a screening of 15L. sakeistrains revealed that arginine, inosine, and adenosine could be used as energy sources by all strains. However, no glycerol catabolism occurred in any of theL. sakeistrains tested. A detailed kinetic analysis of inosine and adenosine catabolism in the presence of arginine byL. sakeiCTC 494, a fermented-meat starter culture, was performed. It showed that nucleoside catabolism occurred as a mixed-acid fermentation in a pH range (pH 5.0 to 6.5) relevant for sausage fermentation. This resulted in the production of a mixture of acetic acid, formic acid, and ethanol from ribose, while the nucleobase (hypoxanthine and adenine in the case of fermentations with inosine and adenosine, respectively) was excreted into the medium stoichiometrically. This indicates that adenosine deaminase activity did not take place. The ratios of the different fermentation end products did not vary with environmental pH, except for the fermentation with inosine at pH 5.0, where lactic acid was produced too. In all cases, no other carbon-containing metabolites were found; carbon dioxide was derived only from arginine catabolism. Arginine was cometabolized in all cases and resulted in the production of both citrulline and ornithine. Based on these results, a pathway for inosine and adenosine catabolism inL. sakeiCTC 494 was presented, whereby both nucleosides are directly converted into their nucleobase and ribose, the latter entering the heterolactate pathway. The present study revealed that the pentose moiety (ribose) of the nucleosides inosine and adenosine is an effective fermentable substrate forL. sakei. Thus, the ability to use these energy sources offers a competitive advantage for this species in a meat environment.

scholarly journals Molecular and Metabolic Adaptations of Lactococcus lactis at Near-Zero Growth Rates

2014 ◽  
Vol 81 (1) ◽  
pp. 320-331 ◽  
Author(s):  
Onur Ercan ◽  
Michiel Wels ◽  
Eddy J. Smid ◽  
Michiel Kleerebezem
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Growth Rates ◽  
Low Carbon ◽  
Content Type ◽  
Growth State ◽  
Mixed Acid ◽  
Metabolic Adaptations ◽  
Zero Growth ◽  
Growth Adaptation

ABSTRACTThis paper describes the molecular and metabolic adaptations ofLactococcus lactisduring the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes. During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY inL. lactisKF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero-growth cultures. The present integrated transcriptome and metabolite (amino acids and previously reported fermentation end products) study provides molecular understanding of the adaptation ofL. lactisto conditions supporting low growth rates and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates toward gene regulation patterns involved in zero-growth adaptation.

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 Metabolic Engineering ofEscherichia colifor Poly(3-hydroxybutyrate) Production under Microaerobic Condition

2015 ◽  
Vol 2015 ◽  
pp. 1-5 ◽  
Author(s):  
Xiao-Xing Wei ◽  
Wei-Tao Zheng ◽  
Xue Hou ◽  
Jian Liang ◽  
Zheng-Jun Li
Keyword(s):  
Escherichia Coli ◽  
Carbon Metabolism ◽  
Batch Fermentation ◽  
Dry Weight ◽  
Microaerobic Condition ◽  
Acid Fermentation ◽  
Pyruvate Formate Lyase ◽  
E Coli ◽  
Mixed Acid ◽  
Theoretical Yield

The alcohol dehydrogenase promoterPadhEand mixed acid fermentation pathway deficient mutants ofEscherichia coliwere employed to produce poly(3-hydroxybutyrate) (P3HB) under microaerobic condition. TheE. colimutant withackA-pta, poxB, ldhA, andadhEdeletions accumulated 0.67 g/L P3HB, up to 78.84% of cell dry weight in tube cultivation. The deletion of pyruvate formate-lyase genepflBdrastically decreased P3HB production and P3HB content to 0.09 g/L and 24.44%, respectively. OverexpressingpflBvia the plasmid in its knocked out mutant restored cell growth and P3HB accumulation, indicating the importance of the pyruvate formate-lyase in microaerobic carbon metabolism. The engineeredE. coliBWapld (pWYC09) produced 5.00 g/L P3HB from 16.50 g/L glucose in 24 h batch fermentation, and P3HB production yield from glucose was 0.30 g/g, which reached up to 63% of maximal theoretical yield.

Sign in / Sign up

Export Citation Format

Share Document