Deletion Of Lactate Dehydrogenase Research Articles

Preprint Highlight: Disruption of lactate metabolism in the peripheral nervous system leads to motor selective deficits.

While the role of the lactate shuttle from glia to neurons in the central nervous system has been associated with neurodegenerative disease, the role of this shuttle from Schwann cells to peripheral neurons during nerve degeneration remains unknown. Here the authors report that deletion of lactate dehydrogenase, the enzyme that catalyzes the interconversion between pyruvate and lactate in motor neurons or Schwann cells, causes progressive motor neuron axon degeneration. However, sensory neurons remain unaffected. The role of lactate dehydrogenase in motor neuron degeneration presents a novel target for rescuing metabolic dysfunction in motor-dominated neuropathies such as amyotrophic lateral sclerosis.

Engineering the cellulolytic extreme thermophile Caldicellulosiruptor bescii to reduce carboxylic acids to alcohols using plant biomass as the energy source.

Caldicellulosiruptor bescii is the most thermophilic cellulolytic organism yet identified (Topt 78°C). It grows on untreated plant biomass and has an established genetic system thereby making it a promising microbial platform for lignocellulose conversion to bio-products. Here, we investigated the ability of engineered C. bescii to generate alcohols from carboxylic acids. Expression of aldehyde ferredoxin oxidoreductase (aor from Pyrococcus furiosus) and alcohol dehydrogenase (adhA from Thermoanaerobacter sp. X514) enabled C. bescii to generate ethanol from crystalline cellulose and from biomass by reducing the acetate produced by fermentation. Deletion of lactate dehydrogenase in a strain expressing the AOR-Adh pathway increased ethanol production. Engineered strains also converted exogenously supplied organic acids (isobutyrate and n-caproate) to the corresponding alcohol (isobutanol and hexanol) using both crystalline cellulose and switchgrass as sources of reductant for alcohol production. This is the first instance of an acid to alcohol conversion pathway in a cellulolytic microbe.

Escherichia coli genome-scale metabolic gene knockout of lactate dehydrogenase (ldhA), increases succinate production from glycerol

Genome-scale metabolic model (GEM) of Escherichia coli has been published with applications in predicting metabolic engineering capabilities on different carbon sources and directing biological discovery. The use of glycerol as an alternative carbon source is economically viable in biorefinery. The use of GEM for predicting metabolic gene deletion of lactate dehydrogenase (ldhA) for increasing succinate production in Escherichia coli from glycerol carbon source remained largely unexplored. Here, I hypothesized that metabolic gene knockout of ldhA in E. coli from glycerol could increase succinate production. A proof-of-principle strain was constructed and designated as E. coli BMS5 (ΔldhA), by predicting increased succinate production in E. coli GEM and confirmed the predicted outcomes using wet cell experiments. The mutant GEM (ΔldhA) predicted 11% increase in succinate production from glycerol compared to its wild-type model (iAF1260), and the E. coli BMS5 (ΔldhA) showed 1.05 g/l and its corresponding wild-type produced .05 g/l (23-fold increase). The proof-of-principle strain constructed in this study confirmed the aforementioned hypothesis and further elucidated the fact that E. coli GEM can prospectively and effectively predict metabolic engineering interventions using glycerol as substrate and could serve as platform for new strain design strategies and biological discovery.

Lactate dehydrogenase activity drives hair follicle stem cell activation.

SummaryWhile normally dormant, Hair Follicle Stem Cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allow them to remain dormant and yet quickly respond to appropriate proliferative stimuli.

Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains

NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2-haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.

Engineered Bacillus subtilis 168 produces l-malate by heterologous biosynthesis pathway construction and lactate dehydrogenase deletion

In the present work, Bacillus subtilis was engineered to produce L-malate. Initially, the study revealed that the slight fumarase activity under anaerobic conditions is extremely favourable for L-malate one-step fermentation accumulation. Subsequently, an efficient heterologous biosynthesis pathway formed by Escherichia coli phosphoenolpyruvate carboxylase and Saccharomyces cerevisiae malate dehydrogenase was introduced into B. subtilis, which led to 6.04 ± 0.19 mM L-malate production. Finally, the L-malate production was increased 1.5-fold to 9.18 ± 0.22 mM by the deletion of lactate dehydrogenase. Under two-stage fermentation conditions, the engineered B. subtilis produced up to 15.65 ± 0.13 mM L-malate, which was 86.3 % higher than that under anaerobic fermentation conditions. Though the L-malate production by the recombinant was low, this is the first attempt to produce L-malate in engineered B. subtilis and paves the way for further improving L-malate production in B. subtilis.

Genetic modifications and introduction of heterologous pdc genes in Enterococcus faecalis for its use in production of bioethanol

Genetically-modified Enterococcus faecalis has a potential of survival and can be used in ethanolic fermentations. Fermentation profiles of E. faecalis JH2-2 were assessed using glucose and lactose as carbon sources. Deletion of lactate dehydrogenase (ldh) genes increased the ethanol production from 0.25 to 0.82 g/l, which was further increased to 0.96 g/l by the insertion of a pyruvate decarboxylase (pdc) gene (from Sarcina ventriculi or Clostridium acetobutylicum) in place ldh1. When grown on lactose, the pdcSv and pdcCa showed 13.6 and 17.6 U mg(-1) of pdc specific activity, respectively. Highest activity (47 U mg(-1)) and ethanol concentration (2.3 g/l) were obtained with pdcCa using an expression plasmid. Formate and acetate were also produced in high quantities. Transcriptional analysis showed that aldehyde alcohol dehydrogenase gene was upregulated up to 16-fold. Further optimizations are required for higher ethanol production.

Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production

2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.

Hydrogen production and metabolic flux analysis of metabolically engineered Escherichia coli strains

Abstract Escherichia coli can produce H2 from glucose via formate hydrogen lyase (FHL). In order to improve the H2 production rate and yield, metabolically engineered E. coli strains, which included pathway alterations in their H2 production and central carbon metabolism, were developed and characterized by batch experiments and metabolic flux analysis. Deletion of hycA, a negative regulator for FHL, resulted in twofold increase of FHL activity. Deletion of two uptake hydrogenases (1 (hya) and hydrogenase 2 (hyb)) increased H2 production yield from 1.20 mol/mol glucose to 1.48 mol/mol glucose. Deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdAB) further improved the H2 yield; 1.80 mol/mol glucose under high H2 pressure or 2.11 mol/mol glucose under reduced H2 pressure. Several batch experiments at varying concentrations of glucose (2.5–10 g/L) and yeast extract (0.3 or 3.0 g/L) were conducted for the strain containing all these genetic alternations, and their carbon and energy balances were analyzed. The metabolic flux analysis revealed that deletion of ldhA and frdAB directed most of the carbons from glucose to the glycolytic pathway leading to H2 production by FHL, not to the pentose phosphate pathway.

The effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in Escherichia coli.

In previous studies, we showed that cofactor manipulations can potentially be used as a tool in metabolic engineering. In this study, sugars similar to glucose, that can feed into glycolysis and pyruvate production, but with different oxidation states, were used as substrates. This provided a simple way of testing the effect of manipulating the NADH/NAD+ ratio or the availability of NADH on the metabolic patterns of Escherichia coli under anaerobic conditions and on the production of 1,2-propanediol (1,2-PD), which requires NADH for its synthesis. Production of 1,2-PD was achieved by overexpressing the two enzymes methylglyoxal synthase from Clostridium acetobutylicum and glycerol dehydrogenase from E. coli. In addition, the effect of eliminating a pathway competing for NADH by using a ldh(-) strain (without lactate dehydrogenase activity) on the production of 1,2-PD was investigated. The oxidation state of the carbon source significantly affected the yield of metabolites, such as ethanol, acetate and lactate. However, feeding a more reduced carbon source did not increase the yield of 1,2-PD. The production of 1,2-PD with glucose as the carbon source was improved by the incorporation of a ldh(-) mutation. The results of these experiments indicate that our current 1,2-PD production system is not limited by NADH, but rather by the pathways following the formation of methylglyoxal.

Evidence that L-(+)-lactate dehydrogenase deficiency is lethal in Streptococcus mutans

In order to construct an effector strain for the replacement therapy of dental caries, we wished to combine the properties of low-level acid production and high-level colonization potential in a strain of Streptococcus mutans. To this end, we made a deletion in the lactate dehydrogenase (LDH) gene cloned from the bacteriocin-producing S. mutans strain JH1000. However, we were unable to substitute the mutant for the wild-type allele by transformation with linear DNA fragments. The mutated gene, carried on a suicide vector, was shown by Southern analysis to integrate into the JH1000 chromosome to yield transformants carrying both the wild-type gene and mutated LDH gene. Three spontaneous self-recombinants of one heterodiploid strain were isolated by screening 1,500 colonies for a loss of the tetracycline resistance encoded by the gene used to mark the LDH deletion. In all three cases, Southern analysis showed that a loss of tetracycline resistance was accompanied by a loss of the mutated LDH gene, resulting in restoration of the wild-type genotype. However, screening the same number of colonies for self-recombinants that did not make lactic acid during anaerobic growth in Todd-Hewitt broth failed to identify clones in which the wild-type allele was lost. A second, simpler screening of more than 80,000 colonies grown aerobically on glucose tetrazolium medium to identify low-level-acid-producing colonies was also unsuccessful. These results are interpreted as indicating that LDH deficiency is lethal in S. mutans under the cultivation conditions used in these experiments. The physiological bases for this hypothesis are described.