ATP Regeneration Research Articles

AGE, PHYSICAL ACTIVITY AND MITOCHONDRIAL FUNCTION IN THE STUDY OF MUSCLE MOBILITY AND AGING (SOMMA)

Abstract We studied whether lower physical activity (PA) in older adults may partly explain the age-related decline in muscle mitochondrial function in the SOMMA cohort of 879 men and women, aged 70+. PA was measured both subjectively (CHAMPS questionnaire) and objectively (wrist-worn ActiGraph Link) classifying PA levels as sedentary, light, or moderate to vigorous (MVPA, ≥3 METS). Muscle mitochondrial function was assessed ex vivo via respirometry in permeabilized fiber bundles and as in vivo by 31Phosphorus magnetic resonance spectroscopy (31P-MRS) of the maximal rate of muscle ATP regeneration (ATPMAX in the quadriceps after contraction. Of 809 with respirometry or 31P-MRS, mean age was 76.4, 58.3% were women, 12.7% Black, 85.2% White and 2.1% other race/ethnicity. Mean OXPHOS and ATPMAX values were 1.91 pmol/(s*mg) and 0.016 mM/sec lower per 5-year age increment. Men self-reported more MVPA than women, but women recorded more MVPA with actigraphy. For both self-reported and recorded activity, associations with maximal tissue oxidative phosphorylation (OXPHOS) as well as ATPMAX were stronger for MVPA than for sedentary or light activity. After adjustment for age, sex, and clinic site, OXPHOS was lower for each standard deviation) fewer minutes/week of MVPA [reported, SD=408 min/wk: -3.66 pmol/(s*mg) (95% CI -4.93, -2.39); recorded, SD=600 min/wk: -1.93 pmol/(s*mg) (95% CI -3.31, -0.55)]. Similar associations were noted for ATPMAX. MVPA attenuated the association of age with mitochondrial function by 31–50% for both OXPHOS and ATPMAX, suggesting that MVPA could offset the lower mitochondrial function with age or that lower mitochondrial function impedes MVPA at older ages.

OPA1-Exon4b modulates the migration and invasion of hepatocellular carcinoma cells by ATP regulation

Optic Atrophy 1 (OPA1), a mitochondrial inner protein, is involved in both mitochondrial fusion dynamic and cell apoptosis. OPA1 Exon4b (OPA1-Exon4b) was reported to be downregulated in hepatocellular carcinoma (HCC). However, the relationship between OPA1-Exon4b and HCC remains unclear. Here we demonstrated that OPA1-Exon4b is related with migration using genome-wide transcriptome profiling. OPA1-Exon4b overexpression suppresses the migration and invasion, and cellular ATP production in HCC cells. The inhibition of migration and invasion by OPA1-Exon4b overexpression could be rescued by ATP addition, showing that OPA1-Exon4b suppresses the migration and invasion by decreasing ATP. We further demonstrated OPA1 overexpression induces the enlargement of mtDNA nucleoids in HCC cells. Thus, our study demonstrated a key role of OPA1-Exon4b to regulate the migration and invasion in HCC, which could provide a new prospect for the clinical diagnosis and therapy of HCC.

GhHSP24.7 mediates mitochondrial protein acetylation to regulate stomatal conductance in response to abiotic stress in cotton

During seed germination, the cotton chaperone protein HSP24.7 regulates the release, from the mitochondrial electron transport chain, of reactive oxygen species (ROS), a stimulative signal regulating germination. The function of HSP24.7 during vegetative stages remains largely unknown. Here we propose that suppression of GhHSP24.7 in cotton seedlings increases tolerance to heat and drought stress. Elevation of GhHSP24.7 was found to be positively associated with endogenous levels of ROS. We identified a new client protein of GhHSP24.7, cotton lysine deacetylase (GhHDA14), which is involved in mitochondrial protein modification. Elevated levels of GhHSP24.7 suppressed deacetylase activity in mitochondria, leading to increased acetylation of mitochondrial proteins enriched in the subunit of F-type ATPase, V-type ATPase, and cytochrome C reductase, ultimately reducing leaf ATP content. Consequently, in combination with altered ROS content, GhHSP24.7 transgenic lines were unable to coordinate stomatal closure under stress. The regulation circuit composed of GhHSP24.7 and GhHDA14 represents a post-translation level mechanism in plant abiotic stress responses that integrates the regulation of ROS and ATP.

Evolutionary approach for improved proton pumping activity of heterologous rhodopsin expressed in Escherichia coli

A light-driven ATP regeneration system using rhodopsin has been utilized as a method to improve the production of useful substances by microorganisms. To enable the industrial use of this system, the proton pumping rate of rhodopsin needs to be enhanced. Nonetheless, a method for this enhancement has not been established. In this study, we attempted to develop an evolutionary engineering method to improve the proton-pumping activity of rhodopsins. We first introduced random mutations into delta-rhodopsin (dR) from Haloterrigena turkmenica using error-prone PCR to generate approximately 7000 Escherichia coli strains carrying the mutant dR genes. Rhodopsin-expressing E.coli with enhanced proton pumping activity have significantly increased survival rates in prolonged saline water. Considering this, we enriched the mutant E.coli cells with higher proton pumping rates by selecting populations able to survive starvation under 50μmolm-2s-1at 37°C. As a result, we successfully identified two strains, in which proton pumping activity was enhanced two-fold by heterologous expression in E.coli in comparison to wild-type strains. The combined approach of survival testing using saline water and evolutionary engineering methods used in this study will contribute greatly to the discovery of a novel rhodopsin with improved proton pumping activity. This will facilitate the utilization of rhodopsin in industrial applications.

Computational Model of the Effect of Mitochondrial Dysfunction on Excitation–Contraction Coupling in Skeletal Muscle

It has become well established that mitochondria not only regulate myoplasmic calcium in skeletal muscle, but also use that calcium to stimulate oxidative phosphorylation (OXPHOS). While experimental approaches have allowed for imaging of mitochondrial calcium and membrane potentials in isolated fibers, capturing the role of mitochondria and the impact of mitochondrial impairments on excitation–contraction coupling (ECC) remains difficult to explore in intact muscle. Computational models have been widely used to examine the structure and function of skeletal muscle contraction; however, models of ECC to date lack communication between the myoplasm and mitochondria for regulating calcium and ATP during sustained contractions. To address this, a mathematical model of mitochondrial calcium handling and OXPHOS was integrated into a physiological model of ECC incorporating action potential propagation, calcium handling between the sarcoplasmic reticulum (SR) and the myoplasm, and crossbridge cycling. The model was used to examine the protective role of mitochondria during repeated stimulation and the impact of mitochondrial dysfunction on ECC resulting from progressive OXPHOS inhibition. Pathological myoplasmic calcium accumulation occurred through distinct mechanisms in the model in the case of either electron transport chain, F1F0 ATP synthase, or adenine nucleotide transporter impairments. To investigate the effect of each impairment on force, a model of calcium-stimulated apoptosis was utilized to capture dysfunction-induced reductions in muscle mass, driving whole muscle force loss. The model presented in this study can be used to examine the role of mitochondria in the regulation of calcium, ATP, and force generation during voluntary contraction.

Dopamine activates astrocytes in prefrontal cortex via α1-adrenergic receptors.

SUMMARYThe prefrontal cortex (PFC) is a hub for cognitive control, and dopamine profoundly influences its functions. In other brain regions, astrocytes sense diverse neurotransmitters and neuromodulators and, in turn, orchestrate regulation of neuroactive substances. However, basic physiology of PFC astrocytes, including which neuromodulatory signals they respond to and how they contribute to PFC function, is unclear. Here, we characterize divergent signaling signatures in mouse astrocytes of the PFC and primary sensory cortex, which show differential responsiveness to locomotion. We find that PFC astrocytes express receptors for dopamine but are unresponsive through the Gs/Gi-cAMP pathway. Instead, fast calcium signals in PFC astrocytes are time locked to dopamine release and are mediated by α1-adrenergic receptors both ex vivo and in vivo. Further, we describe dopamine-triggered regulation of extracellular ATP at PFC astrocyte territories. Thus, we identify astrocytes as active players in dopaminergic signaling in the PFC, contributing to PFC function though neuromodulator receptor crosstalk.

Co-Immobilization of RizA Variants with Acetate Kinase for the Production of Bioactive Arginyl Dipeptides.

The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an l-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides.

Exploration and validation of Taraxacum mongolicum anti-cancer effect

Taraxacum mongolicum gained a lot of concern and was applied in 93 formulas in China due to its fame as a traditional Chinese medicine. The earliest recorded application of Taraxacum mongolicum was traced back to the Han dynasty. Generations of doctors boosted the usage and enriched the pharmacological mechanism. Clinical application of the Taraxacum mongolicum is flourishing as it treats multiple diseases. This study aims to explore the anti-cancer effect, retrieve the active ingredients and screen the key targets of Taraxacum mongolicum in cancer therapy. We collected and evaluated 10 key active compounds to investigate the anti-cancer effect via 69 significant targets and a variety of biological processes and pathways. Gene Ontology (GO) enrichment analysis uncovered targets associated with protein phosphorylation, cell proliferation and apoptotic processes via regulation of kinases, ATP and enzyme binding activities. Half of the top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were directly involved in cancer. Based on standard selection criteria, seven hub targets were obtained. These targets functioned through distinct patterns and pathways in realizing the anti-cancer effect. Molecular docking was conducted to validate the potential combination between compounds and hub targets to explore the pharmacological mechanism of key compounds in Taraxacum mongolicum against cancer. In summary, our findings indicate that the famous and widely used Chinese herb, Taraxacum mongolicum, shows good anti-cancer effect through its active compounds, targeted genes, and multiple involved biological processes. The results may provide a theoretical basis for subsequent experimental validation and drug development of Taraxacum mongolicum extract against cancer.

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation.

D-Allulose is an ultra-low calorie sweetener with broad market prospects. As an alternative to Izumoring, phosphorylation-dephosphorylation is a promising method for D-allulose synthesis due to its high conversion of substrate, which has been preliminarily attempted in enzymatic systems. However, in vitro phosphorylation-dephosphorylation requires polyphosphate as a phosphate donor and cannot completely deplete the substrate, which may limit its application in industry. Here, we designed and constructed a metabolic pathway in Escherichia coli for producing D-allulose from D-fructose via in vivo phosphorylation-dephosphorylation. PtsG-F and Mak were used to replace the fructose phosphotransferase systems (PTS) for uptake and phosphorylation of D-fructose to fructose-6-phosphate, which was then converted to D-allulose by AlsE and A6PP. The D-allulose titer reached 0.35 g/L and the yield was 0.16 g/g. Further block of the carbon flux into the Embden-Meyerhof-Parnas (EMP) pathway and introduction of an ATP regeneration system obviously improved fermentation performance, increasing the titer and yield of D-allulose to 1.23 g/L and 0.68 g/g, respectively. The E. coli cell factory cultured in M9 medium with glycerol as a carbon source achieved a D-allulose titer of ≈1.59 g/L and a yield of ≈0.72 g/g on D-fructose.

Homeostasis of extracellular ATP in uninfected RBCs from a Plasmodium falciparum culture and derived microparticles

Plasmodium falciparum, a dangerous parasitic agent causing malaria, invades human red blood cells (RBCs), causing hemolysis and microvascular obstruction. These and other pathological processes of malaria patients are due to metabolic and structural changes occurring in uninfected RBCs. In addition, infection activates the production of microparticles (MPs).ATP and byproducts are important extracellular ligands modulating purinergic signaling within the intravascular space.Here, we analyzed the contribution of uninfected RBCs and MPs to the regulation of extracellular ATP (eATP) of RBCs, which depends on the balance between ATP release by specific transporters and eATP hydrolysis by ectonucleotidases.RBCs were cultured with P. falciparum for 24–48 h prior to experiments, from which uninfected RBCs and MPs were purified. On-line luminometry was used to quantify the kinetics of ATP release. Luminometry, colorimetry and radioactive methods were used to assess the rate of eATP hydrolysis by ectonucleotidases. Rates of ATP release and eATP hydrolysis were also evaluated in MPs.Uninfected RBCs challenged by different stimuli displayed a strong and transient activation of ATP release, together with an elevated rate of eATP hydrolysis. MPs contained ATP in their lumen, which was released upon vesicle rupture, and were able to hydrolyze eATP.Results suggest that uninfected RBCs and MPs can act as important determinants of eATP regulation of RBCs during malaria.The comparison of eATP homeostasis in infected RBCs, ui-RBCs, and MPs allowed us to speculate on the impact of P. falciparum infection on intravascular purinergic signaling and the control of the vascular caliber by RBCs.

Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.

Enzymatic synthesis of high-titer nicotinamide mononucleotide with a new nicotinamide riboside kinase and an efficient ATP regeneration system

Backgroundβ-Nicotinamide mononucleotide (NMN) is the direct precursor of nicotinamide coenzymes such as NAD+ and NADP+, which are widely applied in industrial biocatalysis especially involving cofactor-dependent oxidoreductases. Moreover, NMN is a promising candidate for medical uses since it is considered to be beneficial for improving health of aged people who usually suffer from an insufficient level of NAD+. To date, various methods have been developed for the synthesis of NMN. Chemical phosphorylation of nicotinamide riboside (NR) to NMN depends on excessive phosphine oxychloride and delicate temperature control, while fermentation of NMN is limited by low product titers, making it unsuitable for industrial-scale NMN production. As a result, the more efficient synthesis process of NMN is still challenging.AimThis work attempted to construct an eco-friendly and cost-effective biocatalytic process for transforming the chemically synthesized NR into the highly value-added NMN.ResultsA new nicotinamide riboside kinase (Klm-NRK) was identified from Kluyveromyces marxianus. The specific activity of purified Klm-NRK was 7.9 U·mg–1 protein, ranking the highest record among the reported NRKs. The optimal pH of Klm-NRK was 7.0 in potassium phosphate buffer. The purified Klm-NRK retained a half activity after 7.29 h at 50 °C. The catalytic efficiencies (kcat/KM) toward ATP and nicotinamide riboside (NR) were 57.4 s−1·mM−1 and 84.4 s−1·mM−1, respectively. In the presence of an external ATP regeneration system (AcK/AcP), as much as 100 g·L–1 of NR could be completely phosphorylated to NMN in 8 h with Klm-NRK, achieving a molar isolation yield of 84.2% and a space–time yield of 281 g·L−1·day−1. These inspiring results indicated that Klm-NRK is a promising biocatalyst which provides an efficient approach for the bio-manufacturing of NMN in a high titer.Graphical

Autophagy and the Energy Status of Plant Cells

In plant cells the homeostatic control of energy balance involves the production and recycling of adenylates with macroergic bonds, ATP and ADP. The maintenance of anabolic processes requires the relative saturation of the adenylate pool with high energy phosphoanhydride bonds. The bulk of ATP synthesis is carried out both in mitochondria and in chloroplasts while optimal ATP levels within other cell compartments are maintained by adenylate kinases (AK). AK activity was recently found in cytosol, mitochondria, plastids and the nucleus. ATP synthesis in energy-producing organelles, as well as redistribution of nutrients among cellular compartments, requires fine-tuned regulation of ion homeostasis. A special role in energy metabolism is played by autophagy, a process of active degradation of unwanted and/or damaged cell components and macromolecules within the central lytic vacuole. So-called constitutive autophagy controls the quality of cellular contents under favorable conditions, i.e., when the cellular energy status is high. Energy depletion can lead to the activation of the pro-survival process of autophagic removal and utilization of damaged structures; the breakdown products are then used for ATP regeneration and de novo synthesis of macromolecules. Mitophagy and chlorophagy maintain the populations of healthy and functional energy-producing “stations”, preventing accumulation of defective mitochondria and chloroplasts as potential sources of dangerous reactive oxygen species. However, the increase of autophagic flux above a threshold level can lead to the execution of the vacuolar type of programmed cell death (PCD). In this case autophagy also contributes to preservation of energy through support of the outflow of nutrients from dying cells to healthy neighboring tissues. In plants, two central protein kinases, SnRK1 (Snf1-related protein kinase 1) and TOR (target of rapamycin), are responsible for the regulation of the metabolic switch between anabolic and catabolic pathways. TOR promotes the energy-demanding metabolic reactions in response to nutrient availability and simultaneously suppresses catabolism including autophagy. SnRK1, the antagonist of TOR, senses a decline in cellular energy supply and reacts by inducing autophagy through several independent pathways. Here, we provide an overview of the recent knowledge about the interplay between SnRK1 and TOR, autophagy and PCD in course of the regulation of energy balance in plants.

Improved methylation in E. coli via an efficient methyl supply system driven by betaine

Methylation reactions are involved in the biosynthesis of various natural molecules, in which S-adenosyl-L-methionine (SAM) acts as the principal biological methyl donor. The limited availability of SAM often affects the biosynthesis of methylated metabolites in cells, especially when heterologous SAM-mediated methyltransferases are employed. To solve this problem, a methyl supply system driven by betaine was developed in this study to enhance SAM availability in cells. A reconstructed methionine cycle was designed in E. coli using betaine as the methyl source by introducing betaine-homocysteine methyltransferase. Ferulic acid served as a model product was used to test the efficiency of methyl supply system. ATP is a co-factor for SAM biosynthesis and a pathway for ATP regeneration from adenosine was introduced to maintain the stability of the adenylate pool. After testing two different S-adenosyl-L-homocysteine (SAH) hydrolysis pathways, the optimized SAHase pathway was adopted for converting SAH back to homocysteine (Hcy). Thus, a methyl supply system was developed which increased SAM availability and therefore improved the titer and productivity of ferulic acid by 12.6-fold and 15.9-fold, respectively. The system was also applied successfully for other methyltransferase-catalyzed reactions. This work provides an efficient methyl supply system for enhanced production of methylated chemicals using betaine as the methyl source.

Polyphosphate polymerizing and depolymerizing activity of VTC4 protein in an arbuscular mycorrhizal fungus

ABSTRACT Arbuscular mycorrhizal (AM) fungi form symbiotic associations with land plants and supply soil minerals including phosphorus to their hosts. AM fungi accumulate polyphosphate (polyP), a linear phosphate polymer, in their mycelia, which functions in phosphorus storage and translocation. In the budding yeast Saccharomyces cerevisiae, it has been demonstrated that the vacuolar transporter chaperone 4 (VTC4) protein, a subunit of the VTC complex, is responsible for polyP synthesis. Here, we conducted a comprehensive survey of VTC proteins in eight AM fungal genomes by Blast analysis and characterized the biochemical properties of the Rhizophagus irregularis VTC4. The genomes of AM fungal species encode VTC1, VTC2, and VTC4. The recombinant protein RiVTC4* (RiVTC4183–474) containing the catalytic tunnel domain was expressed in E. coli cells and purified. RiVTC4* is capable of catalyzing polyP polymerization using ATP as a substrate. Pyrophosphate enhanced polyP-polymerizing activity >10-fold. RiVTC4* exhibited maximum activity at neutral pH and required divalent metal ions, preferentially Mn2+. In the presence of high concentrations of ADP, the reverse reaction (the regeneration of ATP from polyP) by RiVTC4* occurred. In the range of 0.2–5 mM ADP, polyP depolymerization by the reverse reaction was observed at the ATP/ADP ratio of less than 2–5. These results suggest that AM fungal VTC4 not only synthesizes polyP but also regenerates ATP from polyP and ADP, which has potential implications for the modulation of polyP and ATP levels in AM fungi.

Assimilatory deficit and energy regulation in young Handroanthus chrysotrichus plants under flooding stress.

Flooding negatively influences the growth and development of several plant species. Here, we show that the flood tolerance of young Handroanthus chrysotrichus plants involves growth deficit, carbon assimilation reductions, starch remobilization, and energy regulation. The effect of hypoxia was evaluated in a controlled experiment consisting of plants subjected to normoxia and water-logging, with later recovery. We measured morphological changes, gas exchange, photosynthetic pigments, soluble carbohydrates and starch contents, the activity of the enzymes alcohol dehydrogenase (ADH), and pyruvate decarboxylase (PDC), and ATP and ADP levels. While control plants showed normal appearance and growth, flooded plants exhibited a drastic decrease in growth, necrosis of some root tips, hypertrophic lenticels on the stems, and foliar chlorosis. Oxygen deprivation in root cells led to a significant decrease in stomatal conductance. The low Amax rates caused a decline in foliar soluble sugar content at 20days and a subsequent increase in the leaves and roots, coinciding with starch degradation at 40days. We also observed increases of 220.5% in ADH and 292% in PDC activities in the roots at 20 and 40days of flooding. The activation of anaerobic metabolism in stressed plants was an essential mechanism for ATP regulation in both tissues used to maintain a minimal metabolism to cope with hypoxia to the detriment of growth. The post-stress recovery process in H. chrysotrichus occurred slowly, with gas exchange gradually resumed and anaerobic metabolism and sugar content maintained to improve energy regulation.

Synthetic metabolism for in vitro acetone biosynthesis driven by ATP regeneration.

In vitro ketone production continues to be a challenge due to the biochemical features of the enzymes involved-even when some of them have been extensively characterized (e.g. thiolase from Clostridium acetobutylicum), the assembly of synthetic enzyme cascades still face significant limitations (including issues with protein aggregation and multimerization). Here, we designed and assembled a self-sustaining enzyme cascade with acetone yields close to the theoretical maximum using acetate as the only carbon input. The efficiency of this system was further boosted by coupling the enzymatic sequence to a two-step ATP-regeneration system that enables continuous, cost-effective acetone biosynthesis. Furthermore, simple methods were implemented for purifying the enzymes necessary for this synthetic metabolism, including a first-case example on the isolation of a heterotetrameric acetate:coenzyme A transferase by affinity chromatography.

Metabolomic Evaluation of the Central Metabolic Pathways of Mannosylerythritol Lipid Biosynthesis in Moesziomyces antarcticus T-34.

Moesziomyces antarcticus is a basidiomycetous yeast that produces mannosylerythritol lipids (MELs), which have potential applications as bio-based functional materials in various oleochemical industries, the cosmetics, toiletry, agriculture, and pharmaceutical industries. To better understand the MEL producer, we characterized the central metabolic pathways of M. antarcticus strain T-34 grown on glucose or olive oil via metabolomics. The relative fatty acid content was higher in the cells cultured in olive oil compared to glucose, while the acetyl-CoA content was lower in cells cultured in olive oil. The levels of the tricarboxylic acid cycle metabolites citrate/isocitrate, α-ketoglutarate, and succinate were lower in olive oil compared to glucose, while fumarate and malate levels exhibited the opposite pattern. Pyruvate was not detected in olive oil compared to glucose culture. The levels of glycerol, as well as trehalose, myo-inositol, threitol/erythritol, and mannitol/sorbitol, were higher in olive oil compared to glucose cultures. The ATP level was lower in olive oil compared to glucose culture, although the assimilation of fatty acids produced by digestion of olive oil should promote large amounts of ATP production. The possibility that ATP regeneration by respiratory chain complex promote oil utilization and MEL production in M. antarcticus T-34 was found based on the results of this metabolomic analysis.

Muscle Quality Revisited: Biopsy Energetics, MR Spectroscopy, and Muscle Power and Strength

With age, strength may decline faster than muscle mass pointing to a deterioration in muscle quality. Aspects of muscle quality and function are being measured in SOMMA; we hypothesized that in vitro and in vivo bioenergetics capacity of muscle would be related to muscle strength and power. Associations differed between men and women. In men (n=48, ATPMAX, 70, max OXPHOS and max ETS) but not women (n=68, ATPMAX, n=103, max OXPHOS and max ETS), muscle ATP regeneration by 31P MR spectroscopy was correlated with leg power (r = 0.27, p= 0.05). Energy production in tissue was similarly more strongly correlated with power in men than women, though not statistically significant. Correlations between the tissue measures and strength were also stronger in men than women. In ongoing follow-up, we will be able to determine what role that muscle tissue energetics plays in explaining the loss of strength and power with aging.

Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System

Compared with low-yield extraction from plants and environmentally unfriendly chemical synthesis, biocatalysis by asparagine synthetase (AS) for preparation of L-asparagine (L-Asn) has become a potential synthetic method. However, low enzyme activity of AS and high cost of ATP in this reaction restricts the large-scale preparation of L-Asn by biocatalysis. In this study, gene mining strategy was used to search for novel AS with high enzyme activity by expressing them in Escherichia coli BL21 (DE3) or Bacillus subtilis WB600. The obtained LsaAS-A was determined for its enzymatic properties and used for subsequent preparation of L-Asn. In order to reduce the use of ATP, a class III polyphosphate kinase 2 from Deinococcus ficus (DfiPPK2-Ⅲ) was cloned and expressed in E. coli BL21 (DE3), Rosetta (DE3) or RosettagamiB (DE3) for ATP regeneration. A coupling reaction system including whole cells expressing LsaAS-A and DfiPPK2-Ⅲ was constructed to prepare L-Asn from L-aspartic acid (L-Asp). Batch catalytic experiments showed that sodium hexametaphosphate (>60 mmol L−1) and L-Asp (>100 mmol L−1) could inhibit the synthesis of L-Asn. Under fed-batch mode, L-Asn yield reached 90.15% with twice feeding of sodium hexametaphosphate. A final concentration of 218.26 mmol L−1 L-Asn with a yield of 64.19% was obtained when L-Asp and sodium hexametaphosphate were fed simultaneously.