Rebalancing microbial carbon distribution for L-threonine maximization using a thermal switch system

In metabolic engineering, unbalanced microbial carbon distribution has long blocked the further improvement in yield and productivity of high-volume natural metabolites. Current studies mostly focus on regulating desired biosynthetic pathways, whereas few strategies are available to maximize L-threonine efficiently. Here, we present a strategy to guarantee the supply of reduced cofactors and actualize L-threonine maximization by regulating cellular carbon distribution in central metabolic pathways. A thermal switch system was designed and applied to divide the whole fermentation process into two stages: growth and production. This system could rebalance carbon substrates between pyruvate and oxaloacetate by controlling the heterogenous expression of pyruvate carboxylase and oxaloacetate decarboxylation that responds to temperature. The system was tested in an L-threonine producer Escherichia coli TWF001, and the resulting strain TWF106/pFT24rp overproduced L-threonine from glucose with 111.78% molar yield. The thermal switch system was then employed to switch off the L-alanine synthesis pathway, resulting in the highest L-threonine yield of 124.03%, which exceeds the best reported yield (87.88%) and the maximum available theoretical value of L-threonine production (122.47%). This inducer-free genetic circuit design can be also developed for other biosynthetic pathways to increase product conversion rates and shorten production cycles.     

MPS: An algorithm and data base for metabolic pathway synthesis

Summary MPS is a software package for synthesizing metabolic pathways. Based on the concepts of artificial intelligence, it is designed to find the possible pathways to transform a given metabolite to a given target metabolite. MPS can be used to predict on a qualitative basis the effects of adding or deleting enzymatic activity from the cellular environment and to classify pathways with respect to cellular objectives.     

Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.     

Room-temperature synthesis of L-alanine using the alanine dehydrogenase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Alanine dehydrogenase from the hyperthermophilic archaeon Archaeoglobus fulgidus was used at room temperature for batch synthesis of L-alanine by the reductive amination of pyruvate. The reaction mixture included yeast formate dehydrogenase for regeneration of NADH with formate as electron donor. The synthesis of L-alanine at room temperature was accompanied by no detectable loss of alanine dehydrogenase activity over 139 h and > or =99% consumption of pyruvate. The total number of enzyme turnovers was 5.1 million. This work demonstrates the potential utility of novel hyperthermostable enzymes that can be both very active and highly stable at moderate temperature.     

Nonlinear dynamics of eucaryotic pyruvate dehydrogenase multienzyme complex: decarboxylation rate,oscillations, and multiplicity

Pyruvate conversion to acetyl-CoA by the pyruvate dehydrogenase (PDH) multienzyme complex is known as a key node in affecting the metabolic fluxes of animal cell culture. However, its possible role in causing possible nonlinear dynamic behavior such as oscillations and multiplicity of animal cells has received little attention. In this work, the kinetic and dynamic behavior of PDH of eucaryotic cells has been analyzed by using both in vitro and simplified in vivo models. With the in vitro model the overall reaction rate (nu(1)) of PDH is shown to be a nonlinear function of pyruvate concentration, leading to oscillations under certain conditions. All enzyme components affect nu(1) and the nonlinearity of PDH significantly, the protein X and the core enzyme dihydrolipoamide acyltransferase (E2) being mostly predominant. By considering the synthesis rates of pyruvate and PDH components the in vitro model is expanded to emulate in vivo conditions. Analysis using the in vivo model reveals another interesting kinetic feature of the PDH system, namely, multiple steady states. Depending on the pyruvate and enzyme levels or the operation mode, either a steady state with high pyruvate decarboxylation rate or a steady state with significantly lower decarboxylation rate can be achieved under otherwise identical conditions. In general, the more efficient steady state is associated with a lower pyruvate concentration. A possible time delay in the substrate supply and enzyme synthesis can also affect the steady state to be achieved and leads to oscillations under certain conditions. Overall, the predictions of multiplicity for the PDH system agree qualitatively well with recent experimental observations in animal cell cultures. The model analysis gives some hints for improving pyruvate metabolism in animal cell culture.     

Targeting cellular metabolism to improve cancer therapeutics

The metabolic properties of cancer cells diverge significantly from those of normal cells. Energy production in cancer cells is abnormally dependent on aerobic glycolysis. In addition to the dependency on glycolysis, cancer cells have other atypical metabolic characteristics such as increased fatty acid synthesis and increased rates of glutamine metabolism. Emerging evidence shows that many features characteristic to cancer cells, such as dysregulated Warburg-like glucose metabolism, fatty acid synthesis and glutaminolysis are linked to therapeutic resistance in cancer treatment. Therefore, targeting cellular metabolism may improve the response to cancer therapeutics and the combination of chemotherapeutic drugs with cellular metabolism inhibitors may represent a promising strategy to overcome drug resistance in cancer therapy. Recently, several review articles have summarized the anticancer targets in the metabolic pathways and metabolic inhibitor-induced cell death pathways, however, the dysregulated metabolism in therapeutic resistance, which is a highly clinical relevant area in cancer metabolism research, has not been specifically addressed. From this unique angle, this review article will discuss the relationship between dysregulated cellular metabolism and cancer drug resistance and how targeting of metabolic enzymes, such as glucose transporters, hexokinase, pyruvate kinase M2, lactate dehydrogenase A, pyruvate dehydrogenase kinase, fatty acid synthase and glutaminase can enhance the efficacy of common therapeutic agents or overcome resistance to chemotherapy or radiotherapy.     

Suppression of cytotoxic T-lymphocyte activation by pyruvate and L-alanine

The activation of cytotoxic T lymphocytes (CTL) in allogeneic mixed-lymphocyte cultures was found to be strongly inhibited if 1-3 X 10(-2) M L-alanine or the structurally and biochemically related substance pyruvate was present in the period from 7 to 19 or from 19 to 120 hr. The cytotoxic response was not inhibited when L-alanine or pyruvate was present during the first 7 hr of the culture period. L-Alanine produced also little or no suppression, if added on Day 3 of the culture. L-Lactate or D-alanine at similar concentrations was not suppressive during the entire culture period. The suppression by pyruvate and L-alanine was strongly reduced by 1 X 10(-4) M adenosine. Adenosine in combination with an interleukin-2 (IL-2)-containing EL-4-cell supernatant was even more effective. Pyruvate and alanine (1-3 X 10(-2) M) also inhibited the DNA synthesis in mixed-lymphocyte cultures on Day 5 by about 50%, but both substances had practically no effect on DNA synthesis in cultures that had been supplemented with an IL-2-containing EL-4 supernatant. They had also no effect on the IL-2-dependent proliferation of several T-cell clones or of concanavalin A-activated thymocytes. These relatively selective regulatory effects of pyruvate and L-alanine may be useful for the analysis of the biochemical pathways during lymphocyte activation and/or for a selective manipulation of the immune response.     

Regulation of the G1 leads to S phase transition in chick embryo fibroblasts with alpha-keto acids and L-alanine.

Temporal inhibition of protein synthesis with cycloheximide prevents subsequent insulin, but not serum-stimulated DNA synthesis in G1-arrested chick embryo fibroblasts (CEF). The inhibition is measured by the incorporation of 3H-thymidine into acid insoluble material and confirmed by chemical estimate of the DNA content of inhibited and uninhibited cells. Cycloheximide treatment is without effect if the cell cultures are maintained at 4 degrees C while exposed to the drug. Several alpha-keto acids (pyruvate, oxaloacetate, alpha-ketobutyrate) at 0.5-1 mM concentrations restore DNA synthesis in previously inhibited cells when combined with insulin. L-alanine (D-alanine is inert) is even more effective than the keto acids in stimulating DNA synthesis after cycloheximide treatment. Glucose transport was unaffected by cycloheximide treatment while lactate levels in medium from inhibited, insulin-stimulated CEF were reduced 70% compared to uninhibited counterparts. We speculate that cycloheximide treatment may lead to the decay of a glycolytic enzyme which compromises the ability of inhibited cells to synthesize pyruvate from glucose, and thus induces an exogenous requirement for alpha-keto acid or L-alanine. A serum component(s) with a molecular weight of about 100 permitted insulin-stimulated DNA synthesis in inhibited cells.     

Purification and properties of L-alanine dehydrogenase of the phototrophic bacterium Rhodobacter capsulatus E1F1.

In the phototrophic nonsulfur bacterium Rhodobacter capsulatus E1F1, L-alanine dehydrogenase aminating activity functions as an alternative route for ammonia assimilation when glutamine synthetase is inactivated. L-Alanine dehydrogenase deaminating activity participates in the supply of organic carbon to cells growing on L-alanine as the sole carbon source. L-Alanine dehydrogenase is induced in cells growing on pyruvate plus nitrate, pyruvate plus ammonia, or L-alanine under both light-anaerobic and dark-heterotrophic conditions. The enzyme has been purified to electrophoretic and immunological homogeneity by using affinity chromatography with Red-120 agarose. The native enzyme was an oligomeric protein of 246 kilodaltons (kDa) which consisted of six identical subunits of 42 kDa each, had a Stokes' radius of 5.8 nm, an s20.w of 10.1 S, a D20,w of 4.25 x 10(-11) m2 s-1, and a frictional quotient of 1.35. The aminating activity was absolutely specific for NADPH, whereas deaminating activity was strictly NAD dependent, with apparent Kms of 0.25 (NADPH), 0.15 (NAD+), 1.25 (L-alanine), 0.13 (pyruvate), and 16 (ammonium) mM. The enzyme was inhibited in vitro by pyruvate or L-alanine and had two sulfhydryl groups per subunit which were essential for both aminating and deaminating activities.     

L-丙氨酸对海南粗榧悬浮细胞合成三尖杉酯类碱的影响

该研究在海南粗榧悬浮细胞培养的不同阶段(5、10、15、20 d),分别添加不同剂量的L-丙氨酸(10、30、50、100 mg·L~(-1)),测定细胞生长、细胞活力及产物含量,确定L-丙氨酸最佳的添加时间及添加剂量。结果表明:添加L-丙氨酸对细胞生长和细胞活力均有抑制作用;在海南粗榧悬浮培养第15天、添加30 mg·L~(-1)L-丙氨酸时,产物含量最高(4.853 6 mg·L~(-1)),是对照(2.853 8 mg·L~(-1))的1.7倍。同时,为了探讨添加L-丙氨酸对海南粗榧悬浮细胞糖代谢的影响,对培养基糖耗程度、细胞内糖酵解途径(glycolytic pathway,EMP途径)关键酶丙酮酸激酶(Pyruvate kinase,PK)活力、磷酸戊糖途径(hexose monophosphate pathway,HMP途径)关键酶6-磷酸葡萄糖脱氢酶(glucose 6-phosphate dehydrogenase,G6PDH)活力进行了测定,结果显示添加L-丙氨酸后,植物细胞培养液中总耗糖速度与对照相比无明显差异,丙酮酸激酶(PK)活力与对照(25.37 U·g~(-1))相比下降了29.10%,G6DPH活力是对照组(53.49 U·g~(-1))的1.33倍。以上结果说明,糖代谢途径中碳通量在一定程度上由EMP途径转向了HMP途径,三尖杉酯类碱合成的前体物PEP积累,E4P合成量增加,均有利于产物三尖杉酯类碱含量的增加。     

L-alanine: 4,5-dioxovalerate aminotransferase from Pseudomonas riboflavina: purification and inactivation by methylglyoxal

L-Alanine:4,5-dioxovalerate aminotransferase, which catalyzes transamination between L-alanine and 4,5-dioxovalerate to yield delta-aminolevulinate and pyruvate, has been purified from Pseudomonas riboflavina IFO 3140. The enzyme had a molecular weight of 190,000 and consisted of four identical subunits. It was crystallized as pale yellow needles. The enzyme used L-alanine (relative activity 100), beta-alanine (39), and L-ornithine (14) as amino donors. gamma-aminobutyrate (55) and epsilon-aminocaproate (34) were also effective as amino donors. The reaction proceeded according to a ping-pong mechanism and the Km values for L-alanine and 4,5-dioxovalerate were 1.7 and 0.75 mM, respectively. The activity of the enzyme is strongly inhibited by pyruvate, hemin, and methylglyoxal. Methylglyoxal interacted with the enzyme and brought about a complete inactivation.     

Purification and properties of L-alanine aminotransferase from Chlamydomonas reinhardtii.

An enzyme which catalyzes the transamination of L-alanine with 2-oxoglutarate has been purified 157-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. The enzyme showed maximal activity at pH 7.3 and 50 degrees C, has an apparent molecular mass of 105 kDa as estimated by gel filtration, and consists of two identical subunits of 45 kDa each as deduced from PAGE/SDS studies. A stoichiometry of two moles pyridoxal 5-phosphate/mole enzyme was calculated. The enzyme has an isoelectric point of 8.3 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-alanine. Pyridoxal 5-phosphate protected activity against heat inactivation and, to a minor extent, L-alanine and 2-oxoglutarate, but not L-glutamate. Spectral data and activity inhibition and protection studies strongly support the involvement of pyridoxal 5-phosphate in enzyme catalysis through a Schiff's base formation. The purified enzyme was able to transaminate only L-alanine and L-glutamate with glyoxylate out of ten amino acids tested. L-Alanine aminotransferase exhibited hyperbolic kinetic for 2-oxoglutarate, pyruvate, and L-glutamate, and nonhyperbolic behaviour for L-alanine. Apparent Km values were 0.054 mM for 2-oxoglutarate, 0.52 for L-glutamate, 0.24 mM for pyruvate, and 2.7 mM for L-alanine. Transamination of L-alanine in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

End products of glucose and glutamine metabolism by L929 cells

Products of glucose and glutamine metabolism by L929 cells were detected and quantitated by gas chromatography and mass spectrometry of the oxime-trimethylsilyl derivatives. This method allowed detection and identification of all major carboxylic and amino acids produced in the system. Although lactic acid was expected to be the major product, alanine, citric, glutamic, aspartic, and pyruvic acids were also released into the culture medium at significant rates. Incorporation of labeled carbon from D-[U-13C]glucose showed that the alanine, lactic, and pyruvic acids were derived from glucose as was one-third of the citric acid carbon. The rate of glucose utilization for production of these end products was 29-fold greater than the rate of glucose oxidation to CO2, and calculated ATP production from alanine and pyruvate synthesis exceeded that from lactate synthesis by nearly 2-fold. Utilization of glutamine for synthesis of aspartic, glutamic, and citric acids also exceeded the rate of glutamine oxidation, thereby making end-product synthesis from glucose and glutamine the dominant cellular metabolic activity. In the absence of glucose, synthesis and intracellular levels of aspartic and glutamic acids increased, whereas synthesis and cell content of the other acids decreased markedly. This response is consistent with the metabolic pattern proposed by Moreadith and Lehninger (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221) in which much of the glutamine used by these cells is converted to aspartate in the absence of a pyruvate source and to aspartate or citrate in the presence of pyruvate.     

13C-NMR analysis of Aspergillus mutants disturbed in pyruvate metabolism

The metabolic consequences of two defects in pyruvate metabolism of the hyphal fungus Aspergillus nidulans have been investigated by natural abundance 13C-NMR spectroscopy. A pyruvate dehydrogenase complex (pdh) mutant, grown on acetate, accumulates alanine upon starvation which is derived from mannitol reserves. The L-alanine level increases further upon incubation with the non-permissive substrate D-glucose. L-Glutamate is absent from these spectra as it is required both for the transamination of pyruvate and as a reaction on an impaired energy metabolism in such a pdh-deficient strain. A pyruvate carboxylase (pyc) mutant, grown upon acetate, only starts to accumulate alanine after a long incubation period with D-glucose, due to the long-lasting presence of phosphoenolpyruvate carboxykinase and malic enzyme, which are both induced by growth on acetate. When this strain is grown on D-fructose and L-glutamate, alanine also accumulates within 3 h upon transfer to D-glucose.     

Transamination pathways influencing L-glutamine and L-glutamate oxidation by rat enterocyte mitochondria and the subcellular localization of L-alanine aminotransferase and L-aspartate aminotransferase

Using analytical subcellular fractionation techniques, 12% of the total L-alanine aminotransferase activity and 26% of the total L-aspartate aminotransferase activity was localized in enterocyte mitochondria. Alanine and aspartate were products from the oxidation of glutamine and glutamate by enterocyte mitochondria. At low concentrations, malate stimulated aspartate synthesis but was inhibitory at higher concentrations. The malate inhibition of aspartate synthesis, which increased in the presence of pyruvate, was accompanied by an increase in alanine synthesis. With glutamine as substrate in the presence of pyruvate and malate, alanine synthesis was increased by 127% on addition of purified L-alanine aminotransferase, in spite of large amounts of glutamate generated. It was concluded that when pyruvate is available the important route for glutamine or glutamate oxidation by transamination was via L-alanine:2-oxoglutarate aminotransferase and not via L-aspartate:2-oxoglutarate aminotransferase. Results suggested that mitochondria may account for 50% of alanine production from glutamine in the enterocyte despite the relatively low activity of L-alanine aminotransferase therein.     

Metabolic function of Corynebacterium glutamicum aminotransferases AlaT and AvtA and impact on L-valine production

Aminotransferases (ATs) interacting with L-alanine are the least studied bacterial ATs. Whereas AlaT converts pyruvate to L-alanine in a glutamate-dependent reaction, AvtA is able to convert pyruvate to L-alanine in an L-valine-dependent manner. We show here that the wild type of Corynebacterium glutamicum with a deletion of either of the corresponding genes does not exhibit an explicit growth deficiency. However, a double mutant was auxotrophic for L-alanine, showing that both ATs can provide L-alanine and that they are the only ATs involved. Kinetic studies with isolated enzymes demonstrate that the catalytic efficiency, k(cat)/K(m), of AlaT is higher than 1 order of magnitude in the direction of L-alanine formation (3.5 x 10(4) M(-1) s(-1)), but no preference was apparent for AvtA, suggesting that AlaT is the principal L-alanine-supplying enzyme. This is in line with the cytosolic L-alanine concentration, which is reduced in the exponential growth phase from 95 mM to 18 mM by a deletion of alaT, whereas avtA deletion decreases the L-alanine concentration only to 76 mM. The combined data show that the presence of both ATs has subtle but obvious consequences on balancing intracellular amino acid pools in the wild type. The consequences are more obvious in an L-valine production strain where a high intracellular drain-off of the L-alanine precursor pyruvate prevails. We therefore used deletion of alaT to successfully reduce the contaminating L-alanine in extracellular accumulated L-valine by 80%.     

Modeling of hollow-fiber capillary reactor for the production of L-Alanine with coenzyme regeneration

Continuous production of L-alanine with conjugated enzyme systems of alanine dehydrogenase (AlaDH) and lactate dehydrogenase (LDH) or alcohol dehydrogenase (ADH) was carried out with NAD regeneration in an ultrafiltration hollow-fiber capillary reactor (HFCR) which was proposed as a test bioreactor with very small scale. In the AlaDH/LDH system, pyruvate is the intermediate product for L-alanine so that an optimal point existed in pyruvate concentration for the production rate of L-alanine. NAD cycling number of 4850 and L-alanine productivity of 61.7 mmol/L h were obtained at the best condition. In the AlaDH/ADH system, however, the substrate inhibition in the AlaDH reaction by pyruvate should be considered and the best results of NAD cycling number and (L)-alanine productivity were 2700 and 13.5 mmol/L h, respectively. In consideration of concentration distribution and mixing in the axial direction on an HFCR, performance of the reactor was theoretically analyzed with a multistage stirred tank reactor model combined with the kinetic model based on all the elementary reactions involved. Although quantitative discrepancy existed in some cases, the present theoretical model could explain experimental results and is expected to be generally applicable to standard hollow fiber reactors.     

乙醇合成途径的计算机重构

目的:用计算机重构乙醇合成途径,为合成生物燃料乙醇提供理论依据。方法:利用KEGG反应、化合物数据提取反应等式,过滤掉42个通用代谢物参与的反应,然后利用剩下的反应构建反应矩阵;利用广度优先搜索算法在反应矩阵中搜索生成乙醇的代谢途径。结果:计算机重构了23 108条乙醇合成途径,以大肠杆菌作为产乙醇基因工程菌为例,通过限制改构菌整合的关键酶数目,分别得到了78条以酒精O-乙酰基转移酶为关键酶的乙醇合成通路和89条以丙酮酸脱羧酶和乙醇脱氢酶为关键酶的乙醇合成通路,并构建了相应的乙醇合成网络图,标注每个反应的酶及编码该酶的基因。结论:通过计算机方法重构了多种乙醇合成途径,可以为利用微生物工业化生产乙醇提供理论依据。     

Rhythmic regulation of retinal melatonin: Metabolic pathways,neurochemical mechanisms,and the ocular circadian clock

1. Current knowledge of the mechanisms of circadian and photic regulation of retinal melatonin in vertebrates is reviewed, with a focus on recent progress and unanswered questions. 2. Retinal melatonin synthesis is elevated at night, as a result of acute suppression by light and rhythmic regulation by a circadian oscillator, or clock, which has been localized to the eye in some species. 3. The development of suitable in vitro retinal preparations, particularly the eyecup from the African clawed frog, Xenopus laevis, has enabled identification of neural, cellular, and molecular mechanisms of retinal melatonin regulation. 4. Recent findings indicate that retinal melatonin levels can be regulated at multiple points in indoleamine metabolic pathways, including synthesis and availability of the precursor serotonin, activity of the enzyme serotonin N-acetyltransferase, and a novel pathway for degradation of melatonin within the retina. 5. Retinal dopamine appears to act through D2 receptors as a signal for light in this system, both in the acute suppression of melatonin synthesis and in the entrainment of the ocular circadian oscillator. 6. A recently developed in vitro system that enables high-resolution measurement of retinal circadian rhythmicity for mechanistic analysis of the circadian oscillator is described, along with preliminary results that suggest its potential for elucidating general circadian mechanisms. 7. A model describing hypothesized interactions among circadian, neurochemical, and cellular mechanisms in regulation of retinal melatonin is presented.