微生物细胞工厂的代谢调控

代谢调控是构建微生物细胞工厂的重要技术手段.随着合成生物学技术的不断突破,挖掘和人工设计的高质量调控元件大幅度提升了对细胞代谢网络的改造能力;代谢调控研究也已从单基因的静态调控发展到系统水平上的智能精确动态调控.文中简要综述了近30年来代谢途径表达调控技术在代谢工程领域的研究进展.     

Cell surface engineering and application in cell delivery to heart diseases

Cell-based therapy has expanded its influence in cancer immunotherapy, regenerative medicine, and tissue engineering. Due to their secretory functions, differentiation capabilities, specific homing effects through chemotaxis, distinctive therapeutic potentials, and ex vivo expandability, cells have become an attractive reagent for advanced therapeutic strategies. Therefore, the ability to modify cells and manipulate their functions according to intended therapeutic designs has been the central scientific interest in the field of biomedical research. Many innovative methods have been developed with genetic modification of cells being the most advanced cell surface engineering technique. Although genetic modification is a powerful tool, it has a limited applicability due to the permanent modifications made on cells. Alternatively, many endeavors have been made to develop surface engineering techniques that can circumvent the limitations of genetic modification. In this review, current methods of non-genetic cell surface modification, including chemical conjugations, polymeric encapsulation, hydrophobic insertion, enzymatic and metabolic addition, will be introduced. Moreover, cell surface engineering plausible for cardiac remodeling and the future prospective will be discussed at the end.     

The effect of sustained compression on oxygen metabolic transport in the intervertebral disc decreases with degenerative changes

Intervertebral disc metabolic transport is essential to the functional spine and provides the cells with the nutrients necessary to tissue maintenance. Disc degenerative changes alter the tissue mechanics, but interactions between mechanical loading and disc transport are still an open issue. A poromechanical finite element model of the human disc was coupled with oxygen and lactate transport models. Deformations and fluid flow were linked to transport predictions by including strain-dependent diffusion and advection. The two solute transport models were also coupled to account for cell metabolism. With this approach, the relevance of metabolic and mechano-transport couplings were assessed in the healthy disc under loading-recovery daily compression. Disc height, cell density and material degenerative changes were parametrically simulated to study their influence on the calculated solute concentrations. The effects of load frequency and amplitude were also studied in the healthy disc by considering short periods of cyclic compression. Results indicate that external loads influence the oxygen and lactate regional distributions within the disc when large volume changes modify diffusion distances and diffusivities, especially when healthy disc properties are simulated. Advection was negligible under both sustained and cyclic compression. Simulating degeneration, mechanical changes inhibited the mechanical effect on transport while disc height, fluid content, nucleus pressure and overall cell density reductions affected significantly transport predictions. For the healthy disc, nutrient concentration patterns depended mostly on the time of sustained compression and recovery. The relevant effect of cell density on the metabolic transport indicates the disturbance of cell number as a possible onset for disc degeneration via alteration of the metabolic balance. Results also suggest that healthy disc properties have a positive effect of loading on metabolic transport. Such relation, relevant to the maintenance of the tissue functional composition, would therefore link disc function with disc nutrition.     

The serine 814 of TRPC6 is phosphorylated under unstimulated conditions

TRPC are nonselective cation channels involved in calcium entry. Their regulation by phosphorylation has been shown to modulate their routing and activity. TRPC6 activity increases following phosphorylation by Fyn, and is inhibited by protein kinase G and protein kinase C. A previous study by our group showed that TRPC6 is phosphorylated under unstimulated conditions in a human embryonic kidney cells line (HEK293). To investigate the mechanism responsible for this phosphorylation, we used a MS/MS approach combined with metabolic labeling and showed that the serine at position 814 is phosphorylated in unstimulated cells. The mutation of Ser(814) into Ala decreased basal phosphorylation but did not modify TRPC6 activity. Even though Ser(814) is within a consensus site for casein kinase II (CK2), we showed that CK2 is not involved in the phosphorylation of TRPC6 and does not modify its activity. In summary, we identified a new basal phosphorylation site (Ser(814)) on TRPC6 and showed that CK2 is not responsible for the phosphorylation of this site.     

Activation and infection of B cells by Epstein-Barr virus. Role of calcium mobilization and of protein kinase C translocation

Both the activation and the transformation of human B cells by EBV were inhibited by either the Ca2+ channel blocking agent verapamil or the combination of theophylline and dibutyryl cAMP: the day 4 and day 20 peaks of [3H]TdR incorporation were abolished; the EBNA marker was not expressed by day 10; lymphoblastoid cell lines did not arise. Short term incubation of B cells with EBV or verapamil showed that the effect of verapamil was reversible and took place early in the interaction between EBV and B cells. The effect of EBV on the early metabolic events of B cell response was thus examined in the presence and in the absence of the drugs. Compared to anti-mu stimulation, supernatant of the transforming B95-8 strain as well as that of the non-transforming P3HR1 strain induced a drug sensitive increase of the free cytosolic Ca2+ concentration. This increase was associated with a protein kinase C translocation from the cytosol to a membrane bound compartment. Moreover, B95-8 supernatant induced phosphatidyl inositol metabolism by human B cells but at least four times less than that induced by anti-mu antibody. These metabolic events induced by EBV were significantly inhibited by anti-CD21 antibodies whereas anti-mu induced metabolic events were not. The infection of EBV negative Ramos cell line was prevented by verapamil or by theophylline + dibutyryl cAMP. Verapamil did not modify the density of EBV receptors but negatively interfered with the penetration of the virus into B cells. Thus B cell activation through the EBV receptor and virus penetration share a common metabolic pathway which is also used for transduction of the signal delivered through the membrane Ig.     

Cooperation between human cells sensitive to UVA radiations: a clue to the mechanism of cellular hypersensitivity associated with different clinical conditions

Six fibroblast strains sensitive to long wavelength ultraviolet radiation (UVA) and one control strain were used to see if cooperation between the different cell strains could modify the abnormally high yield of single-strand DNA breaks (SSB) in the sensitive strains caused by UVA irradiation in complete Dulbecco's MEM. The sensitive strains were established from individuals showing proneness to different types of light-induced skin damage (actinic reticuloid, familial actinic keratoses with internal malignancies, and unusual frequency of basal cell carcinomata). When sensitive and normal cells were cocultivated, the UVA-induced SSB decreased in the sensitive cells and increased in the normal ones by amounts proportional to the ratio of the two types of cells in the mixtures. Furthermore the regression of SSB, in the sensitive cells, on the proportion of normal cells in the mixture extrapolated to normal levels of SSB when the proportion of normal cells increased to one. Cocultivation of different sensitive cells did not reduce the UVA-induced SSB to levels below those of the less sensitive cell strains. From these results we conclude that substances, present in limiting amounts, even in normal cells, can be transferred from cell to cell, presumably by metabolic cooperation, and modify the yield of SSB caused by UVA radiation. The abnormal yields of SSB in the sensitive cells appear to be entirely attributable to deficits in the substances responsible for the intercellular cooperation. We suggest that such substances are small molecular weight scavengers of active oxygen species.     

CD8+ T cell metabolic changes in breast cancer

Immunometabolism has advanced our understanding of how the cellular environment and nutrient availability regulates immune cell fate. Not only are metabolic pathways closely tied to cell signaling and differentiation, but can induce different subsets of immune cells to adopt unique metabolic programs, influencing disease progression. Dysregulation of immune cell metabolism plays an essential role in the progression of several diseases including breast cancer (BC). Metabolic reprogramming plays a critical role in regulating T cell functions. CD8⁺ T cells are an essential cell type within the tumor microenvironment (TME). To induce antitumor responses, CD8⁺ T cells need to adapt their metabolism to fulfill their energy requirement for effective function. However, different markers and immunologic techniques have made identifying specific CD8⁺ T cells subtypes in BC a challenge to the field. This review discusses the immunometabolic processes of CD8⁺ T cell in the TME in the context of BC and highlights the role of CD8⁺ T cell metabolic changes in tumor progression.     

Mechanisms Orchestrating Mitochondrial Dynamics for Energy Homeostasis

To maintain homeostasis, every cell must constantly monitor its energy level and appropriately adjust energy, in the form of ATP, production rates based on metabolic demand. Continuous fulfillment of this energy demand depends on the ability of cells to sense, metabolize, and convert nutrients into chemical energy. Mitochondria are the main energy conversion sites for many cell types. Cellular metabolic states dictate the mitochondrial size, shape, function, and positioning. Mitochondrial shape varies from singular discrete organelles to interconnected reticular networks within cells. The morphological adaptations of mitochondria to metabolic cues are facilitated by the dynamic events categorized as transport, fusion, fission, and quality control. By changing their dynamics and strategic positioning within the cytoplasm, mitochondria carry out critical functions and also participate actively in inter-organelle cross-talk, assisting metabolite transfer, degradation, and biogenesis. Mitochondrial dynamics has become an active area of research because of its particular importance in cancer, metabolic diseases, and neurological disorders. In this review, we will highlight the molecular pathways involved in the regulation of mitochondrial dynamics and their roles in maintaining energy homeostasis.     

Activation of TNF-R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-α has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling. © 1994 Wiley-Liss, Inc.     

CHOmpact: A reduced metabolic model of Chinese hamster ovary cells with enhanced interpretability

Metabolic modeling has emerged as a key tool for the characterization of biopharmaceutical cell culture processes. Metabolic models have also been instrumental in identifying genetic engineering targets and developing feeding strategies that optimize the growth and productivity of Chinese hamster ovary (CHO) cells. Despite their success, metabolic models of CHO cells still present considerable challenges. Genome-scale metabolic models (GeMs) of CHO cells are very large (>6000 reactions) and are difficult to constrain to yield physiologically consistent flux distributions. The large scale of GeMs also makes the interpretation of their outputs difficult. To address these challenges, we have developed CHOmpact, a reduced metabolic network that encompasses 101 metabolites linked through 144 reactions. Our compact reaction network allows us to deploy robust, nonlinear optimization and ensure that the computed flux distributions are physiologically consistent. Furthermore, our CHOmpact model delivers enhanced interpretability of simulation results and has allowed us to identify the mechanisms governing shifts in the anaplerotic consumption of asparagine and glutamate as well as an important mechanism of ammonia detoxification within mitochondria. CHOmpact, thus, addresses key challenges of large-scale metabolic models and will serve as a platform to develop dynamic metabolic models for the control and optimization of biopharmaceutical cell culture processes.     

Metabolic rate does not scale with body mass in cultured mammalian cells

The allometric scaling of metabolic rate with organism body mass can be partially accounted for by differences in cellular metabolic rates. For example, hepatocytes isolated from horses consume almost 10-fold less oxygen per unit time as mouse hepatocytes [Porter and Brand, Am J Physiol Regul Integr Comp Physiol 269: R226-R228, 1995]. This could reflect a genetically programmed, species-specific, intrinsic metabolic rate set point, or simply the adaptation of individual cells to their particular in situ environment (i.e., within the organism). We studied cultured cell lines derived from 10 mammalian species with donor body masses ranging from 5 to 600,000 g to determine whether cells propagated in an identical environment (media) exhibited metabolic rate scaling. Neither metabolic rate nor the maximal activities of key enzymes of oxidative or anaerobic metabolism scaled significantly with donor body mass in cultured cells, indicating the absence of intrinsic, species-specific, cellular metabolic rate set points. Furthermore, we suggest that changes in the metabolic rates of isolated cells probably occur within 24 h and involve a reduction of cellular metabolism toward values observed in lower metabolic rate organisms. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms. To examine the effect of oxygen on steady-state cellular respiration rates, we grew cells under a variety of physiologically relevant oxygen regimens. Long-term exposure to higher medium oxygen levels increased respiration rates of all cells, consistent with the hypothesis that higher rates of oxygen delivery in smaller mammals might increase cellular metabolic rates.     

Evolution of Associative Learning in Chemical Networks

Organisms that can learn about their environment and modify their behaviour appropriately during their lifetime are more likely to survive and reproduce than organisms that do not. While associative learning – the ability to detect correlated features of the environment – has been studied extensively in nervous systems, where the underlying mechanisms are reasonably well understood, mechanisms within single cells that could allow associative learning have received little attention. Here, using in silico evolution of chemical networks, we show that there exists a diversity of remarkably simple and plausible chemical solutions to the associative learning problem, the simplest of which uses only one core chemical reaction. We then asked to what extent a linear combination of chemical concentrations in the network could approximate the ideal Bayesian posterior of an environment given the stimulus history so far? This Bayesian analysis revealed the ‘memory traces’ of the chemical network. The implication of this paper is that there is little reason to believe that a lack of suitable phenotypic variation would prevent associative learning from evolving in cell signalling, metabolic, gene regulatory, or a mixture of these networks in cells.     

Ontogenic growth of the haemopoietic stem cell pool in humans

Recently, the size of the active stem cell pool has been predicted to scale allometrically with the adult mass of mammalian species with a 3/4 power exponent, similar to what has been found to occur for the resting metabolic rate across species. Here we investigate the allometric scaling of human haemopoietic stem cells (HSCs) during ontogenic growth and predict a linear scaling with body mass. We also investigate the allometric scaling of resting metabolic rate during growth in humans and find a linear scaling with mass similar to that of the haemopoietic stem cell pool. Our findings suggest a common underlying organizational principle determining the linear scaling of both the stem cell pool and resting metabolic rate with mass during ontogenic growth within the human species, combined with a 3/4 scaling with adult mass across mammalian species. It is possible that such common principles remain valid for haemopoiesis in other mammalian species.     

From cancer metabolism to new biomarkers and drug targets

Great interest is presently given to the analysis of metabolic changes that take place specifically in cancer cells. In this review we summarize the alterations in glycolysis, glutamine utilization, fatty acid synthesis and mitochondrial function that have been reported to occur in cancer cells and in human tumors. We then propose considering cancer as a system-level disease and argue how two hallmarks of cancer, enhanced cell proliferation and evasion from apoptosis, may be evaluated as system-level properties, and how this perspective is going to modify drug discovery. Given the relevance of the analysis of metabolism both for studies on the molecular basis of cancer cell phenotype and for clinical applications, the more relevant technologies for this purpose, from metabolome and metabolic flux analysis in cells by Nuclear Magnetic Resonance and Mass Spectrometry technologies to positron emission tomography on patients, are analyzed. The perspectives offered by specific changes in metabolism for a new drug discovery strategy for cancer are discussed and a survey of the industrial activity already going on in the field is reported.     

Importance of various types of metabolic inhibition for cell damage caused by direct membrane damage

Cell damage is caused by energy depletion or by direct membrane damage, or a combination when a direct membrane damage affects energy depleted cells. In this report it was investigated whether the extent of direct membrane damage induced by lysophosphatidyl choline (LPC) or phospholipase C (PhC) on quiescent fibroblasts depended on the metabolic state of the cells. When glycolysis was inhibited cell damage was always extensively increased, whereas cell damage was also increased to a minor degree when exposed to PhC during sole inhibition of oxidative phosphorylation. Acceleration of glycolysis in cells with a low rate of glycolysis resulted in a dramatic improvement of the membrane susceptibility within a few minutes. Thus, susceptibility of the cell membrane to direct membrane damage depends on the metabolic state. The results also emphasize previous findings that glycolysis has a special role in maintaining membrane function and integrity.     

Metabolic flux analysis in mammalian cell culture

Mammalian cell culture metabolism is characterized by glucoglutaminolysis, that is, high glucose and glutamine uptake combined with a high rate of lactate and non-essential amino acid secretion. Stress associated with acid neutralization and ammonia accumulation necessitates complex feeding schemes and limits cell densities achieved in fed-batch culture. Conventional and constraint-based metabolic flux analysis has been successfully used to study the metabolic phenotype of mammalian cells in culture, while ¹³C tracer analysis has been used to study small network models and validate assumptions of metabolism. Large-scale ¹³C metabolic flux analysis, which is required to improve confidence in the network models and their predictions, remains a major challenge. Advances in both modeling and analytical techniques are bringing this challenge within sight.     

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.     

Relationship between dietary fiber and cancer: metabolic, physiologic, and cellular mechanisms

The relationships between fiber consumption and human cancer rates have been examined, together with an analysis of the effects of individual dietary fibers on the experimental induction of large bowel cancer. The human epidemiology indicates an inverse correlation between high fiber consumption and lower colon cancer rates. Cereal fiber sources show the most consistent negative correlation. However, human case-control studies in general fail to confirm any protective effect due to dietary fiber. Case-control studies indicate that if any source of dietary fiber is possibly antineoplastic then it is probably vegetables. These results may mean that purified fibers alone do not inhibit tumor development, whereas it is likely that some other factors present in vegetables are antineoplastic. Experiments in laboratory animals, using chemical induction of large bowel cancer, have in general shown a protective effect with supplements of poorly fermentable fibers such as wheat bran or cellulose. In contrast, a number of fermentable fiber supplements including pectin, corn bran, oat bran, undegraded carageenan, agar, psyllium, guar gum, and alfalfa have been shown to enhance tumor development. Possible mechanisms by which fibers may inhibit colon tumorigenesis include dilution and adsorption of any carcinogens and/or promoters contained within the intestinal lumen, the modulation of colonic microbial metabolic activity, and biological modification of intestinal epithelial cells. Dietary fibers not only bind carcinogens, bile acids, and other potential toxins but also essential nutrients, such as minerals, which can inhibit the carcinogenic process. Fermentation of fibers within the large bowel results in the production of short chain fatty acids, which in vivo stimulate cell proliferation, while butyrate appears to be antineoplastic in vitro. Evidence suggests that if dietary fibers stimulate cell proliferation during the stage of initiation, then this may lead to tumor enhancement. Fermentation also lowers luminal pH, which in turn modifies colonic microbial metabolic acidity, and is associated with increased epithelial cell proliferation and colon carcinogenesis. Because dietary fibers differ in their physiochemical properties it has been difficult to identify a single mechanism by which fibers modify colon carcinogenesis. Clearly, more metabolic and physiological studies are needed to fully define the mechanisms by which certain fibers inhibit while others enhance experimental colon carcinogenesis.     

Glutamine regulates mitochondrial uncoupling protein 2 to promote glutaminolysis in neuroblastoma cells

Mitochondrial uncoupling protein 2 (UCP2) is highly abundant in rapidly proliferating cells that utilize aerobic glycolysis, such as stem cells, cancer cells, and cells of the immune system. However, the function of UCP2 has been a longstanding conundrum. Considering the strict regulation and unusually short life time of the protein, we propose that UCP2 acts as a “signaling protein” under nutrient shortage in cancer cells. We reveal that glutamine shortage induces the rapid and reversible downregulation of UCP2, decrease of the metabolic activity and proliferation of neuroblastoma cells, that are regulated by glutamine per se but not by glutamine metabolism. Our findings indicate a very rapid (within 1?h) metabolic adaptation that allows the cell to survive by either shifting its metabolism to the use of the alternative fuel glutamine or going into a reversible, more quiescent state. The results imply that UCP2 facilitates glutamine utilization as an energetic fuel source, thereby providing metabolic flexibility during glucose shortage. The targeting UCP2 by drugs to intervene with cancer cell metabolism may represent a new strategy for treatment of cancers resistant to other therapies.