Targeting amino acid metabolism in cancer growth and anti-tumor immune response

Recent advances in amino acid metabolism have revealed that targeting amino acid metabolic enzymes in cancer therapy is a promising strategy for the development of novel therapeutic agents. There are currently several drugs in clinical trials that specifically target amino acid metabolic pathways in tumor cells. In the context of the tumor microenvironment,however,tumor cells form metabolic relationships with immune cells,and they oftencompete for common nutrients. Many tumors evolved to escape immune surveillance by taking advantage of their metabolic flexibility and redirecting nutrients for their own advantage. This review outlines the most recent advances in targeting amino acid metabolic pathways in cancer therapy while giving consideration to the impact these pathways may have on the anti-tumor immune response.     

Metabolic regulation in progression to autoimmune diabetes

Recent evidence from serum metabolomics indicates that specific metabolic disturbances precede β-cell autoimmunity in humans and can be used to identify those children who subsequently progress to type 1 diabetes. The mechanisms behind these disturbances are unknown. Here we show the specificity of the pre-autoimmune metabolic changes, as indicated by their conservation in a murine model of type 1 diabetes. We performed a study in non-obese prediabetic (NOD) mice which recapitulated the design of the human study and derived the metabolic states from longitudinal lipidomics data. We show that female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, normoglycemia, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum group. Together, the findings indicate that autoimmune diabetes is preceded by a state of increased metabolic demands on the islets resulting in elevated insulin secretion and suggest alternative metabolic related pathways as therapeutic targets to prevent diabetes.     

Cellular stress responses and cancer: new mechanistic insights on anticancer effect by phytochemicals

During the tumorigenesis, cancer cells are frequently exposed to metabolic stress which is derived from altered cancer cell metabolism as well as unfavorable tumor microenvironment, such as hypoxia and glucose deprivation. Cancer cells need to respond to these stress stimuli properly through inducing cellular stress responses, such as unfolded protein response and autophagy, for cell survival. Therefore, modulation of these stress responses has been investigated as an alternative anticancer strategy, although their therapeutic clinical roles remain to be determined. In this review, we will discuss the cellular stress responses in cancer cells, the alternative anticancer strategy targeting unfolded protein response and/or autophagy, and the role of phytochemicals, which include resveratrol, genistein, curcumin, epigallocatechin-3-gallate and quercetin, in modulating the cellular stress responses.     

Metabolic pathways and fermentative production of L-aspartate family amino acids

The L -aspartate family amino acids (AFAAs), L -threonine, L -lysine, L -methionine and L -isoleucine have recently been of much interest due to their wide spectrum of applications including food additives, components of cosmetics and therapeutic agents, and animal feed additives. Among them, L -threonine, L -lysine and L -methionine are three major amino acids produced currently throughout the world. Recent advances in systems metabolic engineering, which combine various high-throughput omics technologies and computational analysis, are now facilitating development of microbial strains efficiently producing AFAAs. Thus, a thorough understanding of the metabolic and regulatory mechanisms of the biosynthesis of these amino acids is urgently needed for designing system-wide metabolic engineering strategies. Here we review the details of AFAA biosynthetic pathways, regulations involved, and export and transport systems, and provide general strategies for successful metabolic engineering along with relevant examples. Finally, perspectives of systems metabolic engineering for developing AFAA overproducers are suggested with selected exemplary studies.     

Differential Utilization of Dietary Fatty Acids in Benign and Malignant Cells of the Prostate

Tumor cells adapt via metabolic reprogramming to meet elevated energy demands due to continuous proliferation, for example by switching to alternative energy sources. Nutrients such as glucose, fatty acids, ketone bodies and amino acids may be utilized as preferred substrates to fulfill increased energy requirements. In this study we investigated the metabolic characteristics of benign and cancer cells of the prostate with respect to their utilization of medium chain (MCTs) and long chain triglycerides (LCTs) under standard and glucose-starved culture conditions by assessing cell viability, glycolytic activity, mitochondrial respiration, the expression of genes encoding key metabolic enzymes as well as mitochondrial mass and mtDNA content. We report that BE prostate cells (RWPE-1) have a higher competence to utilize fatty acids as energy source than PCa cells (LNCaP, ABL, PC3) as shown not only by increased cell viability upon fatty acid supplementation but also by an increased ß-oxidation of fatty acids, although the base-line respiration was 2-fold higher in prostate cancer cells. Moreover, BE RWPE-1 cells were found to compensate for glucose starvation in the presence of fatty acids. Of notice, these findings were confirmed in vivo by showing that PCa tissue has a lower capacity in oxidizing fatty acids than benign prostate. Collectively, these metabolic differences between benign and prostate cancer cells and especially their differential utilization of fatty acids could be exploited to establish novel diagnostic and therapeutic strategies.     

Natural products from synthetic biology

DNA sequencing has uncovered microbial secondary metabolic potential that never surfaced in fermentation based screens. Deep and cheap sequencing of a genus such as Streptomyces can rapidly expose hundreds of metabolic genes and operons. Meanwhile, synthetic biologists, in their quest to engineer advanced biofuels, are mastering metabolic engineering. Natural products, a reliable source of new therapeutic leads for many years, have fallen into disfavor with drug discoverers partly because these molecules are rarely available as pure compounds and sourcing is often problematic. The convergence of next generation sequencing and synthetic biology, along with less spectacular progress in analytic technologies such as mass spectroscopy, opens the door to the creation of large, reliable libraries of pure natural products for drug discovery.     

Physiological significance of heme oxygenase in hypertension

The last decade has witnessed an explosion in the elucidation of the role that the heme oxygenase system plays in human physiology. This system encompasses not only the heme degradative pathway, including heme oxygenase and biliverdin reductase, but also the products of heme degradation, carbon monoxide, iron, and biliverdin/bilirubin. Their role in diabetes, inflammation, heart disease, hypertension, transplantation, and pulmonary disease are areas of burgeoning research. The research has focused not only on heme itself but also on its metabolic products as well as endogenous compounds involved in a vast number of genetic and metabolic processes that are affected when heme metabolism is perturbed. It should be noted, however, that although the use of carbon monoxide and biliverdin/bilirubin as therapeutic agents has been successful, these agents can be toxic at high levels in tissue, e.g., kernicterus. Care must be used to ensure that when these compounds are used as therapeutic agents their deleterious effects are minimized or avoided. On balance, however, the strategies to target heme oxygenase-1 as described in this review offer promising therapeutic approaches to clinicians for the effective management of hypertension and renal function. The approaches detailed may prove to be seminal in the development of a new therapeutic strategy to treat hypertension.     

Evaluation of target-specific natural compounds for drug discovery against Leishmaniasis

Leishmaniasis is a parasitic disease with no effective vaccine still now. Globally, it has affected millions of people, precisely in the undeveloped and developing countries. The control strategy for leishmaniasis depends only on chemotherapeutic methods that are associated with several side effects. Therefore, to overcome these negative impacts natural products are the best alternative for developing effective herbal-based drugs, which can act as one of the safest and effective alternative options to treat this particular disease. Leishmania, the causative agent of this disease possesses unique enzymes and metabolic pathways that are different from its mammalian host. Moreover, these unique enzymes, along with the signaling molecules and metabolic pathways that are crucial for its survival, serve as a suitable drug target for the evaluation of specific natural inhibitors to overcome leishmaniasis. Hence, in this review, we have discussed various specific targets of Leishmania, along with their natural inhibitors which can play a significant role in anti-leishmanial drug discovery.     

Production of isoprenoid pharmaceuticals by engineered microbes

Throughout human history, natural products have been the foundation for the discovery and development of therapeutics used to treat diseases ranging from cardiovascular disease to cancer. Their chemical diversity and complexity have provided structural scaffolds for small-molecule drugs and have consistently served as inspiration for medicinal design. However, the chemical complexity of natural products also presents one of the main roadblocks for production of these pharmaceuticals on an industrial scale. Chemical synthesis of natural products is often difficult and expensive, and isolation from their natural sources is also typically low yielding. Synthetic biology and metabolic engineering offer an alternative approach that is becoming more accessible as the tools for engineering microbes are further developed. By reconstructing heterologous metabolic pathways in genetically tractable host organisms, complex natural products can be produced from inexpensive sugar starting materials through large-scale fermentation processes. In this Perspective, we discuss ongoing research aimed toward the production of terpenoid natural products in genetically engineered Escherichia coli and Saccharomyces cerevisiae.     

Towards systems metabolic engineering of microorganisms for amino acid production

Microorganisms capable of efficient production of amino acids have traditionally been developed by random mutation and selection method, which might cause unwanted physiological changes in cellular metabolism. Rational genome-wide metabolic engineering based on systems and synthetic biology tools, which is termed 'systems metabolic engineering', is rising as an alternative to overcome these problems. Recently, several amino acid producers have been successfully developed by systems metabolic engineering, where the metabolic engineering procedures were performed within a systems biology framework, and entire metabolic networks, including complex regulatory circuits, were engineered in an integrated manner. Here we review the current status of systems metabolic engineering successfully applied for developing amino acid producing strains and discuss future prospects.     

柠檬烯和红没药烯的微生物代谢工程

柠檬烯和红没药烯均为植物天然产物,分别属于单萜类和倍半萜类化合物,能够预防和治疗癌症等多种疾病。以其作为前体物,还可以转化合成多种具有高附加值的工业产品,例如药品、保健品、化妆品及生物燃料等。目前柠檬烯和红没药烯的工业生产主要是通过植物提取法实现的,但从植物组织中提取柠檬烯和红没药烯存在着产物含量低和分离纯化困难等缺点。微生物代谢工程的快速发展为这些植物天然产物的生产提供了一条更具潜力的生物合成路线。利用微生物代谢工程技术构建生产这些有价值的植物天然产物的微生物细胞工厂具有绿色清洁、可持续发展和经济效益好等独特优势。文中系统综述了近年来代谢工程技术在微生物合成柠檬烯和红没药烯过程中的应用进展,包括所涉及的宿主菌株、关键酶、代谢途径及其改造等,并探讨了其未来发展方向。     

Tailoring of microbes for the production of high value plant-derived compounds: From pathway engineering to fermentative production

Plant natural products have been an attracting platform for the isolation of various active drugs and other bioactives. However large-scale extraction of these compounds is affected by the difficulty in mass cultivation of these plants and absence of strategies for successful extraction. Even though, synthesis by chemical method is an alternative method; it is less efficient as their chemical structure is highly complex which involve enantio-selectivity. Thus an alternate bio-system for heterologous production of plant natural products using microbes has emerged. Advent of various omics, synthetic and metabolic engineering strategies revolutionised the field of heterologous plant metabolite production. In this context, various engineering methods taken to synthesise plant natural products are described with an additional focus to fermentation strategies.     

One carbon metabolism and its implication in health and immune functions

One carbon (1C) metabolism is critical for cellular viability and physiological homeostasis. Starting from its crucial involvement in purine biosynthesis to posttranslational modification of proteins, 1C metabolism contributes significantly to the development and cellular differentiation through methionine and folate cycles that are pivotal for cellular function. Genetic polymorphisms of several genes of these pathways are implicated in disease pathogenesis and drug metabolism. Metabolic products of 1C metabolism have significant roles in epigenetic modifications through DNA and histone protein methylation. Homocysteine is a product that has clinical significance in the diagnosis and prognosis of several critical illnesses, including chronic immune diseases and cancers. Regulation of the function and differentiation of immune cells, including T-cells, B-cells, macrophages, and so forth, are directly influenced by 1C metabolism and thus have direct implications in several immune disease biology. Recent research on therapeutic approaches is targeting nuclear, cytoplasmic, and mitochondrial 1C metabolism to manage and treat metabolic (i.e., type 2 diabetes), neurodegenerative (i.e., Alzheimer's disease), or immune (i.e., rheumatoid arthritis) diseases. 1C metabolism is being explored for therapeutic intervention as a common determinant for a spectrum of immune and metabolic diseases. Identifying the association or correlation between essential metabolic products of this pathway and disease onset or prognosis would further facilitate the clinical monitoring of diseases.     

Quality control of gene therapy products: approach of the French Agency for the Safety of Health Products

Gene therapy is a new therapeutic strategy which can constitute in some diseases a true alternative or a complement to the "classical treatments". Regarding the innovative features, the complexity and the extreme diversity of the gene therapy products (naked DNA, synthetic vectors, viral vectors, genetically modified cells), these new products presently in clinical trials have to be precisely evaluated and controlled for their medicine quality as well as their biological origin and/or their specific characteristics of genetically modified organisms. The French Agency for the Safety of Health Products engaged an in-depth scientific review concerning the control of this very heterogeneous class of potential therapeutics through the creation of a working group. The objectives of this group were to determine the testings to be performed by a national authority for each type of gene therapy products and to select the appropriate techniques or methods to be developed. Controls considered as essential are listed and include the verification of the identity, the purity, the transfer and expression efficiency as well as the microbiological and viral safety of the products. This implies the development of diverse techniques of molecular biology, cellular biology, physico-chemistry, animal testing, histology and microbiology. Finally, in order to define the basis of testings of these emerging products, the marketing of which should be effective for some of them in the next years, it appears extremely important to harmonize the quality, efficiency and safety criteria, to develop specific references and standards and to create specific guidelines for the control of gene therapy products.     

Engineering Escherichia coli for production of functionalized terpenoids using plant P450s

Terpenoids are a highly diverse class of natural products that have historically provided a rich source for discovery of pharmacologically active small molecules, such as paclitaxel (Taxol) and artemisinin. Unfortunately, these secondary metabolites are typically produced in low abundance in their host organism, and their isolation consequently suffers from low yields and high consumption of natural resources. Furthermore, chemical synthesis of terpenoids can also be difficult to scale for industrial production. For these reasons, an attractive alternative strategy is to engineer metabolic pathways for production of pharmaceuticals or their precursors in a microbial host such as Escherichia coli. A key step is developing methods to carry out cytochrome P450 (P450)-based oxidation chemistry in vivo. Toward this goal, we have assembled two heterologous pathways for the biosynthesis of plant-derived terpenoid natural products, and we present the first examples of in vivo production of functionalized terpenoids in E. coli at high titer using native plant P450s.     

Potential therapeutic drug target identification in Community Acquired-Methicillin Resistant Staphylococcus aureus (CA-MRSA) using computational analysis

The emergence of multidrug-resistant strain of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strain has highlighted the urgent need for the alternative and effective therapeutic approach to combat the menace of this nosocomial pathogen. In the present work novel potential therapeutic drug targets have been identified through the metabolic pathways analysis. All the gene products involved in different metabolic pathways of CA-MRSA in KEGG database were searched against the proteome of Homo sapiens using the BLASTp program and the threshold of E-value was set to as 0.001. After database searching, 152 putative targets were identified. Among all 152 putative targets, 39 genes encoding for putative targets were identified as the essential genes from the DEG database which are indispensable for the survival of CA-MRSA. After extensive literature review, 7 targets were identified as potential therapeutic drug target. These targets are Fructose-bisphosphate aldolase, Phosphoglyceromutase, Purine nucleoside phosphorylase, Uridylate kinase, Tryptophan synthase subunit beta, Acetate kinase and UDP-N-acetylglucosamine 1-carboxyvinyltransferase. Except Uridylate kinase all the identified targets were involved in more than one metabolic pathways of CA-MRSA which underlines the importance of drug targets. These potential therapeutic drug targets can be exploited for the discovery of novel inhibitors for CA-MRSA using the structure based drug design (SBDD) strategy.     

Transgenic approaches to improve the nutritional quality of plant proteins

Summary Plant proteins, when used as dietary protein, are generally incomplete in nutrition due to their deficiency in several essential amino acids, for example, lysine and tryptophan in cereals and methionine and cysteine in legumes. Attempts to breed crops with increased levels of lysine and methionine have been less than satisfactory. Modern biotechnology offers alternative approaches for rectifying this nutrition deficiency. In the past decade, several transgenic strategies aimed at modifying the amino acid composition of plant proteins and enhancing the content of specific essential amino acid(s) for nutrition improvement have been developed and tested. These include synthetic proteins, modification of protein sequences, over-expression of heterologous or homologous proteins, and metabolic engineering of the free essential amino acid pool and protein sink. The progress and potential of these approaches and studies are reviewed. As plant proteins are the primary source of all dietary protein consumed by humans and animals and are inexpensive to produce in comparison with meat, improving their quality will make a significant contribution to our future food needs. The research and development in this area of interest is making promising progress towards this endeavor.     

Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers

Actinobacillus succinogenes is a promising candidate for industrial succinate production. However, in addition to producing succinate, it also produces formate and acetate. To understand carbon flux distribution to succinate and alternative products we fed A. succinogenes [1-(13)C]glucose and analyzed the resulting isotopomers of excreted organic acids, proteinaceous amino acids, and glycogen monomers by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The isotopomer data, together with the glucose consumption and product formation rates and the A. succinogenes biomass composition, were supplied to a metabolic flux model. Oxidative pentose phosphate pathway flux supplied, at most, 20% of the estimated NADPH requirement for cell growth. The model indicated that NADPH was instead produced primarily by the conversion of NADH to NADPH by transhydrogenase and/or by NADP-dependent malic enzyme. Transhydrogenase activity was detected in A. succinogenes cell extracts, as were formate and pyruvate dehydrogenases, which the model suggested were contributing to NADH production. Malic enzyme activity was also detected in cell extracts, consistent with the flux analysis results. Labeling patterns in amino acids and organic acids showed that oxaloacetate and malate were being decarboxylated to pyruvate. These are the first in vivo experiments to show that the partitioning of flux between succinate and alternative fermentation products can occur at multiple nodes in A. succinogenes. The implications for designing effective metabolic engineering strategies to increase A. succinogenes succinate production are discussed.     

Biochemical production capabilities of Escherichia coli

Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products. (c) 1993 John Wiley & Sons, Inc.