How will bioinformatics influence metabolic engineering?

Ten microbial genomes have been fully sequenced to date, and the sequencing of many more genomes is expected to be completed before the end of the century. The assignment of function to open reading frames (ORFs) is progressing, and for some genomes over 70% of functional assignments have been made. The majority of the assigned ORFs relate to metabolic functions. Thus, the complete genetic and biochemical functions of a number of microbial cells may be soon available. From a metabolic engineering standpoint, these developments open a new realm of possibilities. Metabolic analysis and engineering strategies can now be built on a sound genomic basis. An important question that now arises; how should these tasks be approached? Flux-balance analysis (FBA) has the potential to play an important role. It is based on the fundamental principle of mass conservation. It requires only the stoichiometric matrix, the metabolic demands, and some strain specific parameters. Importantly, no enzymatic kinetic data is required. In this article, we show how the genomically defined microbial metabolic genotypes can be analyzed by FBA. Fundamental concepts of metabolic genotype, metabolic phenotype, metabolic redundancy and robustness are defined and examples of their use given. We discuss the advantage of this approach, and how FBA is expected to find uses in the near future. FBA is likely to become an important analysis tool for genomically based approaches to metabolic engineering, strain design, and development.     

NADPH metabolism: a survey of its theoretical characteristics and manipulation strategies in amino acid biosynthesis

Reduced nicotinamide adenine nucleotide phosphate (NADPH), which is one of the key cofactors in the metabolic network, plays an important role in the biochemical reactions, and physiological function of amino acid-producing strains. The manipulation of NADPH availability and form is an efficient and easy method of redirecting the carbon flux to the amino acid biosynthesis in industrial strains. In this review, we survey the metabolic mode of NADPH. Furthermore, we summarize the research developments in the understanding of the relationship between NADPH metabolism and amino acid biosynthesis. Detailed strategies to manipulate NADPH availability are addressed based on this knowledge. Finally, the uses of NADPH manipulation strategies to enhance the metabolic function of amino acid-producing strains are discussed.     

Mammalian catalase: a venerable enzyme with new mysteries

Mammalian catalase has been the subject of many classic biochemical studies. Despite our detailed knowledge of its functional mechanisms and its three-dimensional structure, however, several unexpected features of mammalian catalase have been recently discovered. For example, some mammalian catalases seem to have oxidase activity and produce reactive oxygen species when exposed to UVB light. In addition, bovine catalase uses unbound NAD(P)H to prevent substrate inactivation without displacing catalase-bound NADP(+). Coupled with the earlier discovery of catalase-bound NADPH, these developments indicate that serendipity and new investigative approaches can reveal unexpected features, even for an enzyme that has been studied for over 100 years.     

Gene discovery via metabolic profiling

Biochemical analysis is adding a new dimension to the process of gene discovery. Two major developments have recently taken place in the emerging science of biochemical genomics. The first is an approach that uses a combination of tagged fusion proteins and simple pooling strategies in order to efficiently and directly assign biochemical function to the products of open reading frames (ORFs) expressed in yeast. The second is the application of metabolic profiling technologies to the study of mutant and transgenic plants. The latter approach has the potential not only to discover novel genes but also to ascribe a function to them in the context of the organism from which they are derived.     

Fermentative butanol production by Clostridia

Butanol is an aliphatic saturated alcohol having the molecular formula of C(4)H(9)OH. Butanol can be used as an intermediate in chemical synthesis and as a solvent for a wide variety of chemical and textile industry applications. Moreover, butanol has been considered as a potential fuel or fuel additive. Biological production of butanol (with acetone and ethanol) was one of the largest industrial fermentation processes early in the 20th century. However, fermentative production of butanol had lost its competitiveness by 1960s due to increasing substrate costs and the advent of more efficient petrochemical processes. Recently, increasing demand for the use of renewable resources as feedstock for the production of chemicals combined with advances in biotechnology through omics, systems biology, metabolic engineering and innovative process developments is generating a renewed interest in fermentative butanol production. This article reviews biotechnological production of butanol by clostridia and some relevant fermentation and downstream processes. The strategies for strain improvement by metabolic engineering and further requirements to make fermentative butanol production a successful industrial process are also discussed.     

Opportunities for yeast metabolic engineering: Lessons from synthetic biology

Constant progress in genetic engineering has given rise to a number of promising areas of research that facilitated the expansion of industrial biotechnology. The field of metabolic engineering, which utilizes genetic tools to manipulate microbial metabolism to enhance the production of compounds of interest, has had a particularly strong impact by providing new platforms for chemical production. Recent developments in synthetic biology promise to expand the metabolic engineering toolbox further by creating novel biological components for pathway design. The present review addresses some of the recent advances in synthetic biology and how these have the potential to affect metabolic engineering in the yeast Saccharomyces cerevisiae. While S. cerevisiae for years has been a robust industrial organism and the target of multiple metabolic engineering trials, its potential for synthetic biology has remained relatively unexplored and further research in this field could strongly contribute to industrial biotechnology. This review also addresses are general considerations for pathway design, ranging from individual components to regulatory systems, overall pathway considerations and whole-organism engineering, with an emphasis on potential contributions of synthetic biology to these areas. Some examples of applications for yeast synthetic biology and metabolic engineering are also discussed.     

Evolutionary engineering of Saccharomyces cerevisiae for improved industrially important properties

This article reviews evolutionary engineering of Saccharomyces cerevisiae. Following a brief introduction to the 'rational' metabolic engineering approach and its limitations such as extensive genetic and metabolic information requirement on the organism of interest, complexity of cellular physiological responses, and difficulties of cloning in industrial strains, evolutionary engineering is discussed as an alternative, inverse metabolic engineering strategy. Major evolutionary engineering applications with S. cerevisiae are then discussed in two general categories: (1) evolutionary engineering of substrate utilization and product formation and (2) evolutionary engineering of stress resistance. Recent developments in functional genomics methods allow rapid identification of the molecular basis of the desired phenotypes obtained by evolutionary engineering. To conclude, when used alone or in combination with rational metabolic engineering and/or computational methods to study and analyze processes of adaptive evolution, evolutionary engineering is a powerful strategy for improvement in industrially important, complex properties of S. cerevisiae.     

Engineered biosynthetic pathways and biocatalytic cascades for sustainable synthesis

Nature exploits biosynthetic cascades to construct numerous molecules from a limited set of starting materials. A deeper understanding of biosynthesis and extraordinary developments in gene technology has allowed the manipulation of natural pathways and construction of artificial cascades for the preparation of a range of molecules, which would be challenging to access using traditional synthetic chemical approaches. Alongside these metabolic engineering strategies, there has been continued interest in developing in vivo and in vitro biocatalytic cascades. Advancements in both metabolic engineering and biocatalysis are complementary, and this article aims to highlight some of the most exciting developments in these two areas with a particular focus on exploring those that have the potential to advance both pathway engineering and more traditional biocatalytic cascade development.     

Mitochondrial function in vivo: spectroscopy provides window on cellular energetics

Mitochondria integrate the key metabolic fluxes in the cell. This role places this organelle at the center of cellular energetics and, hence, mitochondrial dysfunction underlies a growing number of human disorders and age-related degenerative diseases. Here we present novel analytical and technical methods for evaluating mitochondrial metabolism and (dys)function in human muscle in vivo. Three innovations involving advances in optical spectroscopy (OS) and magnetic resonance spectroscopy (MRS) permit quantifying key compounds in energy metabolism to yield mitochondrial oxidation and phosphorylation fluxes. The first of these uses analytical methods applied to optical spectra to measure hemoglobin (Hb) and myoglobin (Mb) oxygenation states and relative contents ([Hb]/[Mb]) to determine mitochondrial respiration (O₂ uptake) in vivo. The second uses MRS methods to quantify key high-energy compounds (creatine phosphate, PCr, and adenosine triphosphate, ATP) to determine mitochondrial phosphorylation (ATP flux) in vivo. The third involves a functional test that combines these spectroscopic approaches to determine mitochondrial energy coupling (ATP/O₂), phosphorylation capacity (ATP_max) and oxidative capacity (O_2max) of muscle. These new developments in optical and MR tools allow us to determine the function and capacity of mitochondria noninvasively in order to identify specific defects in vivo that are associated with disease in human and animal muscle. The clinical implication of this unique diagnostic probe is the insight into the nature and extent of dysfunction in metabolic and degenerative disorders, as well as the ability to follow the impact of interventions designed to reverse these disorders.     

Targeted gene delivery: the role of peptide nucleic acid

Receptor-mediated endocytosis can be exploited to achieve efficient cell-specific gene delivery. Our laboratory has used two approaches for targeted gene delivery. One uses polycation as a carrier for plasmid DNA and the other uses peptide nucleic acid (PNA) as a carrier. Targeted gene delivery using polycation carriers has been widely utilized with some success. This approach mainly suffers from large particle size and non-specific interaction with blood components. These drawbacks have limited use of this type of vector forin vivo applications. Using PNA as a carrier, on the other hand, allows for smaller particle size and less non-specific interactions. The stability of this vector in the circulation may be a limiting factor. In addition, both types of vector lack mechanisms for endosome escape and nuclear transport. In this chapter, current developments and uses for targeted gene delivery of each approach are reviewed.     

Targeted gene delivery: The role of peptide nucleic acid

Receptor-mediated endocytosis can be exploited to achieve efficient cell-specific gene delivery. Our laboratory has used two approaches for targeted gene delivery. One uses polycation as a carrier for plasmid DNA and the other uses peptide nucleic acid (PNA) as a carrier. Targeted gene delivery using polycation carriers has been widely utilized with some success. This approach mainly suffers from large particle size and non-specific interaction with blood components. These drawbacks have limited use of this type of vector for in vivo applications. Using PNA as a carrier, on the other hand, allows for smaller particle size and less non-specific interactions. The stability of this vector in the circulation may be a limiting factor. In addition, both types of vector lack mechanisms for endosome escape and nuclear transport. In this chapter, current developments and uses for targeted gene delivery of each approach are reviewed.     

Targeted gene delivery: the role of peptide nucleic acid

Receptor-mediated endocytosis can be exploited to achieve efficient cell-specific gene delivery. Our laboratory has used two approaches for targeted gene delivery. One uses polycation as a carrier for plasmid DNA and the other uses peptide nucleic acid (PNA) as a carrier. Targeted gene delivery using polycation carriers has been widely utilized with some success. This approach mainly suffers from large particle size and non-specific interaction with blood components. These drawbacks have limited use of this type of vector for in vivoapplications. Using PNA as a carrier, on the other hand, allows for smaller particle size and less non-specific interactions. The stability of this vector in the circulation may be a limiting factor. In addition, both types of vectorlack mechanisms for endosome escape and nuclear transport. In this chapter, current developments and uses for targeted gene delivery of each approach are reviewed.     

Biosynthesis and Catabolism of L-Ascorbic Acid in Plants

L-Ascorbic acid (AsA) is a vital antioxidant compound that plays a critical role in the cellular metabolism of plants and animals. Research on plant AsA metabolism experienced a significant resurgence after 1998 following the identification of AsA-deficient Arabidopsis mutants and the elucidation of a biosynthetic pathway accepted by the overwhelming majority of the plant science community. The identification and cloning of novel biosynthetic genes and the ensuing metabolic engineering of plant AsA content has however revealed a more complex picture. Additional biosynthetic routes have been identified and unexpected biochemical phenotypes were observed upon expression of animal AsA biosynthetic genes. The isolation of novel AsA conjugates from plant tissues and the evidence for long distance transport of AsA in plants have provided additional facets to its functionality. Although some progress has been made regarding the impact of AsA recycling on pool size, we still do not have a clear picture of the biochemistry of AsA degradation. This communication comprehensively reviews new developments in the AsA metabolic system and prompts directions for future research.     

Mechanisms of action of isoniazid

For decades after its introduction, the mechanisms of action of the front-line antituberculosis therapeutic agent isoniazid (INH) remained unclear. Recent developments have shown that peroxidative activation of isoniazid by the mycobacterial enzyme KatG generates reactive species that form adducts with NAD(+) and NADP(+) that are potent inhibitors of lipid and nucleic acid biosynthetic enzymes. A direct role for some isoniazid-derived reactive species, such as nitric oxide, in inhibiting mycobacterial metabolic enzymes has also been shown. The concerted effects of these activities - inhibition of cell wall lipid synthesis, depletion of nucleic acid pools and metabolic depression - drive the exquisite potency and selectivity of this agent. To understand INH action and resistance fully, a synthesis of knowledge is required from multiple separate lines of research - including molecular genetic approaches, in vitro biochemical studies and free radical chemistry - which is the intent of this review.     

Mycobacterial persistence: adaptation to a changing environment

Mycobacterium tuberculosis is a bacterial pathogen that can persist within an infected individual for extended periods of time without causing overt, clinical disease, in a state normally referred to as latent or chronic tuberculosis. Although the replicative state of the bacterium during this period is a matter of some conjecture, recent developments have indicated that the bacterium requires the regulated expression of a set of genes and metabolic pathways to maintain a persistent infection in an immunocompetent host. The characterization of these gene products and their role in bacterial metabolism and physiology is starting to provide insights into the mechanisms that M. tuberculosis has evolved to adopt its highly successful mode of pathogenicity.     

Cancer cell metabolism: Rewiring the mitochondrial hub

To adapt to tumoral environment conditions or even to escape chemotherapy, cells rapidly reprogram their metabolism to handle adversities and survive. Given the rapid rise of studies uncovering novel insights and therapeutic opportunities based on the role of mitochondria in tumor metabolic programing and therapeutics, this review summarizes most significant developments in the field. Taking in mind the key role of mitochondria on carcinogenesis and tumor progression due to their involvement on tumor plasticity, metabolic remodeling, and signaling re-wiring, those organelles are also potential therapeutic targets. Among other topics, we address the recent data intersecting mitochondria as of prognostic value and staging in cancer, by mitochondrial DNA (mtDNA) determination, and current inhibitors developments targeting mtDNA, OXPHOS machinery and metabolic pathways. We contribute for a holistic view of the role of mitochondria metabolism and directed therapeutics to understand tumor metabolism, to circumvent therapy resistance, and to control tumor development.