For several decades, various industrial strains have been successfully developed by traditional mutagenesis and selection to improve the yield and productivity of native products synthesized by these strains. The development of molecular biological techniques for DNA recombination introduced a new dimension to metabolic pathway modification. Genetic engineering allowed precise modification of specific enzymatic reactions in metabolic pathways, leading to the construction of well-defined genetic background . Soon after the feasibility of DNA recombination was established, the potential of directed pathway modification became apparent and various terms were coined to express the potential applications of this technology, such as, molecular breeding , in vitro evolution , microbial or metabolic pathway engineering [357, 593], cellular engineering , and metabolic engineering [31, 567]. The advent of recombinant DNA technology has enabled metabolic pathway modification by means of targeted genetic modifications.
Metabolic engineering can be defined as directed modification of cellular metabolism and properties through the introduction, deletion, inhibition and/or modification of metabolic pathways by using recombinant DNA and other molecular biology techniques [31, 331, 564], The analysis aspect of metabolic engineering focuses on the identification of important parameters that define a physiological state, use of this information to elucidate the control architecture of a metabolic network, and propose targets for modification to achieve an appropriate objective . The synthesis aspect of metabolic engineering examines the complete biochemical reaction network, focusing on pathway synthesis, thermodynamic feasibility, pathway flux, and flux control . This multidisciplinary field embraces principles from chemical engineering, computational sciences, biochemistry, and molecular biology. Potential advantages of using genetically engineered organisms instead of natural isolates can be :
• The pathway can be turned on in situations where it would normally be suppressed (e.g. degradation of a hazardous compound to a concentration lower than necessary to induce the pathway in the natural isolate),
• High levels of an enzyme in desired pathways can be obtained by the aid of strong promoters,
• A single promoter can be used to control the pathways moved from lower eucaryotes to bacteria, keeping in mind that each protein is controlled by a separate promoter in lower eucaryotes,
• Several pathways can be combined in a single recombinant organism by recruiting enzymes from more than one organism,
• A pathway can be moved from a slowly growing organism into a more easily cultured organism,
• The genetically engineered cell can be proprietary property.
Product biosynthesis ranging from primary to secondary metabolite production pathways of microorganisms are of highest interest in metabolic engineering. Biopharmaceutical production via plant and mammalian cell cultures are also of immediate interest due to potential uses in pharmaceutical industry.
Several reviews on metabolic engineering cover general ([86, 87,149, 566] and specific organisms such as yeast , plants [114, 127], and Escherichia coli . Bacteria and yeast have numerous applications in metabolic engineering because they are well-studied microorganisms and genetic tools for these are well-developed. Mathematical representations for their growth and substrate utilization, and product synthesis in these organisms are available in literature. Furthermore, their generation times are relatively small, allowing quick experimentation and development. Many practical applications of metabolic engineering are cited in various papers and books [87, 331, 564, 565]:
• Improvement of yield and productivity of native products synthesized by microorganisms. Examples include ethanol production by Escherichia coli , succinic acid production by E. coli , acetone and butanol production by Clostridium acetobutylicum , production of L-lysine, L-phenylalanine, and L-tyrosine by Corynebacterium sp. [143, 249, 611], L-proline production by Serratia marcescens .
• Expansion of the range of substrates for cell growth and product formation. Examples include ethanol production from xylose (and possibly from hemicellulose hydrolysates) by Saccharomyces cerevisiae , ethanol production from lactose (and possibly from whey) by S. cerevisiae , and ethanol production from starch [60, 238].
• Synthesis of products that are new to the host cell. Examples are various modified and novel polyketide antibiotics by Saccharopolyspora erythraea and Streptomyces sp. , production of 1,3 propanediol by E. coli , and polyhydroxyalkanoate production by a small oilseed plant, Arabidopsis thaliana ,
• Design of improved or new metabolic pathways for degradation of various chemicals, especially xenobiotics. Examples are degradation of mixtures of benzene, toluene and xylene (BTX) by Pseudomonas putida  and degradation of polychlorinated biphenyls (PCBs) by Pseudomonas sp. .
• Modification of cell properties that facilitate fermentation and/or product recovery. Examples include better growth of E. coli and other microorganisms under microaerobic conditions , uptake of glucose without consuming phosphoenolpyruvate in E.coli, and ammonia transport without ATP consumption in Methylophilus methylotrophus ,
These examples are a small subset of many success stories of metabolic engineering that have been reported. They illustrate the various types of approaches that can be undertaken experimentally:
• Extending an existing pathway to obtain a new product
• Amplifying a flux-controlling step
• Diverting flux at branch points ("nodes") to a desired product by circumventing a (feedback) control mechanism, amplifying the step initiating the desired branch (or the converse), removing reaction products, or manipulating levels of signal metabolites.
The interest in metabolic engineering is stimulated by potential commercial applications in that improved methods are sought for developing strains which can increase production of useful metabolites. Recent endeavors have focused on the theme of using biologically derived processes as alternatives to chemical processes. Such manufacturing processes pursue goals related to "sustainable development" and "green chemistry" as well as positioning companies to exploit advances in the biotechnology field. Examples of these new processes include the microbial production of indigo (developed by Genencor) and propylene glycol (developed by DuPont) and other improvements in more traditional areas of antibiotic and amino acid production. The extension of metabolic engineering to produce desired compounds in plant tissues and to provide better understanding of genetically determined human metabolic disorders broadens the interest in this field beyond the fermentation industry and bodes well for increasing impact of this approach in the future .
A number of industrially important aromatic compounds, including the aromatic amino acids and other metabolites, can be produced in microorganisms through metabolic engineering of the aromatic pathway. Plastics and other synthetic polymers, whose desirable properties include chemical and biological inertness, have become essential for a multitude of applications in common consumer products. On the other hand, increasing concern about environmental pollution by these non-biodegradable polymers has created interest in the development of completely biodegradable polymers . Polyhydroxyalkanoates (PHAs) are an important class of biodegradable polymers that can be produced by a number of microorganisms. However, the high production cost and some poor material properties are preventing the use of PHAs in a wide range of applications. Continued pressure to provide aromatic compounds with very low production costs will create new challenges to develop competitive biotechnological processes ,
Traditional strain improvement methods as well as metabolic engineering strategies have been used for enhancing the production of antibiotics and production of novel antibiotics. A wide variety of microorganisms synthesize antibiotics while only clinically useful antibiotics are produced by the eubacteria, Actinomycetes, in particular Streptomyces, and the filamentous fungi. Metabolic engineering techniques are applied for strain improvement to increase the final amount of antibiotics produced in fermentation processes. A typical strain improvement program involves generation of genotype variants in the population, either by means of physically or chemically induced mutations or by recombination among strains . A detailed case study in penicillin producing strain improvement is discussed in Nielsen .
Yeasts have been associated in a number of ways with mankind for the production of alcoholic beverages, baker's yeast, and recently for the production of ethanol, pharmaceutical proteins and enzymes. Other metabolites, including pyruvate, xylitol, carotenoids, and inositol, can be produced by metabolically engineered yeasts. Metabolic engineering strategies have been applied to modify the cellular properties of yeast to improve fermentation and product recovery processes as a result of extended range of substrate utilization. Renewable substrates for extension of substrate range include starch, the most abundant and readily extractable plant biomass, and cellulose, hemicellulose and pectin fractions in lignocellulosic materials, and whey lactose . Traditional approaches include using mixed cultures or multistage operations such as physical and enzymatic pretreatment of substrates prior to fermentation. With the development of recombinant DNA technology, the introduction of heterologous genes into a host yeast facilitates one step conversion of substrates into useful end products (e.g., recombinant Saccharomyces cerevisiae containing a-amylase and glucoamy-lase genes that allow the yeast to grow on starch and convert it into ethanol ).
Another area of application of metabolic engineering is in the production of secondary metabolites that can be used as pharmaceuticals, including anticancer drugs vinblastine and more recently, taxol by plant cells . Some major objectives are:
• Improving nutritional value of crops (e.g., essential amino acid supply for storage proteins, modifying lignin amount or type to enhance forage digestibility)
• Creating new industrial crops (e.g., modified fatty acid composition of seed triglycerides, pharmaceuticals, polyhydroxybutyrate synthesis, bioremediation)
• Altering photosynthate partitioning to increase economic yield
• Enhancing resistance to biotic and abiotic stresses
• Reduction of undesired (toxic or unpalatable) metabolites
• Using them as research tool to test basic ideas about metabolic regulation.
No single discipline can bring about the successful development and applications of metabolic engineering . Metabolic engineering offers one of the best ways for meaningfully engaging chemical engineers in biological research for it allows the direct application of the core subjects of kinetics, transport, and thermodynamics to the reactions of metabolic networks . With the advent of genomics and proteomics, enormous amounts of information on the genetic and protein makeup of various microorganisms are becoming available. As a consequence, bioinformatics will play an increasingly significant role in the evolution of metabolic engineering. Also, directed evolution of enzymes will become a powerful tool for the generation of enzymes or even metabolic pathways suitable for given tasks. These improvements in metabolic engineering will lead to reshaping the biotechnology endeavor, giving rise to more precise, more focused and more effective bioprocessing and intervention at the cellular and organismal levels. Furthermore, it will also bring more control at all levels (gene expression and protein translation, protein, metabolite, pathway, and flux levels).
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