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1、CHAPTER 1 The Essence of Metabolic Engineering The concept of metabolic pathway manipulation for the purpose of endowing microorganisms with desirable properties is a very old one indeed. We have many outstanding examples of this strategy in the areas of amino acids, antibiotics, solvents, and vitam
2、in production. These methods rely heavily on the use of chemical mutagens and creative selection techniques to identify superior strains for achieving a certain objective. Despite widespread acceptance and impressive successes, the genetic and metabolic profiles of mutant strains were poorly charact
3、erized and mutagenesis remained a ran- dom process where science was complemented with elements of art. The development of molecular biological techniques for deoxyribonucleic acid (DNA) recombination introduced a new dimension to pathway manipu- lation. Genetic engineering allowed the precise modif
4、ication of specific enzymatic reaction(s) in metabolic pathways and, hence, the construction of well-defined genetic backgrounds. Shortly after the feasibility of recombinant DNA technology was established, various terms were coined to represent the 2 Metabolic Engineering potential applications of
5、this technology to directed pathway modification. Some of the terms suggested were molecular breeding (Kellogg et al., 1981), in vitro evolution (Timmis et al., 1988), (microbial or metabolic) pathway engineering (MacQuitty, 1988; Tong et al., 1991), cellular engineering (Nerem, 1991), and metabolic
6、 engineering (Stephanopoulos and Vallino, 1991; Bailey, 1991). Although the exact definition varies from author to author, all convey similar meanings with respect to the general goals and means of metabolic engineering. Here we define metabolic engineering as the directed improvement of product for
7、mation or cellular properties through the modification of specific biochemical reaction(s) or the introduction of new one(s) with the use of recombinant DNA technology. An essential characteristic of the preceding definition is the specificity of the particular biochemical reaction(s) targeted for m
8、odification or to be newly introduced. Once such reaction targets are identified, established molecular biological techniques are applied in order to amplify, inhibit or delete, transfer, or deregulate the correspond- ing genes or enzymes. DNA recombination in a broader sense is routinely employed a
9、t various steps toward these ends. Although a certain sense of direction is inherent in all strain improvement programs, the directionality of effort is a strong focal point of metabolic engineering compared to random mutagenesis, as it plays a dominant role in enzymatic target selection, experiment
10、al design, and data analysis. On the other hand, direction in cell improvement should not be interpreted as rational pathway design and modification, in the sense that it is totally decoupled from random mutagenesis. In fact, strains that are obtained by random mutation and exhibit superior properti
11、es can be the source of critical information about pathway configuration and control, extracted via reverse metabolic engineering. As with all traditional fields of engineering, metabolic engineering too encompasses the two defining steps of analysis and synthesis. Because metabolic engineering emer
12、ged with DNA recombination as the enabling technology, attention initially was focused, almost exclusively, on the syn- thetic side of this field: expression of new genes in various host cells, amplification of endogenous enzymes, deletion of genes or modulation of enzymatic activity, transcriptiona
13、l or enzymatic deregulation, etc. As such, metabolic engineering was, to a significant extent, the technological manifes- tation of applied molecular biology with very little engineering content. Bioprocess considerations do not qualify as metabolic engineering. A more significant engineering compon
14、ent can be found in the analytical side of metabolic engineering: How does one identify the important parameters that define the physiological state? How does one utilize this information to elucidate the control architecture of a metabolic network and then propose rational targets for modification
15、to achieve a certain objective? How does one 1 The Essence of Metabolic Engineering 3 further assess the true biochemical impact of such genetic and enzymatic modifications in order to design the next round of pathway modifications and so on until the goal is attained? Instead of the mostly ad hoc t
16、arget selection process, can one prescribe a rational process to identify the most promising targets for metabolic manipulation? These are some of the ques- tions that the analytical side of metabolic engineering would address. On the synthetic side, another novel aspect of metabolic engineering is the focus on integrated metabolic pathways instead of individual reactions. As such, it examines complete biochemical reaction networks,
