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Metabolic Engineering Perspectives

Nian Liuand Gregory Stephanopoulos

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

1.1 History and Overview of Metabolic Engineering

Metabolic engineering emerged in the late 1980s primarily to capitalize on the advent of recombinant DNA technologies that allowed, for the first time, the direct genetic modification of microbial cells. Its original manifestation was with the first Metabolic Engineering conference in 1996, which was renamed after the conference of "Recombinant DNA Biotechnology III: The Integration of Biological and Engineering Science." The peer-reviewed scientific journal, Metabolic Engineering, soon followed, and the book, Metabolic Engineering: Principles and Methodologies, completed the essentials of a new discipline. Even prior to its formal establishment, the ideas of metabolic engineering had already emerged. In a sense, the field was preceded by the study of mixed cultures: If a particular conversion from substrate A to a desired final product B could not be accomplished by a single organism, which could only convert A to an intermediate I, then it was logical to complement the organism with another species that completes the route from I to B. Recombinant technology essentially allowed the isolation and transfer of genes comprising the pathway from I to B so that the complete conversion could be accomplished in a single organism. Despite the declining interest in mixed cultures after that, many concepts of coexistence and stability as well as related mathematical methods of nonlinear dynamics and bifurcation theories found their way to the analysis of recombinant cultures in various configurations.

Of course, metabolic engineering would not be in its current position without molecular biology, which lies at the heart of modern biotechnology with numerous applications. In plant sciences, they enable the introduction of new, useful traits in crops such as draught and salinity resistance [1]; in the medical area, they facilitate the identification of genes underlying a disease and the development of gene therapy as a cure [2]; in environmental applications, they are used for the degradation of recalcitrant compounds [3]. In the microbial world, the central objective of metabolic engineering and associated industrial biotechnology applications is the overproduction of chemical and fuel products either native to an organism or newly synthesized through the introduction of a heterologous pathway. A microorganism is thus converted into a "chemical factory" to execute new biochemistry through its numerous native and non-native enzymes. Many parallels can be drawn between this approach and conventional chemical processes. For instance, just like how chemical conversions are determined by the stoichiometry, kinetics, and thermodynamics of reactions, a microbial pathway is also defined by these physical parameters of the constituent enzymes. Similar to the necessity of identifying rate limiting steps in chemical processes, a central goal of metabolic engineering is also the analysis of bottlenecking steps in a biochemical reaction network. A key difference between the two is that, while methods to overcome limiting steps are limited in chemistry, there are molecular biology tools including gene deletion and overexpression that can specifically target bottlenecking enzymes to enhance the overall cell productivity.

At this point, one commonly asked question by many scientists and engineers is "Why should one use microbes instead of chemistry to carry out these reactions?" The answer lies in the unique ability of enzymes to conduct complex chemistry with high specificity. Thus, cell-catalyzed processes will be the preferred methods for making more intricate molecules such as pharmaceuticals, vitamins, proteins, probiotics, and other similar compounds. While biotechnology can make most of these products in a few steps, chemical methods would require a much longer synthesis route including a series of unavoidable protection-deprotection steps to accomplish the same goal. The second class of applications where biotechnology is likely to be superior is sustainable production, which requires the use of renewable feedstocks. Sugars, as a prime example, tend to be highly reactive and attempts at modifying them using organic chemistry techniques will commonly induce many byproducts. On the other hand, these renewable compounds are the perfect substrate for most microorganisms. With the power of molecular biology and metabolic engineering, sugars can be converted to the target product (organic acids, alcohols, biopolymers, solvents, and many other chemical products) with high yield and specificity. Correspondingly, these applications have fueled much interest in metabolic and microbial cell engineering to achieve diverse goals.

Despite the focus of product biosynthesis, it should be noted that the methodology of metabolic engineering is applicable to nearly all areas of biotechnological activity. For example, judicious choice of isotopic tracers and analysis of labeled metabolites identified the function of a reverse TCA cycle in cancer cells under hypoxia [4]. This discovery had profound implications on our understanding of cancer metabolism and its treatment. In plant sciences, transferring genes with unknown functions into yeast cells and characterizing the metabolic steps in microbes has led to the elucidation of a new pathway responsible for cucurbitacin synthesis, which is used by plants for defense against pests [5]. A similar strategy has also been used to identify naturally synthesized herbicides that are highly effective [6]. These are just a few examples illustrating the broad application of metabolic engineering tools developed for the purpose of understanding and manipulating cell physiology, and there is no doubt that these tools will find further applications in the times to come.

Nevertheless, to maintain a tighter focus, in this book we will keep our discussion within processes that use microbial biocatalysts as the enabling element. Hence, this volume is mostly dedicated to microbial systems and reviews the issues and methods related to improving the capability of host cells to produce useful products. Host selection, pathway design and expression, assessment of pathway function, elimination of stoichiometric limitations and kinetic bottlenecks, and evaluation of cell performance in bioreactor environments are all core topics underlying the various chapters. Experimental and mathematical tools that help achieve strain optimization are also discussed.

For the remainder of this chapter in particular, we will briefly touch upon the central ideas of metabolic engineering. First and foremost, it is clear that metabolic engineering is related closely to microbial metabolism. This relationship is further addressed in the next section where computational and experimental methods that dissect cellular physiology are presented. Particular attention is given to methods probing cell-wide and genome-wide properties as they provide a holistic view of the entire cellular metabolism instead of a local one, and this is a hallmark of metabolic engineering. In Section 1.3, we examine the two general approaches to engineering a better cell catalyst, rational and combinatorial, along with systems metabolic engineering which combines the two. In the final few sections, we examine other important topics of metabolic engineering, such as host cell selection, substrate considerations, and synthetic biology, before closing with an assessment of the state of the field and its future directions.

1.2 Understanding Cellular Metabolism and Physiology

In metabolic engineering, cellular metabolism is viewed as a network of biochemical reactions that can be exploited to convert a starting substrate to the final product through a sequence of steps. The traditional approach to metabolic engineering relies on initially developing a systematic understanding of the metabolic network with an eye on kinetic bottlenecks and stoichiometric limitations, and then applying this knowledge to engineer pathways that funnel fluxes toward the desired substance. Earlier efforts that utilized this methodology oftentimes focused on a more "localized" view of metabolic pathways in that only the steps directly connecting the substrate to the product were considered. This paradigm, despite largely simplifying the complexity of biological systems, has seen great success in terms of improving the titer, productivity, and yield of several bioproducts, such as amino acids [7]. Since the number of reactions that needs to be considered is relatively small, it is possible to manually interrogate each enzyme to determine its kinetic and thermodynamic limitations, shedding light on how the properties of individual steps affect flux through the entire pathway. Once this is known, an overall engineering strategy can hence be formulated and subsequently carried out. However, successes in employing this "localized" view are limited to situations where only a few enzymatic steps are relevant, thereby restricting its range of applications. As the field progressed, the biosynthesis of complex molecules with more structural and functional diversity quickly became the focus of many researchers. Correspondingly, new tools and methods have been developed to better understand cellular metabolism and guide engineering on a "global" scale....