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Biotechnol. Prog. 1998, 14, 820

Mathematical Modeling and Analysis in Biochemical Engineering: Past Accomplishments and Future Opportunities
James E. Bailey
Institute of Biotechnology, ETH Zu rich, 8093 Zu rich, Switzerland

This is a personal commentary on the history and future prospects of mathematical modeling and analysis in biochemical engineering. Major transitions in these fields were driven by the appearance of the Aiba, Humphrey, and Millis text, Fredricksons guidance on conceptualizing mathematical representations of cell populations, and Ramkrishnas development of the cybernetic modeling approach. The value of mathematical models to organize data, to consider interactions in complex systems in a rational way, to correct the conventional wisdom, and to understand essential qualitative features of biological systems has been clearly documented in prior research. The impact of this research in biotechnology discovery has so far been limited, but this will change in the future if we are adept in recognizing emerging opportunities and in integrating new concepts and tools into our research. Mathematical structures and methods, allied with extraordinary contemporary computing power, are essential to the emerging field of functional genomics. Important in this quest is a hierarchy of powerful modeling, analysis, and computational tools which can capture essential quantitative features of available experimental data and use these effectively for analysis and design of metabolism.

Foreword
This paper is based upon a keynote lecture given by the author at the conference Biochemical Engineering X following an invitation by the conference chairs to review the... achievements of the last half-century and to bring the younger generation of biochemical engineers into the community. Thus empowered to approach the topic as an old timer, this text is a personal, explicitly opinionated, perspective on the topic and is not intended as a scholarly review. Furthermore, I have extrapolated somewhat the Chairs charge and have devoted considerable attention to the present state of the art and its implications for future contributions of mathematical models and methods in biotechnology, medicine, and the biosciences. Absent the present circumstance requiring single authorship to preserve the province of personal commentary for this work, Vassily Hatzimanikatis would appear here as coauthor. Through our close association in the past 7 years, he has substantially enriched my perspectives of this field. Moreover, he has contributed greatly to the hierarchy of models and methods summarized below. However, full responsibility for the selection of topics, and the opinions, contained herein rests with me.

Introduction
By definition, biochemical engineers are concerned with biochemical systems, often with systems employing growing cells. Even the simplest living cell is a system of such forbidding complexity that any mathematical description of it is an extremely modest approximation. This situation prompts the question: from a formal, logical viewpoint, what is the relationship between a physical system and a mathematical description of it? Contemplation of this question leads, in one direction,
S8756-7938(97)00126-4 CCC: $15.00

into the labyrinths of the philosophy of science. Guidance into this territory from the learned guide Rutherford Aris led me to John Castis two-volume treatise Reality Rules, which explores the general definitions of a mathematical model as well as numerous specific examples of models in different contexts (Casti, 1992a,b). Gratefully for engineers who are (or should be) engaged in discovery and development of useful technology, Casti asserts an inextricable coupling between a model and its intended application: Basically, the point of making models is to be able to bring a measure of order to our experience and observations, as well as to make specific predictions about certain aspects of the world we experience (italics added) (page 2, Casti, 1992a). Therefore, mathematical modeling does not make sense without defining, before making the model, what its use is and what problem it is intended to help to solve. The use of mathematics to provide a rigorous, systematic, and quantitative linkage between molecular and microscopic phenomena on one hand and macroscopic process performance on the other was permanently installed into the pedagogical and research principles of chemical engineering through the pioneering efforts of Bird, Stewart, and Lightfoot (Bird et al., 1960), Amundson and Aris (1958), and others starting around 1960. This chemical engineering science paradigm is evident in a number of publications in the sphere of biological process engineering in a similar time frame but was not explicitly articulated, in a unified and accessible form, until the appearance of the revolutionary textbook Biochemical Engineering by Aiba, Humphrey, and Millis in 1973 (Aiba et al., 1973). This book marks a breakthrough from an earlier descriptive, empirical approach to a modern era in which rigorous mathematical reasoning is applied to achieve understanding of key relationships and identification of defining parameter groups.

1998 American Chemical Society and American Institute of Chemical Engineers Published on Web 01/17/1998

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Figure 1. Classifications introduced by A. G. Fredrickson for mathematical (and other) representations of cell populations.

Arnold Fredrickson contributed a systematic framework for viewing cell populations which has shaped the way that biochemical engineers conceive of, mathematically describe, and experimentally characterize systems involving living cells (Tsuchiya et al., 1966; Fredrickson et al., 1970). Fredrickson introduced the term segregated, to indicate explicit accounting for the presence of heterogeneous individuals in a population of cells, and the term structured, to designate a formulation in which cell material is composed of multiple chemical components. The four different combinations of segregation and structure, or their absence, introduced by Fredrickson (see Figure 1 for a summary) conveniently partition the most common types of mathematical descriptions and of experiments into only four disjoint categories. Most models and most measurements belong to the nonsegregated class, as do all examples presented here. Chemical reaction engineering (CRE) is a tributary of the chemical engineering science watershed which has successfully established a predictive scale-up and optimization capability for complex synthetic catalytic systems by explicit consideration of chemical and physical details at increasingly finer scales. Although the same approach finds useful engagement in biochemical process modeling, a completely different avenue of thinking has emerged. Growing from seeds planted in Minnesota in the 1960s, Ramkrishna and co-workers have created a novel, powerful approach to description of biological phenomena which they have named cybernetic modeling (Dhurjati et al., 1985; Kompala et al., 1986). The unifying and central concept of cybernetic models is the hypothesis that cells function, and allocate limited resources, in a fashion that optimizes the resulting outcome for the cells. Thus, in a cybernetic model, formulation of the hypothesized objective function takes the place of a mechanistic description in terms of molecules which regulate gene expression or determine enzyme activities. Calculation of the solution to an optimization problem replaces numerical solution of conservation equations on these molecules. This is a profound conceptual leap from the reductionist, increasingly detailed, traditional viewpoint of chemical reaction engineering, and one which has not been sufficiently appreciated in biochemical engineering research. Cybernetic models have enjoyed impressive success in describing, quantitatively, complex phenomena such as the dynamics of mixed substrate utilization (Ramakrishna et al., 1996; Straight and Ramkrishna, 1994)) and functioning of storage pathways (Varner and Ramkrishna, 1997a,b). For synthetic catalysts there is no possibility of predicting behavior on the basis of assertion of an inbuilt survival, self-optimizing response,

but Ramkrishnas cybernetic principle is certainly a reasonable component of modeling strategies for biological systemssperhaps even a necessary component in many situations in which details of mechanism are unknown. Adaptation of this cybernetic approach to cells genetically modified for enhanced production (and thus following a human-directed, diverging evolutionary path) is an intriguing challenge. However, until metabolic engineeering (Bailey, 1991) progresses much further in genetically reprogramming cells for appplications, most of the cells functions derive from earlier, natural evolution. Next we consider some examples of different ways that mathematical models in biochemical engineering have been, and can be, used. Although biochemical engineering encompasses many different types of processes including heat and mass transfer and product recovery, with different constituents and dominating phenomena, much of the biochemical engineering mathematical modeling research literature concerns biological reaction processes and, recently, processes in cells. This is my field of experience, and, therefore, the specific examples discussed concern these applications.

Mathematical Models of Biological Systems

There is a zoo of mathematical models in the biochemical engineering and mathematical biology literature. Many of these appear, particularly to the naive reader (and sometimes to the sophisticated one), to have little purpose other than calculating numbers which conform reasonably to experimental data. This is, in itself, not a distinguished endeavor; it is not particularly difficult, and it teaches little. I believe that one reason mathematical biology receives so little respect from biological scientists, and is generally not recognized as a credible research tool in biological science and in biotechnology discovery, is failure to communicate clearly and persuasively the reason for making the model. Sometimes, I fear, this is because the modeler him(her)self has not clearly asked this question while doing the work. As noted in Castis pithy comment above, modeling is relatively meaningless without explicit definition, at the outset, of its purpose. The following examples, all based on biochemical engineering research, illustrate several different good reasons, each important in some venue of biotechnology, for making mathematical models. Why Make Models? Organizing Disparate Information into a Coherent Whole. Molecular biology, and the biotechnology it has created, has thrived in past decades upon the basis of strict reductionism which typically proves a relationship between different molecules, or parts of them, by direct in vitro experimentation with the isolated molecules. This research has generated vast amounts of information about the components involved in various biological process and their properties. However, relatively little synthesis of the resultant encyclopedia of basic biological knowledge has occurred, probably because the intuitive and strategic bases for acquiring this knowledge are, in some respects, contradictory to synthetic, integrative thinking and understanding. Unexpectedly rapid progress in sequencing entire genomes of several microorganisms, and the resulting technologies which will rapidly accelerate other genome sequencing projects, has, in my opinion, created a crisis in the biological sciences. Now, there is no need to dissect a genome and to painstakingly identify assorted genes within it, since the entire pallette of genes is accessible on the Internet. Suddenly, and now inescapably, comes the question: what do these genes do, acting together? Answering this difficult question, which will

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Figure 3. Schematic diagram of important DNA domains, regulatory proteins, and their interactions in controlling replication of the plasmid dv (adapted from Lee and Bailey, 1984). Figure 2. Schematic diagram of the major components and reactions included in the first generation of E. coli single-cell models formulated by Shuler and co-workers (adapted from Domach et al., 1984). A1 ) ammonium ion; A2 ) glucose (and associated compounds in the cell); W ) waste products (CO2 H2O, and acetate) formed from energy metabolism during aerobic growth; P1 ) amino acids; P2 ) ribonucleotides; P3 ) deoxyribonucleotides; P4 ) cell envelope precursors; M1 ) protein (both cytoplasmic and envelope); M2RTI ) immature stable RNA; M2RTM ) mature stable RNA (rRNA and tRNAsassume 85% rRNA throughout); M2M ) messenger RNA; M3 ) DNA; M4 ) nonprotein part of cell envelope (assume 16.7% peptidoglycan, 47.6% lipid, and 35.7% polysaccharide); M5 ) glycogen; PG ) ppGpp; E1 ) enzymes in the conversion of P2 to P3; E2, E3 ) molecules involved in directing cross-wall formation and cell envelope synthesis; GLN ) glutamine; E4 ) glutamine synthetase. * indicates that the material is present in the external environment.

be discussed again later in this commentary, clearly requires a capability for integrated consideration of many interacting components. Michael Shuler and co-workers launched a visionary project around 1980 (Domach et al., 1984; Domach and Shuler, 1984). Their goal was to construct a large-scale computer simulator of growth of a single cell of the bacterium Escherichia coli which was built from, and therefore constrained by, the basic processes of nutrient uptake and catabolism, production of precursor metabolites, synthesis of macromolecules, and cell envelope and cell volume accumulation (Figure 2) and which would calculate, as a function of glucose concentration in the medium, the doubling time and the volume of the cell as an output of this simulation. This was a heroic undertaking at the time because no one before had made the effort to search through a sea of information in the biochemistry, microbiology, physiology, and genetic literature to estimate all of the numbers required to formulate explicit quantitative equations describing the fundamental classes of metabolic reactions described in the textbooks and to integrate this into a single computer code. Furthermore, the whole idea of calculating how long the computer cell would require to reproduce itself, as a natural consequence of the simulation of processes within the cell, was a completely new idea which broached, explicitly, the critical question: do we really understand enough about this most well studied and best known type of cell to calculate, on the basis of all this information, how long it takes to reproduce itself? The Shuler singlecell model introduced, by a substantial margin, a much finer chemical representation of the composition of the cellsor, to use Arnold Fredricksons terminology, greater structuresthan had been considered previously. Scores of parameter values were painstakingly extracted from a like number of diverse references, and the whole was assembled into a computer code which, at its time, was a significant challenge to available computing power. The model successfully achieved its goals and, subsequently,

served as a platform for further development to address other questions, such as simulation of mixed nitrogen source utilization considering different uptake regimes (Shu and Shuler, 1989, 1991) and perturbations in metabolism resulting from introduction of recombinant plasmids (Peretti and Bailey, 1986, 1989). Shulers project was prescient in another fashion: it anticipated the coming explosive expansion of computing power, such that a serious mainframe calculation, running substantially slower than real time in 1984, has become an almost instantaneous desktop result at present. Why Make Models? To Think (and Calculate) Logically about What Components and Interactions Are Important in a Complex System. From DNA Sequence to Phenotype: The Genetically Structured Models of S. B. Lee. The number of plasmids per cell can be an important determinant of cloned protein production by recombinant microorganisms. By the early 1980s, built upon a base of extensive prior research on phage , a number of particular proteins and nucleotide sequences implicated in controlling replication of the plasmid dv had been identified (Figure 3; Lee and Bailey, 1984a,b). Moreover, experimental information was available on, for example, equilibrium constants for binding of particular proteins to particular sites on the DNA, and also effects of mutations in DNA sequences involved in these particular DNA-protein interactions on overall characteristics of the system were known. All of this information fits into a reasonable conceptual, qualitative picture of the overall system. However, a coherent, internally consistent quantitative calculation of the operation of this system is far beyond the capability of the human mind, due to the number of different components and binding sites involved and their coupling with host cell growth and with other processes directly involved on the plasmid. Therefore, the question arises whether, when all of these interactions and relationships are written down systematically, in explicit mathematical language, the resulting mathematical model exhibits behavior consistent with experimental observations? Translating the schematic picture in Figure 3 into a corresponding mathematical formulation involved several new considerations, including efficient calculation of complex repressor-multiple operator equilibria and also formulation of a mathematical representation for transcriptional activation of an origin of replication. S. B. Lee successfully addressed these issues, arrived at a detailed mathematical model based on intracellular material balances for the regulatory proteins and DNA sequences involved, and demonstrated, using mostly previously published parameters, that the characteristics of the wild-type system could be simulated. Thus, this mathematical modeling research proved, arguably for the first time, that the picture of molecular-level interactions and regulation described previously in the molecular biology literature, and schematically illustrated here in

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Figure 3, is in fact consistent with the observed behavior of the system as a whole, operating within a growing cell. Any model is more credible if it can also describe, in a consistent fashion, situations different from the one on which the original model is based. Lee further demonstrated the value of the hypotheses depicted in Figure 3 by showing the capability of the resulting model to simulate both the number of plasmids per cell and the quantity of a key regulatory protein called the cro repressor for a number of mutant plasmids. However, in this respect, Lee went considerably further conceptually and methodologically and established a precedent for a key idea which will undoubtedly become widely embraced in the future. Lee coined the term genetically structured model for this concept, extending the concept of structure, introduced by Fredrickson for chemical composition, to the level of nucleotide sequence. The essence of Lees concept is explicit articulation of a direct linkage between a particular nucleotide sequence (in one of the operator sites) and the affinity of a particular protein for that sequence, which in turn influences a corresponding transcription event, which affects the frequency of initiation of plasmid replication, and, ultimately, which determines the number of plasmids per cell (terms here refer to this particular example, but the genetically structured modeling concept is obviously more general). Thus, with this type of model, there is an explicit, unambiguous, quantitative mapping from nucleotide sequence to overall phenotype. All that is lacking to implement this connection in full, for this example, are equilibrium binding parameters for mutant plasmids with modified operator sequences. However, given the known loci of the mutations for particular mutant plasmids, Lee knew exactly which model parameters should change for a given mutant (and all others should not). Further, he adopted the reasonable constraint that binding affinity of the repressor protein for mutant operator sequences would be reduced by any mutation. Thus constrained, he was able to determine altered binding affinities for each particular mutant plasmid which, when utilized in the mathematical model equations, correctly calculated plasmid number and repressor level. Clearly, as more experimental data become available on protein-nucleic acid interactions, and as theoretical possibilities to predict effects of sequence changes on these parameters improve, such genetically structured models can provide a unique resource for predicting the relationship between nucleotide sequence and complex functions of the organism. Why Make Models: To Discover New Strategies. The environment of a cell determines not only the rate of processes conducted by that cell but also which types of processes operate in that cell. The classic example of this, well-known by bakers and brewers far before arrival of an era of quantitative physiology, is the switch in metabolism exhibited by bakers yeast (Saccharomyces cerevisiae) when external glucose concentration is increased in an aerated medium. At low glucose concentrations, the yeast respires and produces predominantly cell mass and CO2 from consumed glucose. However, if the glucose concentration exceeds about 5 g/L, the yeast instead conducts aerobic fermentation and produces now biomass, ethanol, and more CO2 per glucose consumed than in the respiratory metabolism situation. Notice also in this example that the alteration in metabolism driven by the external environment is manifested in that external environment in terms of ethanol production and greater CO2 production when aerobic fermentation occurs.

Early pioneers in employing digital computers to analyze and control biochemical processes overcame challenges in hardware and software development, and interface construction, which are almost impossible to comprehend from a contemporary perspective of awesome desktop computing power, facile software, and versatile plug-in interfaces. Among this group, L. Nyiri (1972) clearly articulated in general mathematical terms the notion that cell metabolism is reflected in measurable quantities of the culture and, in turn, that metabolism could be controlled by manipulation of environmental parameters such as pH, temperature, dissolved oxygen, aeration rate, and other operating variables. In roughly the same time frame as robust mass spectrometers suitable for continuous on-line monitoring of gas composition under industrial conditions became available, Cooney, Wang, and Wang, in a pioneering study, showed clearly how off-gas measurement, a mathematical model relating off-gas composition to cell metabolism, and computer control could be used together to control glucose feeding for efficient growth of bakers yeast to very high cell densities (Cooney et al., 1977). The basic concept derives simply from the above classic example: the supplied glucose must be matched to the capacity of the yeast to utilize this glucose in respiratory metabolism, and glucose must not be overfed such that its concentration increases to a point which activates aerobic fermentation (since this produces ethanol which is a waste of carbon substrate and which also inhibits growth of the yeast). Although the model used here is not complex from a mathematical point of viewsit is comprised only of the stoichiometric relationships describing carbon conversion under respiratory and fermentative conditionssthese relationships are the crucial link enabling implementation of an algorithm for operating the process effectively based on accessible on-line measurements of the process. Besides its historical significance, this example has had important industrial impact. The concept of balancing carbon source supply with capacity of the organism to use that carbon in an efficient respiratory metabolism applies also to many other processes, such as growth of E. coli (which, in environments of high glucose concentration, produces acetate, which is highly inhibitory to growth and metabolic activity). Production of cloned proteins in bacteria, yeasts, and other hosts has come to rely on this basic principle of balancing to identify medium formulations and feeding protocols which allow propagation of recombinant cells to high cell densities, thereby increasing the volumetric productivity of the cloned protein. Before implementation of this strategy, several early biotechnology companies were achieving final cell densities in E. coli fermentations of about 1 g/L, while, with these metabolically coupled feeding strategies, the cell density and the bioreactor production capacity are increased by more than one order of magnitude. Why Make Models? To Make Important Corrections in the Conventional Wisdom. Many situations encountered in biochemical engineering, and in biological science research, are extremely complicated. In such a situation it is easy to make mistakes, from relatively specific ones, such as misinterpreting results of an experiment, to major conceptual errors. Mathematical modeling, and analysis of the resulting model, can aid substantially in avoiding such mistakes or in identifying errors or omissions in earlier thinking and interpretations. Two important examples of this class of applications of mathematical models are discussed next.

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Shear Damage to Cultured Mammalian Cells. If you find a review or commentary article, or workshop proceedings, from the early 1980s on needs in biochemical engineering research, or how biochemical engineers can contribute to important challenges in the then-emerging biotechnology industry, you will undoubtedly find statements centered on the following proposition: Mammalian cells do not have the strong outer envelopes of microorganisms, and these much more fragile cells therefore are damaged in the agitated bioreactors traditionally employed for microbial cultivations. Special bioreactors, or new types of cultivation media or conditions, are needed in order to avoid shear stress damage to mammalian cells cultured in vitro. (For a definition of shear stress, see Bird et al. (1960), p 4.) Like most common misconceptions, this statement seems reasonable enough, and indeed, it has motivated a large body of research and development work in biochemical engineering since (including some in my lab, then at Caltech!). However, this statement contains several major errors, which can be exposed by systematic, quantitative reasoning following the logic of chemical engineering science. A more rational understanding of mechanical interactions affecting mammalian cells in vitro begins with the question: what forces ultimately act on the cell to distort or disrupt it, thereby killing the cell or altering its function? Conceptual and quantitative guidance for this question comes from a large body of earlier literature dealing with break-up of dispersed phases in multiphase systems. First, this analogous situation teaches us that turbulence can contribute significantly to disruption and break-up of bubbles and droplets, and therefore, perhaps turbulence may damage dispersed mammalian cells, cell aggregates, or mammalian cells adhering to microcarriers. Energy in a turbulent flow is dissipated by transmittal from large-scale fluctuations to smaller-scale fluctuations which finally are dissipated by viscous flow. For convenience in speaking about and visualizing these different length-scale fluctuations, they are often referred to as eddies and their characteristic length scale as eddy size. The simplest mathematical description of a turbulent flow, and one that has found several important applications in mixing and mass transfer in bioreactors, is the theory of isotropic turbulence, which considers the turbulent flow to be essentially uniform everywhere in a vessel and which, according to a theory due to Kolmogorov, states that the smallest turbulent eddy found in such a turbulent flow is dictated by the total power input per unit volume. Croughan and Wang undertook a series of experiments to investigate the relationship between power input per unit volume and viability of cells on microcarriers, using the minimum turbulence length scale provided by Kolmogorov theory as the interpreting parameter connecting the two (Croughan and Wang, 1989). This research showed very clearly that agitation of a microcarrier suspension did not cause any damage to the cells until the mixing intensity was sufficient to generate turbulence with a length scale reaching down to the same small length scale as the size of the microcarriers. Later, for individual hybridoma cells, A. McQueen showed that extremely intense flows were needed to damage these cells and that the intensity required was also consistent with Kolmogorov theory (McQueen and Bailey, 1987). These intensities substantially exceed those expected in a stirred-tank bioreactor. These investigations lead to the conclusion, now widely known and practiced industrially, that mammalian cell populations comprised of suspended individual cells can be easily grown without

damage in a single-phase stirred tank vessel, while with microcarriers or cell aggregates, special attention to mixing may be needed. There are other misconceptions in this territory which are not mathematical and therefore do not receive detailed comment here. In brief, beware the concept and term shear damage, and pay careful attention to gasliquid interactions (especially during bubble formation and disengagement, and in foams) and look carefully at the literature on dispersed droplet breakup in shear and extensional flows. Mitogenic Potency of Growth Factors. Many types of mammalian cells require stimulation by exogenous growth factors to proliferate. From a molecular viewpoint, growth factors exert this effect by binding to a particular receptor on the external cell surface, then being internalized, with consequent biochemical response of the cell (induction or repression of gene expression is included here in this term), activation of DNA replication and entry into the cell cycle, and increase in the number of cells. Growth factor function is similar to that of many other proteins, and smaller molecules including a number of pharmaceuticals, in the sense that initial interaction between the affected cell and the external ligand occurs at a particular receptor, followed by some type of signal transduction which activates the response of the cell. Given this mechanism, pharmaceutical companies have devoted massive resources to identification and smallscale production of new receptors to screen protein, chemical, and natural products libraries upon the basis of their affinity for these receptors. To a large degree, the affinity of binding to a receptor has been accepted, without much critical thought, as being synonymous with increased effectiveness of that ligand in activating or inhibiting the response which is accessed via that receptor (see, for example, Hajduk et al., 1997). Douglas Lauffenberger and his collaborators have, over a period of several years, carefully formulated a mathematical description of the network of processes involved in receptor-mediated phenomena (see, for example, Lauffenberger and Linderman, 1993). As just noted, this network includes ligand interaction with the receptor at the outer cell surface, but also integral to the overall network of ligand effect is trafficking of bound receptors into the cell. Armed with this model, and refined methods for assessing its parameters in different situations, Lauffenberger and his collaborators recently reported the striking result that an epidermal growth factor (EGF) mutant which binds more weakly to the EGF receptor than other forms of EGF can have greater activity to stimulate cell division than do other variants because these mutants exhibit more rapid trafficking kinetics (Reddy et al., 1996). Thus, an effective ligand is one that operates more effectively within the entire system of receptor-mediated response and is not necessarily a ligand that binds with higher affinity to the receptor. This important result should lead to more effective screening procedures, presumably depending more on whole-cell assays which exhibit more of the complete receptor-mediated response. A related concept arose earlier in the field of biological transport, where it was recognized that the effectiveness of a carrier depends not only on its affinity for capturing its substrate but also on the carriers transport characteristics and its ability to release the substrate at its destination. Biochemical engineers have contributed centrally to the theoretical foundations of biological

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Figure 4. Operating diagram for proliferation or quiescence of mammalian cells based on a mathematical model of the molecular system (which includes the proteins cyclin E and E2F) controlling transition from the G1-phase to the S-phase (Hatzimanikatis et al., 1995).

transport (Schultz et al., 1974) and to transfer of biological transport strategies into liquid membrane separations. Why Make Models? To Understand the Essential, Qualitative Features. When analyzing or engineering a complex system, it is often sufficient to have certain qualitative results, without a need for particular numerical values. For a specific example important in biotechnology from several respects, consider the transition from the G1-phase to S-phase in a mammalian cell. This particular checkpoint of the eucaryotic cell cycle is a frequent barrier to cell proliferation and is surmounted only by stimulating the cells with exogenous growth factors. My group has been interested in the possibility of activating cultured mammalian cells into a proliferating state in the absence of such exogenous growth factors by genetic engineering to modify expression of particular proteins involved in control of the G1-S transition. To evaluate this possibility, a mathematical model describing this regulatory network was formulated and simulations were undertaken to determine whether the cells remained in a quiescent state or entered a sustained cycling state as a function of the expression level of two key proteins involved in regulating this checkpoint, namely cyclin E and E2F (Hatzimanikatis et al., 1995). Figure 4 summarizes the results of these calculations, showing those combinations of cyclin E expression rate and E2F level which correspond to cell-cycle arrest and cell proliferation. The important qualitative conclusion from these calculations is the possibility, for certain cell states, of achieving a transition from nonproliferating, growth-arrested behavior to actively dividing and proliferating performance either by increasing the cyclin E expression level or by increasing the amount of E2F in the cells. Both of these qualitative indications from the model have been demonstrated experimentally in CHO cells in separate experiments (Renner et al., 1995; Lee et al., 1996). This result becomes increasingly important as concern mounts that use of animal serum or other animal-derived protein in mammalian cell culture risks contamination of the product with prions or other pathogens.

The Impact of Mathematics in Creating and Implementing Biotechnology

My own experience, and that observed for many colleagues, is an evolution of interests and motivations

which, over the years, moves away from more abstract intellectual challenges to a desire to see a tangible, practical outcome of our research. Whether this is a consequence of mental atrophy or acquisition of wisdom is a valid question, but not the current topic. From my old timer perspective, then, I ask the question: what impact in the inception and creation of biotechnology has been made by research on mathematical models and methods? Compared with recombinant DNA technology, microchemical methods, PCR, and other foundations of contemporary biotechnology discovery, the answer to this question, it seems to me, is clear: relatively little. The emphases on applied mathematical research and on biotechnology discovery in the previous paragraph are intended to differentiate its subject from important aspects of biochemical engineering practice. Mathematical models and calculations based upon them are fundamental to design and operation of development and production facilities in the biotechnology industry. The modern practices of chemical, mechanical, and other engineering fields are built upon mathematical tools and their realization in effective computational algorithms. Hence, the implementation of biotechnology, in terms of process scale-up and operation, would not have been possible without application of the mathematical concepts and methods which lie at the heart of modern engineering education. How Can We Mobilize the Power of Mathematics for Invention and for Technology Creation? I believe that, in order for biochemical engineers to play a more important role in the biotechnology industry, we should try to become more involved in the creation of new technologies which meet pivotal, core needs of this industry at the stage of discovery. What future directions in research on mathematical models and methods might provide this kind of impact? One important possibility is contributing to understanding, and applications of, the relationships among genotype, environment, and phenotype (primarily within the domain of human medicine, but also pertinent in agriculture, antiinfective and antiviral pharmaceutical development, and many other areas). These relationships constitute the essence of functional genomics, an extremely visible, active field which has quickly emerged in the wake of unexpectedly rapid success in genome and expressed gene sequencing. With complete sequences of genomes of many organisms now in the databases and large, possibly comprehensive libraries of expressed human gene sequences also in hand (at least privately), science, and the biotechnology industry, faces the difficult yet crucial question mentioned earlier: what does the product of this gene do, within the context of the entire organism? How do various gene products interact to determine healthy or pathological states of the whole organism? Several recent commentaries decry failures of contemporary biological science to understand life. There is a clearly recognized need now to integrate information on components to understand, and to treat, the entire complex system. This cannot be done, systematically, with minimal error, and with maximal constructive output, without mathematical models and related analytical and computational tools. I do not speak here of the bioinformatics technologies which are now receiving great attention in the general science and trade press; these tools are clearly important to organize, search, and otherwise utilize massive amounts of accumulating sequence information. More on this later. Here, I mean instead formulation and application of mathematical models of different aspects of metabolism, whether at the

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cellular, tissue, or higher level. (The term metabolism here denotes all activities of the living cell, which includes, for example, signal transduction and other information transmittal pathways, regulatory interactions, etc., and not only enzymatic conversion of nutrients into various biochemicals). A Critical Role in Functional Genomics. This critical need to achieve systems-level understanding of complex biological functions offers a tremendous opportunity to bring mathematical modeling and analysis into the heart of modern biological science discovery and future advances in molecular medicine. The basic intuitive framework required, the practitioners experience, and facilities with the necessary tools to do this are inherent in modern biochemical engineering. Conversely, these characteristics are in several respects alien to the background of biological scientists and physicians. However, if biochemical engineers are to realize their potential to achieve a major contribution, we must make the effort to learn the important biology and the main biological and medical problems and we must then devise creative ways to contribute. We must also undertake the often painful and frustrating task, for all involved, to communicate our methods, and our insights, to our colleagues in biology and medicine. Without their understanding, our work will remain where much of it has languished, in a small, closed society of kindred spirits who speak a common dialect shared with few others. A Hierarchy of Methods for Utilizing Information on Metabolism. Prior research by biochemical engineers, and others active in theoretical biology, has created powerful mathematical machinery for understanding, and manipulating effectively, complex biological systems. Vassily Hatzimanikatis and I have developed our own perspective on this machinery, viewed as a hierarchy of increasingly sophisticated experimental information, its mathematical representation, associated analyses, and corresponding conclusions or outputs. This hierarchy develops from top to bottom in Figure 5, where external, in/out, and within refer to boundaries around a particular system defined by the researcher, which could be a particular metabolic network within a cell, a particular organelle, one cell, a collection of cells (the situation assumed here), or a society of many organisms, among many possibilities. Items in italics denote input experimental information, items in shaded boxes indicate the quantitative characterization arising directly from the experimental and direct quantitative characterization above in the diagram, and bold text denotes outputs or analyses that are accessible at a given level of quantitative characterization of the system. To illustrate application of this hierarchy, we tour it briefly, taking as an example nongrowing S. cerevisiae cells conducting batchwise conversion of substrate glucose to ethanol and other products in an anaerobic, isothermal buffer solution. Measurements of the time trajectories of glucose, ethanol, and glycerol concentrations (the top italic text) can be analyzed, taking into account the known stoichiometric structure of the intracellular reaction network and assuming quasi-steady state for intermediate, intracellular metabolites, to obtain estimates of the intracellur fluxes (i.e., reaction rates) of the invloved pathways. Many different tools for undertaking such a metabolic flux analysis, and many results based upon this method, are available in the literature (for some recent applications, see Vallino and Stephanopoulos (1993), Varma and Palsson (1994), Sauer et al. (1996), Weichert and De Graf (1997), Takiguchi et al. (1997), and Schmidt et al. (1997)). Knowing fluxes inside

Figure 5. Hierarchy (with detail increasing from top to bottom) of mathematical structures for utilizing experimental information to derive additional insights into cell function and for design of improved function. Primary experimental information is shown in italics directly derived information in shaded boxes, and results of model analysis in bold text.

the system greatly increases our understanding of it, particularly after examining responses of the internal flux distribution to genetic or environmental changes designed to improve the process or to reveal more information about the metabolic system. Adding experimental data on pertinent intracellular metabolites into the picture, accessible in this situation by in vivo NMR spectroscopy and more generally available by cell fixation followed by diverse assays, a kinetic modelsthat is, reaction rate expressions and their parametersscan be formulated. The simplest such models are linear relations between rates and the concentrations which affect them, and powerful algorithms for parameter estimation (e.g., Schlosser et al., 1993) are available for this case. However, enzyme kinetics are often nonlinear under conditions of interest. Some of this nonlinearity may be retained to good approximation by a transformation of variables (into new variables which are the logarithmic deviations from reference steady-state conditions) which gives rise to material balances which are nonlinear in the original rate and concentration variables but which are linear in the transformed variables. Such a (log)linear model has a number of advantages, summarized elsewhere (Hatzimanikatis and Bailey, 1997). Central among these is its linear mathematical character, which opens the door to a body of extremely powerful general linear systems theory (concerning, for example, stability and robustness) and with sophisticated computational algorithms available for linear systems (such as the mixed-integer linear programming optimization al-

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gorithm; see, for example, Hatzimanikatis et al. (1996)). Also, this class of models can be used to estimate shifts in time-average properties of the system when exposed to temporal oscillations in environment, a common situation, for example, encountered by cells circulating in spatially inhomogeneous large-scale stirred bioreactors. Another feature of the (log)linear formulation is its explicit use of parameters (although in a significantly different context) which are identical with those which are central in a body of prior theoretical and experimental work called metabolic control analysis (MCA), thereby providing an interface with prior studies organized around MCA themes. MCA is a good illustration of the critical importance of effective communication between mathematics and biology. The most famous outputs of MCA are the flux control coefficients, which are the derivatives of the logarithm of a chosen flux (typically to the desired product) with respect to the logarithm of some parameters (typically the amounts of various enzymes in the network of metabolic reactions which produces the product). Thanks to a lovely initial exposition of the physical interpretation of these control coefficients by Kacser and Burns (1973) and aided by a barrage of later refinements and ornaments in subsequent publications and conferences (Heinrich and Rapaport, 1974; Liao and Delgado, 1993; see also the entire October 7, 1996 issue of The Journal of Theoretical Biology), a number of biological scientists have become intuitively comfortable with the control coefficient concept and have begun to communicate in MCA-speak. This is a major accomplishment of the mathematical biology community in bridging an abyss of difficult communication. (This success, however, has a dark side. There are clear signs that MCA has been oversold in a certain sensesthat some relative novices in systems mathematics (not all of whom are biologists) view MCA as the end-all of metabolic systems theorysthat MCA is seen as the theory of everything for metabolic engineering. This is, sadly, far from true. MCA provides characteristic parameters of a particular steady-state condition of a metabolic system which can be useful to know and which can nicely complement information on metabolic fluxes in the same network. However, from a mathematical point of view, control coefficients are no more than logarithmic sensitivity coefficients at one steady state and, as such, clearly do not provide answers to many important questions about metabolic systems.) In a commentary rich in insight, R. Strohman has written The failure (of genetic determinism) is located in the mistaken idea that complex behavior may be traced solely to genetic agents and their surrogate proteins without recourse to the properties originating from the complex and nonlinear interaction of these agents. (italics added) (Strohman, 1996). Building from the nonlinearity of enzyme-catalyzed reaction rates and permease-mediated transport rates with respect to substrate(s) and effector concentrations, and equilibrium constraints among some metabolite concentrations which arise frequently, biological systems are intrinsically nonlinear. Hence it is not surprising that various biological systems exhibit inherently nonlinear phenomena such as multiple steady states in the same environment (observed, for example, in yeast chemostats and nerve networks), sustained oscillations in time (called limit cycles), or patterns in space, which persist in response to small perturbations (e.g., in yeast chemostats, slime mold communities, or circadian rhythms), and complex or chaotic oscillations (e.g., in brain dynamics). To describe these phenomena with a mathematical

model, and therefore, in order for a model to be of any use in interpreting, predicting, or modifying such phenomena, the model must be nonlinear. A nonlinear model can also be important to expand the scope and dependability of engineering analysis. The (log)linear class of models mentioned above, and others based on information at one operating point, can approximate system properties reasonably only in some suitably small neighborhood of the operating point (and, maddening to engineers, there is almost no theoretical or practical guidance as to what suitably small really means!). With a suitable nonlinear model, response of the system to large changes in its characteristics or environment can be well approximated, allowing more reliable metabolic or bioprocess design and optimization. As noted in the hierarchy diagram in Figure 5, nonlinear models can be obtained by employing a larger data set of experimentally observed metabolite concentrations and corresponding intracellular reaction rates. Also, information from prior research on in vitro kinetics for enzymes found in the system can suggest useful functional forms to be used in estimating their in vivo kinetics (see, e.g., Galazzo and Bailey (1990)). Alternatively, nonlinear kinetic models can be formulated by starting directly from this in vitro information or, if no such information is available, by assuming reasonable kinetic expressions based on general experience in enzyme kinetics. (A reasonable question here, and one which has generated some heat in the mathematical biology literature, is the validity of using information derived from in vitro studies of the isolated enzyme for the in vivo situation in which the enzyme functions in an environment crowded with other proteins, membranes, and other compounds which might affect that enzymes turnover rate. If the purpose of the kinetic model is to provide a reasonably accurate mathematical formula for calculating intracellular reaction rates in terms of the variables included in the model, the ability to achieve this representation is the criterion on which the model must be judged. For this use, it is irrelevant whether the general form of that model has been developed on the basis of assumptions which conform to the precise physicochemical situation which exists. If the model is to be used for hypothesis testing, entirely different considerations arise, but there is not room here to discuss this. Remembering Castis essential guidance stated at the outset, no model is inherently right or wrong; a model can only be evaluated in the context of its intended purpose.) Mathematical and computational methods for study and application of nonlinear models are much more limited in scope and power than those available for linear systems. However, extensive research in the fields of bifurcation theory and nonlinear dynamics provides sophisticated guidance to identify conditions under which a system may experience a bifurcation, or basic change in its behavior, such as a transition from one stable steady state to more than one, or from a single stable steady state to a sustained nonlinear oscillation. These methods have been introduced previously into the realm of chemical reactor theory and are now penetrating into biological systems modeling (Varner and Ramkrishna, 1997) and analysis. As an example, V. Hatzimanikatis (personal communication) has employed modern computational algorithms to search for possible bifurcations in the nonlinear model formulated by Schlosser at al. (1994) of the resting yeast cell system introduced above. Prior research suggests that such a study might usefully search for bifurcations which occur when two parameters are

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Figure 6. Operating diagram calculated from a detailed, enzyme-based mathematical model of glucose catabolism in nongrowing cells of S. cerevisiae. Different regions, as labeled, correspond to parameter values which are may give rise to nonlinear (limit cycle) oscillations, more complex oscillations, and chaotic behavior.

varied: the amount of phosphofructokinase in the cells (represented by the parameter Vmax,PFK in the model, and a quantity amenable to modification by genetic engineering) and a characteristic rate at which ATP is utilized for maintenance of the cells (represented in the model by the parameter KATPase, and possible to change by changing the cell environment (e.g., external pH) or possibly by genetic engineering). The results of this analysis are summarized in Figure 6, which identifies regions in a space spanned by values of these two parameters in which different types of behavior are possible (usually this type of computation reveals a possibility for a bifurcation but does not always guarantee that a bifurcation occurs). Both axes here are scaled by the respective parameter values at the reference steady state, about which the analysis is based, so these values correspond to the point (1.0, 1.0) in the diagram. At this point, a unique stable steady state exists, and such behavior is also expected for other parameter values in the associated area. The other areas, defined by a combination of analytical mathematical relationships and computational searches, denote parameter values for which limit cycles and more complicated types of dynamic behavior can arise. With this guidance regarding conditions under which resting yeast cells could exhibit different types of dynamic structures, computer simulations using the complete nonlinear model were undertaken for different parameter values. These calculations revealed not only limit-cycle sustained oscillations, well-known for yeast glycolysis both experimentally and from earlier models, but also more complex oscillations (with approximately three times the period as the simpler limit-cycle oscillations) and also, for the first time for this system, chaotic dynamics (see Figures 7 and 8). There are a number of implications of this result, but the most pertinent for this discussion is the demonstration that we now have the mathematical and computational tools to work in a somewhat systematic way with nonlinear models and to understand what types on nonlinear phenomena a given model can exhibit. Without the mathematical tools which enabled generation of Figure 6 without undue computational effort, determining that the simple yeast glycolysis model considered here could produce chaos would have been very difficult. Following such pathways of modeling and analysis, we

are now empowered to model, and thereby to understand and to modify usefully, a wide class of nonlinear metabolic systems such as oscillating bioreactors or arrhythmic hearts. This example again illustrates an application of mathematical methods to understand important qualitative features of an important biological system. A New Paradigm: Mathematics Allied with Massive Computing Power. Exponential increases in computing capabilities are familiar to everyone, whether by access to complex graphics on the Internet or by press accounts of the chess prowess of Deep Blue (http:\\www.chess.ibm.com\home\html\b.html). While computing power has exploded, and continues to, it has done so in a continuum. This has obscured broad awareness that the arrival of cheap, massive computing power has wrought a basic transformation in the way mathematics can now be done, and used. Proof of Fermats Last Theorem, one of the most sought after but elusive goals of basic mathematics for more than three centuries, was accomplished recently with the aid of a computer (Aczel, 1996). The most basic resources and tools of genomicsshuge sequence databases and ways to search them efficientlysare based on this massive computing capability. Similarly, mathematical models and tools to rationalize functional genomics and to empower metabolic design also depend on massive computing power, although focused with different algorithms based on different theoretical foundations. The metabolic optimization (Hatzimanikatis et al., 1996) and bifurcation analysis (Figure 5) computations mentioned above would not have been feasible without powerful computers. We should be striving in the future to capitalize on this newfound power and to be searching to find the stillhidden doors to the new pathways which massive computing capability provides to describe and understand biological systems. The critical importance of discovering new tools and applying them effectively is also the major theme of the rest of this commentary.

The Good Old Days: Integrating Powerful Tools into Research on Mathematical Modeling and Analysis
Anyone who was not too young or too old at the time will tell you that the 1960s were an exhilarating, creative time (although of course burdened also with controversy, sorrow, and rage). Within chemical engineering, as mentioned earlier, a transformation of how teaching and research was done, and how people thought about things, was rapidly occurring. An important force in this transformation was introduction of a collection of powerful mathematical theories into the toolbox of chemical engineering research. This did not occur by accident. It was an active effort, and it attracted many of the best minds in our field at the time. Perhaps the best way an academic chemical engineer could gain recognition during those years, and some thereafter, was to discover and understand a cool theory in mathematics (often originating in Russian research) and then to introduce and explain it to colleague chemical engineers, showing a powerful application in our field. Thus, Amundson and Aris (1958) empowered stability analysis of reactors based on the theorems of Liapunov; Aris, Jackson, Horn, and others introduced powerful optimization algorithms built on variational calculus and Pontryagins Maximum Principle (Pontryagin et al., 1963); Luss and Gavalas applied functional analysis from Krasnoselskii (Krasnoselski, 1964) and others to study steady-state multi-

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Figure 7. Nonlinear phenomena exhibited by a model of yeast glucose catabolism. Model calculations (1) of the phosphoenolpyruvate (PEP) time trajectories and (2) phase-space trajectories; time is a parameter advancing in the direction indicated by the arrow on the curves shown in a three-dimensional coordinate system spanned by the intracellular concentrations of PEP, ATP, and FdP (fructose 1,6-diphosphate). Two cases are shown corresponding to parameter values identified in the calculation of Figure 6 as candidates for limit cycle (A) and more complex (B) oscillations.

Figure 8. Chaotic dynamics in yeast glucose catabolism. Model calculations of phase-space trajectories for parameter values identified in the calculation of Figure 6 as candidates for chaotic behavior.

plicity (e.g., Balakotaiah and Luss, 1983) and bifurcations (Gavalas, 1968). In the same era, Amundson, Wei, and Prater showed the power of sensitivity analysis and linear algebra to organize and analyze complex chemical systems (Amundson, 1966; Wei and Prater, 1962). Some years later, Lyberatos and Kuszta translated V. Arnolds theories of versal families and unfoldings (Arnold, 1983) to organize systematic searches for bifurcations to com-

plex dynamic behavior in chemical systems (Lyberatos et al., 1995). Although some refinements have been added, almost all of the mathematical methods and modeling concepts discussed earlier in this paper are built on these formidable but now dusty foundations. By basing our work on essentially the same set of concepts and tools for the past decade or two, we risk decay in our potential to contribute significantly. Imagine someone in the molecular biology laboratory without the latest PCR tricks, the latest vectors, genes, and antibodies! An effective researcher identifies interesting new problems to attack in the context of concepts, and research technology, available. This ensures that the project is both potentially important and potentially feasible. Therefore, unless we make a conscious effort to learn new tools and methods, to understand concepts from presently unfamiliar areas, and then to integrate these with creatively chosen new problems in our field, we risk stagnation and irrelevance. Of course any dedicated quest for new tools risks the solution in search of a problem syndrome, which, taken to an extreme, can be as counterproductive as defining our research based on an aging, fixed set of tools. We must also understand, and embed in our conscious and unconscious creative thinking, emerging new knowledge in biology and medicine. The ideal evolution of research involves a constant interplay of new tools with ideas for important new problems and recogni-

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tion of openings for major advances when these elements happen to snap into alignment. A Huge Gap Between Theory and Practice. One way we can try to identify topics which may need our attention or, viewed differently, which may present exceptionally good research opportunities is to discern large discontinuities between developments in biology and medicine and activities of the biochemical engineering research community. There is presently a yawning chasm between genomics and functional genomics researchsmoving now almost at light speed scientifically and commerciallysand mathematically and computationally oriented research in biochemical engineering. How many publications have you seen, in this literature, which contribute to new insights from, or new uses of, nucleotide or amino acid sequence information? Yet the generation of this information is clearly the most significant development in biology in the current decade, and the future implications are probably difficult to overstate. Why this huge disconnection? One answer could be that biochemical engineers think Sequence analysis is the realm of the computer scientist (or, in current jargon, the bioinformatics expert). Another might be Predicting higher-order structures, and intra/intersequence interactions and their energies is the realm of the biochemist. (I, mistakenly, used almost these words in discouraging then-Ph.D. student Alex Seressiotis initiatives in this area more than a decade ago! However, I do not have such opinions now.) I believe that the biochemical engineering research community has not made the effort to understand the statistics and information theory and the molecular physics needed to work on these problems seriously. The word community is critical here because we have adopted for years a systematic bias in hiring, funding, and promotion decisions for faculty and researchers toward experimentalists and, quite strongly, against theoreticians. This has significantly decreased the opportunity and incentive for those who could provide the intellectual and creative leadership into new mathematical and computational methods and their effective application. If we as a community do not assign more value to these types of activities, and nurture the rare emerging stars in theory as much as we do those in experiment, future contributions of biochemical engineers in the areas discussed above will fall far short of their potential. This would be, in my opinion, a great misfortune for our profession, for I believe that we, and perhaps only we, can make a critical impact, via mathematical models and computing, to emerging central problems, and industrial opportunities, in understanding and improvement of complex biological systems. A Provocative Example from the Research of Steve Benner. Partially because the biochemical engineering community does not understand the mathematical and computational tools needed to enter the territories of sequence analysis and application, we have not thought much at all about which of our important problems we might solve, and what technological vistas we might uncover, working within these still (for us) uncharted waters. The possibilities are far from hypothetical, as the following example shows. Prediction of protein three-dimensional (3D) structure from amino acid sequences has been for years a holy grail of protein chemistry. Such a capability would have profound technological consequences, defining binding sites for ligands, substrates, and effectors and thereby immediate targets and leads for drug discovery. Moreover, in a bioprocess context, knowledge of 3D protein

configuration would tell us what sequences to mutagenize to select more stable or active enzymes or where to target mutations to eliminate metabolite inhibition of a key enzyme in a metabolic pathway. Substantial progress toward the goal of structure prediction was made by Steve Benner, a bioorganic chemist, and his collaborator from computer science, Gaston Gonnet. The main breakthrough they achieved was a restructuring of the protein sequence database (specifically, how the sequences are ordered in the database) which made possible much more efficient searching of the database, permitting comparison of each sequence present with every other sequence in the database without undue computation effort. Combined with quantitative measures of relatedness of sequences, Gonnet, Cohen, and Benner (1992) were able to map evolutionary pathways and to suggest structures of primordial ancestors of contemporary proteins. Coincident with this effort, by identifying relationships between an amino acid sequence with unknown 3D structure and peptide sequences with known 3D structures, Benner was able to predict, successfully, the 3D structure of several proteins before these 3D structures were published (e.g., Benner, 1989). In addition, this work engendered reasonable hypotheses about structures of ancient metabolic pathways, information of profound potential importance in understanding the function of complex control circuits in contemporary cells which often confound metabolic engineering efforts to redirect fluxes, especially in central metabolism.

A Final Word
Biochemical and biomedical engineers have the enormous privilege now of working alongside, and interacting with, the largest explosion of knowledge in the history of science. Biology and medicine are advancing at a fantastic pace, revealing new phenomena, mechanisms, molecules, and genes every moment. Benefits to health, agriculture, and the environment will be enormous. To contribute substantially to these developments, we must constantly strive to define important problems, based upon creative application of the best tools available. We should become familiar with presently alien topics such as mining sequence databases, deciphering the programming of development, and managing resource allocation and stress resistance in plants. While this discussion has focused on research, it is perhaps more important that we continuously update our teaching programs, so that bright new minds entering our field are imbued with fresh knowledge and challenged by the incredible opportunities before us.

Acknowledgment
The authors Ph.D. students, postdocs, and collaborators in mathematical modeling and analysis of biological systems have taught him almost everything in this field. Thanks go to Y. Nishimura, D. Sincic, S. Yeung, D. Agar, M. Hjortso, P. Doran, D. Clark, S.-B. Lee, F. Srienc, J. Galazzo, N. Da Silva, J. Shanks, S. Peretti, A. McQueen, K. Reardon, J.-H. Seo, D. D. Do, A. Seressiotis, C. Khosla, G. Lyberatos, B. Kuszta, J.-D. Ricci, N. Dedhia, T. Przybycien, K. D. Wittrup, P. Schlosser, D. D. Axe, P. Licari, W. Chen, T. Holcomb, M. Morari, V. Hatzimanikatis, Ch. Floudas, K. Lee, P. Tsai, P. Uman a, Th. Szyperski, K. Wu thrich, M. Dauner, U. Sauer, D. Cameron, A. Rosell Uriz, and J. Varner. The authors research in metabolic engineering is supported by the Swiss Federal Technical University (the ETH) and the Swiss Priority Program in Biotechnology (SPP BioTech). Our

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activities with several projects in the biotechnology programs of the European Commission Fourth Framework Program are supported by the Swiss Bundesamt fu r Bildung und Wissenschaft (BBW). All opinions stated above are those of the author.

References and Notes

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Accepted December 1, 1997. BP9701269