koffas1999

Annu. Rev. Biomed. Eng. 1999.
01:535–557
Copyright q 1999 by Annual Reviews. All rights reserved
Metabolic Engineering
M. Koffas, C. Roberge, K. Lee, and G. Stephanopoulos
Department of Chemical Engineering, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139; e-mail: mattheos@mit.edu
Key Words metabolism, flux, pathway

Abstract Metabolic engineering is the science that combines systematic analysis
of metabolic and other pathways with molecular biological techniques to improve
cellular properties by designing and implementing rational genetic modifications. As
such, metabolic engineering deals with the measurement of metabolic fluxes and elu-
cidation of their control as determinants of metabolic function and cell physiology.
A novel aspect of metabolic engineering is that it departs from the traditional reduc-
Annu. Rev. Biomed. Eng. 1999.1:535-557. Downloaded from www.annualreviews.org
tionist paradigm of cellular metabolism, taking instead a holistic view. In this sense,
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metabolic engineering is well suited as a framework for the analysis of genome-wide

differential gene expression data, in combination with data on protein content and in
vivo metabolic fluxes. The insights of the integrated view of metabolism generated
by metabolic engineering will have profound implications in biotechnological appli-
cations, as well as in devising rational strategies for target selection for screening
candidate drugs or designing gene therapies. In this article we review basic concepts
of metabolic engineering and provide examples of applications in the production of
primary and secondary metabolites, improving cellular properties, and biomedical
engineering.
CONTENTS
Introduction .....................................................................................
535
Metabolic Fluxes ..............................................................................
537
Primary Metabolites ..........................................................................539
Secondary Metabolites ....................................................................... 542
Cell and Tissue Engineering ............................................................... 545
Biomedical Applications .................................................................... 546
Inborn Errors of Metabolism ............................................................... 547
Cell and Organ Physiology ................................................................. 548
Concluding Remarks ......................................................................... 550
INTRODUCTION
Altering metabolic pathways to improve cell properties and the chances of cell
survival is as old as nature itself. The genomic and metabolic evolution of extre-
mophiles is an example of such a natural adaptation process in bacteria (96). The
1523–9829/99/0820–0535$08.00 535
536 KOFFAS ET AL
intentional manipulation of metabolic pathways by humans to improve the prop-

erties and productivity of microorganisms is similarly an established concept.
Techniques such as genetic modifications via random mutagenesis have yielded,
for example, improved strains of Corynebacterium glutamicum and its related
species Brevibacterium lactofermentum and Brevibacterium flavum that excrete
large amounts of amino acids into the fermentation medium (52, 80, 106). Ran-
dom mutagenesis relies heavily on chemical mutagens and creative selection tech-
niques to identify superior strains for achieving a certain objective. Such
traditional genetic approaches for strain improvement have been applied exten-
sively in the past also in the areas of antibiotics, solvents, and vitamin production
among others.
Since the past decade, the development of recombinant DNA techniques has
introduced a new dimension to pathway manipulation by offering, for the first
time, the capability to construct specific metabolic configurations with novel,
beneficial characteristics. Genetic engineering allows precise modification of spe-
cific enzymatic reactions in metabolic pathways, leading to the construction of
well-defined genetic backgrounds. The redirection of cellular metabolism to cre-

ate or enhance desirable attributes has been accomplished with a variety of novel
techniques and applied towards an even greater variety of goals.
In this context, metabolic engineering has emerged as the technological and
scientific discipline dealing with the introduction of specific modifications to met-
abolic pathways to improve cellular properties. Metabolic engineering involves
manipulation of enzymatic, transport, and regulatory functions of the cell by using
recombinant DNA technology (5, 86). First, various analytical techniques are used
to identify and subsequently determine fluxes through critical metabolic pathways
in the cell or tissue of interest. This knowledge provides the rational basis for
applying, in the second step, molecular biological techniques to enhance meta-
bolic flux through a pathway of interest and minimize metabolic flow to undesired
biosynthetically related products. 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 with random mutagenesis, because this
directionality plays a dominant role in enzymatic target selection, experimental
design, and data analysis.
Although various terms have been coined over the past two decades to rep-
resent the increasing activity in pathway modification (pathway engineering, cel-
lular engineering, in vitro evolution, etc) (58, 65), the term metabolic engineering
has succeeded in capturing the ever growing interest in this area. Furthermore,
although initially embodied as a collection of examples from the chemical indus-
try and biomedical research, metabolic engineering is quickly becoming a distinct
scientific field. Its novel contribution lies in its emphasis on complete metabolic
networks rather than individual reactions. To elaborate, as with all traditional
fields of engineering, metabolic engineering too encompasses the two defining
steps of analysis and synthesis. Because metabolic engineering emerged with
DNA recombination as the enabling technology, its initial focus was on synthesis
METABOLIC ENGINEERING 537
in the form of new pathway construction. As such, differentiation from genetic

engineering was initially diffuse, and metabolic engineering could be considered
as the technological manifestation of applied molecular biology. The real contri-
bution of metabolic engineering emerged as soon as a need for a more rational
approach to identifying promising targets of metabolic manipulation was articu-
lated, replacing the previous, mostly ad hoc target selection process. In this sense
the contribution of metabolic engineering emanates from pathway analysis, which
yields an enhanced perspective on metabolism and cellular function, including
consideration of reactions in their entirety rather than in isolation. Thus, metabolic
engineering seeks to analyze and then synthesize and design, using techniques
and information developed from extensive reductionist research.
Metabolic engineering has found many applications, especially in microbial
fermentation. It has been applied to increase the production of chemicals that are
already produced by the host organism (e.g. 13, 15, 22, 34, 92), to produce desired
chemical substances from less expensive feedstocks (e.g. 6, 53, 107), and to
generate products that are new to the host organism (e.g. 67, 68). Other challenges
associated with metabolic engineering are the biosynthesis of secondary metab-

olites, the generation of organisms with desirable growth characteristics, and the
manipulation of pathways for the production of chiral compounds as intermedi-
ates in the synthesis of pharmaceutical products (38). Finally, although less widely
appreciated, metabolic engineering techniques can also be applied for studying
physiological systems and isolated whole organs in vivo to elucidate the metabolic
patterns that occur in different physiological states, such as ‘‘fed’’ or ‘‘fasted,’’
as well as in disease (105).
METABOLIC FLUXES
Because metabolic pathways and fluxes are at the core of metabolic engineering,
it is important to elaborate on their definition. A metabolic pathway is defined as
any sequence of feasible and observable biochemical-reaction steps connecting a
specified set of input and output metabolites. The pathway flux is the rate at which
input metabolites are processed to form output metabolites (84). The importance
of feasibility and observability should be noted in the definition, in view of the
diversity and complexity of various metabolic maps. If some metabolic fluxes in
a pathway or metabolic network cannot be determined independently, it is better
to lump these reactions together, because their inclusion provides no additional
information.
The determination of intracellular fluxes, along with analysis of factors affect-
ing flux distributions, is collectively referred to as metabolic flux analysis (MFA).
MFA combines data on uptake and secretion rates, biosynthetic requirements,
metabolic stoichiometry, and quasi–steady-state mass balances on metabolic
intermediates to determine intracellular metabolic fluxes (84). MFA has been the
focus of attention of many researchers in the past and has yielded important
538 KOFFAS ET AL
information on flux distribution and its control in many bacteria. The combination
of analytical methods to quantify fluxes and their control with molecular bio-
logical techniques to implement suggested genetic modifications is the essence
of metabolic engineering. An iterative cycle of genetic change followed by an
analysis of its consequences and design of further modifications, analogous to
that articulated for protein engineering, can also be applied in the development
of an optimized strain (3). The flux thus becomes a focal point of metabolic
engineering and justifies further research for the development of methods for its
determination in vivo.
MFA also reveals the degree of pathway engagement in the overall metabolic
process. Furthermore, elucidation of the control of flux provides a mechanistic
basis for rationalizing observed fluxes and flux distributions at key metabolic
branch points (86). As these fluxes are determined under in vivo conditions, MFA
also allows valid comparisons to be made between in vivo and in vitro enzymatic
behavior. Finally, the flux is a fundamental determinant of cell physiology and a
critical parameter to use when comparing the behavior of strain variants. Even if
the fermentation characteristics of such strains differ, such differences may be

relatively unimportant if the flux distributions around key branch points have not
been altered.
Intracellular fluxes are calculated by MFA with a stoichiometric model for the
internal reactions and mass balances around intracellular metabolites (98).
Required data are obtained from measured extracellular fluxes, such as substrate
uptake rates and metabolite secretion rates (85). Intracellular fluxes from reactions
that are cyclical or that split and later converge may be determined by assaying
asymmetries in the distribution of radio-labeled atoms of intermediate metabolites
(77, 83). The metabolic fluxes determined by MFA provide a comprehensive
perspective of the control system at work in metabolic networks. This control
system can be described in terms of metabolic control coefficients (30, 41). Flux
control coefficients are measures of the degree of control exercised by an enzyme
on the overall network flux and can be determined by measuring flux responses
to metabolic perturbations.
As mentioned in the previous paragraphs, elucidation of the flux control struc-
ture of metabolic pathways offers tremendous opportunities for the rational design
of the optimal genotype of a cellular catalyst. This activity should be viewed as
complementary to molecular biological toolboxes for implementing gene transfers
and other similar modifications. In fact, recombinant technology has advanced so
rapidly in recent years that rational analysis of metabolic pathways for the iden-
tification of target genes and enzymes is the limiting component in the directed
optimization of cellular function. Evidence for this assertion is the observation
that currently, almost 20 years after the pioneering developments in genetic engi-
neering, we have hardly begun to harness the potential of modern biotechnology
in the areas of fuels, chemicals, or materials production.
In the following sections, we review a few illustrative applications of meta-
bolic-pathway manipulation. We organize the various applications of metabolic
engineering into four basic groups: (a) improving primary metabolite production,
(b) improving secondary metabolite and biopharmaceutical production, (c)
improving cell properties, and (d) improving the biomedical field. We have three
goals in reviewing these applications: first, to provide a sample of the truly broad
range of possibilities for biocatalyst improvement afforded by pathway manipu-
lation and metabolic engineering; second, to alert the reader to the complexity of
metabolic pathways, along with their regulation and need of coordination with
overall metabolism; and, third, to underscore the methods used for effecting
desired changes in cellular systems for industrial use or medical reasons. This
review concludes with some ideas and suggestions for future directions of the
field.
PRIMARY METABOLITES
A large number of mainly industrial applications can be classified as primary
metabolites. A central goal of metabolic engineering is to achieve the production

of commodity chemicals by overexpressing key metabolic pathways that already

exist in the host organism or by introducing new routes of metabolism. We next
review efforts in metabolic engineering to improve the yield and productivity of
ethanol, amino acids, and solvents.
A major challenge of using biotechnology in the industrial production of fuels
has long been the construction of microorganisms that are able to ferment inex-
pensive and abundant carbon resources, such as lignocellulosic materials, into
ethanol for use as a biofuel, among other applications. The most commonly used
ethanol producer, Saccharomyces cerevisiae, cannot ferment pentoses, which may
constitute 8%–28% of lignocellulose. On the other hand, other bacteria such as
Erwinia chrysanthemi, Klebsiella planticola, and Escherichia coli can grow effi-
ciently on a wide range of carbon substrates that includes five-carbon sugars, but
competing pathways divert carbon flow away from ethanol production. Initial
studies were only partially successful in redirecting fermentative metabolism to
ethanol production in these bacteria, by amplifying their pyruvate decarboxylase
activity (7, 91, 92). A further improvement of the process was achieved by cloning
and overexpressing the Zymomonas mobilis adhB gene, yielding recombinants of
E coli (34) and Klebsiella oxytoca (67, 68, 104) that efficiently ferment a variety
of sugars to ethanol. Ohta et al investigated the expression of the pyruvate decar-
boxylase and alcohol dehydrogenase genes of Z. mobilis in a related enteric bac-
terium, K. oxytoca (67, 68). Klebsiella strains have two additional fermentation
pathways not present in E. coli, which are used to convert pyruvate to succinate
and butanediol. As for E. coli, it was possible to divert .90% of the carbon flow
from sugar catabolism away from the native fermentative pathways and toward
ethanol.
Amino acid production is also a heavily researched area. Tryptophan synthesis
in E. coli is highly regulated by a complex set of feedback mechanisms. By
540 KOFFAS ET AL
transducing each of several mutations one at a time, researchers combined, within

a single strain, a long list of alterations to these mechanisms, thus creating a
tryptophan overproducer (1, 82). A Corynebacterium glutamicum strain able to
produce 18 g liter11 of tryptophan has been altered to produce large amounts of
tyrosine (26 g liter11) by overexpressing deregulated 3-deoxy-D-arabino-heptu-
losonate-7-phosphate synthase and chorismate mutase (33). Overexpression of an
additional gene, prephenate dehydratase, in the previous construct led to the pre-
dominant production of phenylalanine (28 g liter11). In a similar way, significant
progress was made recently in efforts to construct efficient threonine-producing
strains by metabolic engineering. The genes that are involved in the threonine
production pathway of C. glutamicum were cloned, and the regulatory properties
of the enzymes encoded by them were well characterized (e.g. 20, 22, 39). The
cloning of a deregulated (threonine-insensitive) homoserine dehydrogenase, along
with modulation of the activity of homoserine kinase relative to that of homo-
serine dehydrogenase (the first two enzymes in the threonine pathway), yielded
threonine-secreting strains (14). Similarly, overexpression of ilvA, the first gene
in the isoleucine pathway, allowed significant isoleucine accumulation by B. lac-

tofermentum strains (15).

Another industrially significant primary metabolite is 1,3-propanediol (1,3-
PD), an intermediate in chemical and polymer synthesis, for example, in the
synthesis of polyurethanes and polyesters. 1,3-PD is currently derived from petro-
leum, and it is expensive to produce relative to similar diols. Tong & Cameron
(93) constructed an E. coli propanediol-producing strain carrying genes from the
Klebsiella pneumoniae dihydroxyacetone (dha) regulon. These genes allow the
strain to grow anaerobically on glycerol and produce 1,3-PD. A further process
improvement via metabolic engineering in the same field is in production of 1,3-
PD by using sugars as a carbon source, because sugars are significantly less
expensive than glycerol. No known natural organism ferments sugars directly to
1,3-PD. One way to replace, at least partially, the need for glycerol is by cofer-
mentation of glycerol and a sugar such as glucose. Cofermentation is not possible
with native 1,3-PD producers because glucose represses the 1,3-PD pathway. E.
coli that has been transformed with the K. pneumoniae 1,3-PD oxidoreductase
gene (dhaT) and the glycerol dehydratase gene (dhaB) of the same organism is
able to coferment glycerol and glucose (93, 94). The glycerol is converted pri-
marily to 1,3-PD, and the glucose is used for growth and regeneration of reducing
potential. Until today the production of 1,3-PD by fermenting sugars alone has
not been possible to a commercially advantageous extent. The initial success with
the metabolic engineering of 1,3-PD production, coupled with the complexity and
difficulty of rational process improvement with Thermoanaerobacterium ther-
mosaccharolyticum, the best naturally occurring organism for the fermentation of
sugars to 1,2-propanediol, led to the construction of pathways in E. coli similar
to those used by T. thermosaccharolyticum. This was accomplished by overex-
pressing the native glycerol dehydrogenase of E. coli or cloning the aldolase
reductase gene from rat lens cDNA into E. coli. Either of these two strains led to
the production of 1,2-PD in equal amounts in batch and continuous culture. These
results provide the first example of recombinant organisms able to ferment sugars
to 1,2-PD and demonstrate that various strategies for the fermentation of sugars
to 1,3-PD are possible (9).
Another example of converting native metabolic intermediates to desirable end
products is the production of the b-carotene precursor to vitamin A. By intro-
ducing the three carotenogenic genes required for lycopene synthesis from farenyl
diphosphate under the control of Candida utilis, a C. utilis strain producing 1.1
mg of lycopene/g (dry weight) of cells has been generated. By using concepts of
metabolic engineering with C. utilis, carbon flux in this yeast strain was redirected
away from ergosterol formation for potential use by the carotenoid pathway. The
influential steps in the pathway that were manipulated were 3-hydroxy methyl-
glutaryl (HMG)-coenzyme A (CoA) reductase, encoded by the HMG gene, and
squalene synthase, encoded by the ERG9 gene. A combination of ERG9 gene
disruption and the overexpression of the HMG catalytic domain gave the highest
lycopene yield. These findings illustrate how modifications in related biochemical
pathways can be used to enhance the production of commercially desirable com-

pounds such as carotenoids (81).

Scientists are now beginning to elucidate the pathway of vitamin C biosyn-
thesis in higher plants. Several enzymes and precursors at work in this pathway
have recently been identified (103), and MFA may in the near future allow for
their rational modification. To this end, Sauer et al (76) investigated fluxes in a
riboflavin-producing Bacillus subtilis strain and found that generation of the
desired riboflavin was limited by the biosynthetic pathway fluxes and not by the
fluxes associated with central carbon metabolism.
Another group of metabolic engineering applications is the engineering of
organisms that can use abundant byproducts of various industries as nutrients by
extending the range of their substrates. Whey, with high lactose and protein con-
tent, is a nutrient-rich byproduct of the dairy industry that can provide an inex-
pensive carbon and nitrogen source in biotechnological processes. Although a
variety of microbes can use whey, some of the most industrially prominent organ-
isms are unable to do so. The E. coli lacZY operon (coding for b-galactosidase
and lactose permease) was inserted into Xanthomonas campestris, a bacterium
that is used for xanthan gum production (24). The recombinant stain expressed
high levels of b-galactosidase and grew well in a medium containing lactose as
the sole carbon source. In another approach, an S. cerevisiae recombinant strain
was constructed that expressed the gene for a secreted b-galactosidase (lacA) from
Aspergillus niger (53). This approach offers significant advantages over earlier
processes for the fermentation of whey by S. cerevisiae, which used either b-
galactosidase–prehydrolyzed whey or yeast coimmobilized with b-galactosidase.
Sucrose is another abundant and inexpensive carbon source found in, for example,
cane molasses. A successful attempt to create a recombinant sucrose-metabolizing
strain involved the cloning of the scrA gene, which codes for sucrase, from E.
coli B-62 onto a plasmid and then transferring the cloned DNA fragment onto
542 KOFFAS ET AL
the chromosome of E. coli K-12. Tryptophan producer derivatives of E. coli

K-12 expressing the scrA gene grew well in sucrose medium and excreted
amounts of tryptophan comparable to these from similar strains grown on glucose
(95). Starch, derived from renewable sources such as corn and cereals, is a very
important carbon and energy source in biotechnological processes. It is a mixture
of linear and branched homopolymers of D-glucose. Because most microorgan-
isms are unable to degrade this glucose biopolymer, work has focused on cloning
genes for enzymatic starch hydrolysis into various organisms (49). Along these
lines, an S. cerevisiae strain was constructed that contained a glucoamylase gene
from Aspergillus sp. (35). The recombinant strain was able to grow on amylodex-
trins, albeit at a lower rate than occurs when glucoamylases are added to the
fermentation medium.
Cofactor engineering is a novel approach to metabolic engineering. The clon-
ing of the Streptococcus mutant nox-2 gene, coding for the H2O-forming (non-
toxic) NADH oxidase, under the control of the nisA promoter in Lactococcus
lactis, provides a powerful tool with which to attempt the modulation of the
metabolism. The main effect of overproducing the NADH oxidase was an

observed decrease in the NADH/NAD ratio under aerobic conditions. This engi-
neered system could be used to provoke a shift from homolactic to mixed-acid
fermentation during aerobic glucose catabolism. The magnitude of this shift was
directly dependent on the level of NADH oxidase overproduced. These results
indicate that the observed shift from homolactic to mixed-acid fermentation under
aerobic conditions is mainly modulated by the level of NADH oxidation resulting
in low NADH/NAD ratios in the cells (57).
SECONDARY METABOLITES
The challenges associated with engineering the biosynthesis of secondary metab-
olites are qualitatively different from those associated with the production of
commodity bulk chemicals. Products of interest are often complex molecules that
are necessarily derived from biological sources. In this arena, pathway engineer-
ing may increase the efficiency of existing production methods but may also lead
to the development of new products. The relatively high value of these products
shifts the emphasis from economics and efficiency to innovation.
An interesting example is the production of antibiotics. Antibiotics are made
by secondary metabolic pathways that use common metabolites in less specific
and, sometimes, more intricate ways than metabolites are used in primary metab-
olism. Recently, it has become apparent that yields of secondary metabolites,
including antibiotics, can also be improved by overcoming rate-controlling bio-
synthetic steps through genetic techniques. In addition, metabolic engineering
techniques are applied to modify known antibiotics to improve their properties
and also to synthesize new product forms.
Among various antibiotic producers of industrial importance, Streptomyces
species rank near the top. Actinorhodin biosynthesis genes were transferred from
S. coelicitor, the only species with well-established genetics, to S. lividans,

enabling the latter strain to produce actinorhodin. Later, clustered erythromycin
genes from S. erythreus were transferred to S. lividans, allowing the recombinant
strain to produce erythromycin A. Transformation of the fungi Neurospora crassa
and A niger with a cosmid containing Penicillium chrysogenum penicillin bio-
synthetic genes resulted in the production of penicillin V by these strains (4, 60).
The manufacture of antitumor drugs, many of which are natural products, has
also received significant attention. The undesirable side effects of antitumor drugs,
as well as the development of resistance to them, have fueled the need for the
discovery of novel therapeutic agents and the means for their synthesis. The
manipulation at a genetic level of the enzymes composing drug production path-
ways within cells has emerged as a very powerful tool for achieving these goals.
This method, which has been termed combinatorial biosynthesis, has yielded
derivatives and analogs of drugs such as mithramycin (21, 28), tylosin (2), eryth-
romycin (19, 36, 37, 63, 99), and methymycin (108). Here, as an example, we
focus on techniques used to engineer a biological reaction pathway for the pro-
duction of the cancer chemotherapy drugs epirubicin (48-epidoxorubicin) and 48-

epidaunorubicin. The medical and industrial significance of epirubicin lies in its

antitumor activity, its use as a precursor of the drug candidate 4-iodoxorubicin
(87), and its reduced cardiotoxicity relative to that of doxorubicin (102), a heavily
prescribed chemotherapy medication. The currently used synthetic means of pro-
ducing epirubicin suffers from many complexities. The scheme involves seven
different synthesis steps, which are followed by still more separation and depro-
tection steps. Madduri et al have described an alternative process that generates
48-epidaunorubicin and epirubicin directly and relies on the fermentation of a
Streptomyces peucetius strain to which has been introduced an avermectin or
erythromycin biosynthetic gene (59).
This discovery was made with the use of a S. peucetius strain in which the
dnmV gene has been disrupted (69). This strain accumulates e-rhodomycinone, a
precursor of 48-epidaunorubicin and epirubicin, unless transformed with a plasmid
containing the wild-type dnmV gene. In this case, daunorubicin and doxorubicin,
analogs of 48-epidaunorubicin and epirubicin, respectively, are produced (97).
The function of the DnmV enzyme was hypothesized to be that of a TDP-4-
ketohexulose-reductase (26), much like that of the Saccharopolyspora erythraea
eryBIV (89) and Streptomyces avermitilis avrE (66) gene products. Cloning of
these latter two genes onto plasmids used to transform the dnmV mutant yielded
bacteria that produced 48-epidaunorubicin and epirubicin. The integration of the
avrE gene directly into the S. peucetius chromosome gave results similar to those
seen with the gene acting in trans from a plasmid.
Madduri et al also found that the yield of 48-epidaunorubicin was increased
by introducing a dnrH mutation into the avrE integrant strain (59). It is believed
that the dnrH genes code for enzymes that catalyze side reactions that form gly-
cosides of daunorubicin and its precursors (78). Additional productivity gains
were achieved by providing an overexpressed plasmid copy of the dnmT gene
into the avrE integrant. The enzyme DnmT is hypothesized to catalyze a limiting
544 KOFFAS ET AL
step in the synthesis of the daunorubin precursor, daunosamine. By incorporating

all three findings—the integration of avrE, the mutation of dnrH, and the over-
expression of dnmT—into one strain, 48-epidaunorubicin titers were realized that
approximated those of daunorubicin seen in the wild-type strain. Although these
product concentrations are still too low to effectively compete with the extant
synthetic process results, the gains made by metabolic engineering are significant
and point the way for future improvements that can be implemented once the
entire biosynthetic network has been better characterized. Extensive reviews of
combinatorial biosynthesis advances can be found elsewhere (32, 74).
Biological processes using reactions catalyzed by enantiospecific enzymes are
increasingly being investigated as methods for the manufacture of pharmaceutical
compounds and their intermediates. Because these compounds are often active,
as a treatment, in only one particular chiral form, processes that exclusively gen-
erate the desired chiral form are very advantageous. Although biologically cata-
lyzed reactions, termed biotransformations, do possess this attribute, their
productivity is often economically unfavorable relative to yields seen in organic
synthesis processes. Because of this, metabolic engineering is being called on to

provide the means of increasing the efficiencies and product concentrations of

the pathways of interest, so that they can be more attractive for use in industrial
practice.
One potential application of biotransformations is the manufacture of Crixivan
(indinavir sulfate), a protease inhibitor developed and produced by Merck & Co.
and targeted for the treatment of human immunodeficiency virus (23). The chem-
ical structure of Crixivan contains five chiral centers, two of which are contributed
to the final compound from the intermediate 1-amino-2-(R)-indanol, a derivative
of indene. This drug precursor can be produced by transformation of indene by
the Rhodococcus sp. strain I24 (8). This soil isolate has been shown to grow with
naphthalene and toluene as its only carbon sources, and it is believed to possess
at least three different oxygenases. The presence of the multiple-oxygenase
enzymes results in formation of multiple stereochemical enantiomers of the
desired 1-amino-2-indanol when indene is supplied to I24 cultures. To increase
both the specificity with which I24 produces the 2-(R) form and the final product
titers, the pathway of indene bioconversion has been the subject of recent study.
In particular, the nature of the specific oxygenases in action is being investigated,
and factors controlling flux distribution to the different competing pathways ana-
lyzed. These studies are aided by novel methods of flux determination that make
use of radio-labeled tracer compounds.
The polyketide family is another rich source of bioactive molecules with anti-
biotic and pharmacological properties. Reasons that polyketides are an attractive
study model for metabolic engineering include the following: (a) their complex
structure results from simple units combined in diverse ways; (b) the modular
construction of the enzymatic catalyst allows control of enzyme structure and,
hence, polyketide type at the genetic level. Recent progress in this area has estab-
lished the groundwork to generate novel polyketide structures through genetic
engineering of polyketide synthases and at the same time to derive knowledge

that elucidates the structure-function relationship in polyketide synthases (48, 64).
Moreover the field provides an opportunity to bridge the fields of genetics and
chemistry and, above all, promises to enable scientists to rationally design novel
molecules at the level of DNA.
CELL AND TISSUE ENGINEERING

Metabolic engineering can also be used to construct cells with desirable properties
by altering characteristics such as growth, proliferation, tolerance to exogenous
factors, substrate utilization, etc. These characteristics are the result of complex
biological functions involving multiple gene products. This complexity may
afford greater efficiency or higher quality control or it may exist simply because
a single protein cannot provide the required function. Metabolic engineering strat-
egies that coordinately modify multigene expression therefore have the potential
to achieve previously inaccessible metabolic states.

The hemoglobin (VHb) of the microorganism Vitreoscilla species has been

extensively investigated in recent years as a tool for maintaining metabolic activ-
ity under hypoxic conditions. In its native host, VHb binds oxygen at a low
extracellular concentration and acts as a buffering agent at high intracellular-
oxygen concentrations, allowing the bacteria to survive in the hypoxic environ-
ments to which it is indigenous (100). The gene (vgb) encoding the hemoglobin
was cloned (18, 51) and used to transform a wide range of species in attempts to
improve respiration, growth, and productivity. A few of the most recent advances
have involved increasing the total protein secretion, neutral protease, and a-amy-
lase activities of B. subtilis (46), boosting antibiotic production in S. coelicolor
(16) and S. lividans (60), enhancing lysine production in C. glutamicum (75), and
improving the degradation of benzoic acid by Xanthomonas maltophilia (56). The
effectiveness of VHb in improving cellular processes was compared with that of
two other globins, horse heart myoglobin (HMb) and yeast flavohemoglobin
(YFb), and found to be the superior growth enhancer in E. coli (47). The inves-
tigators theorized that this was so because, of the three globins, VHb was unique
in possessing a slow oxygen on-rate constant and a fast oxygen off-rate constant.
This results in VHb being particularly effective in both scavenging sparse oxygen
molecules and donating them to the respiratory components that require oxygen.
Another recent work has identified a strong effect of growth medium on the
efficacy of improving processes with VHb: Wei et al (101) attempted to alter the
production of acetoin and 2,3-butanediol in Serratia marcescens by transforming
the species with plasmids containing vgb. When the cells were grown on Luria
broth supplemented with 2% glucose, the non–vgb-bearing strains produced 15-
fold as much acetoin and fourfold as much 2,3-butanediol as those cells with vgb.
For growth on Luria broth supplemented with 2% casein acid hydrolysate, though,
a vgb-bearing strain produced significantly more of the two products than did
546 KOFFAS ET AL
strains without the hemoglobin gene. This result clearly demonstrates the com-
plications that can arise in using bacterial hemoglobins as metabolic engineering
tools.
In the area of cell cycle improvements, some of the most complex and impor-
tant regulatory mechanisms of eukaryotic cells are those that govern cell division.
Effective reprogramming of the complex regulatory apparatus to achieve biopro-
cess goals, such as cessation of proliferation at high cell density to allow an
extended period of high production, can require coordinated manipulation of mul-
tiple genes. The overexpression of the cyclin-dependent kinase inhibitors p21 and
p27 has already proven to be effective in cancer therapy. In a stable genetic
configuration, only regulated overexpression of p27 was successful in inducing
a sustained Chinese hamster ovary cell growth arrest in G1 phase, which also
resulted in a 10-fold increase in per-cell secreted alkaline phospatase, a model
heterologous protein used during these studies. Stable overexpression of p21
alone did not result in growth arrest. Recently, by tetracycline-regulated coex-
pression of p21 and the differentiation factor CCAAT/enhancer-binding protein
a (which both stabilizes and induces p21), Fussenegger et al achieved effective

cell cycle arrest. Production of secreted alkaline phospatase has been increased
10- to 15-fold, on a per cell basis, relative to an isogenic control cell line. Because
the activation of apoptosis response is a possible complication in a proliferation-
arrested culture, the survival gene bcl-xL was coexpressed with another CDI, p27,
found to enable CHO cell cycle arrest predominantly in G1 phase. CHO cells
stably transfected with a tricistronic construct containing the genes for these pro-
teins and for secreted alkaline phospatase showed 30-fold higher secreted alkaline
phospatase expression than control cells (25).
BIOMEDICAL APPLICATIONS
Besides manufacturing applications, metabolic engineering is having a significant
impact on the medical field. The main focus here is the design of new therapies
by identifying specific targets for drug development and by contributing to the
design of gene therapies. Such approaches currently target a specific single enzy-
matic step implicated in a particular disease. There is no assurance, however, that
the manipulation of a single reaction will translate to systematic responses in the
human body. Although mammalian intermediary metabolism was defined in bio-
chemical terms many years ago, it is important to remember that much of this
information accrued from studies in vitro. We have less understanding of the
organization in vivo. Furthermore, the intersections of the central pathways of
metabolism, such as glycolysis, gluconeogenesis, urea synthesis, tricarboxylic
acid cycle, etc, cause changes in one pathway, owing to inborn error or disease,
to affect pathways that may seem remote from the initial metabolic defect (73).
In this regard, medical applications are no different from the ones mentioned
earlier in an industrial context, and, as such, they will benefit from developments
in metabolic engineering through a better analysis of experimental results and

applications to the rational selection of targets for medical treatment. In this sec-
tion, some representative examples are highlighted that illustrate the application
of metabolic engineering tools to the study of human disease.
Inborn Errors of Metabolism

One of the earliest applications of MFA to inherited disorders of metabolism
concerned aberrations associated with glycogen storage diseases. In a series of
13
C-tracer experiments, Kalderon et al (42–45) examined the pathways of hepatic
glucose storage and use in glycogen storage disease (GSD) types I (GSD-I) and
III (GSD-III). In children with GSD-I, the isotopomer distributions of infused [U-
13
C]glucose and plasma glucose were identical, indicating absence of glucose
recycling, whereas a significant change in the isotopomer distribution of plasma
glucose was observed in normal and GSD-III subjects. The absence of recycled
glucose in their plasma eliminated a mechanism for glucose production in GSD-
I patients involving gluconeogenesis, suggesting a deficiency of a gluconeogenic

enzyme such as glucose 6-phosphatase. In contrast, gluconeogenesis was sug-

gested as the major route for endogenous glucose synthesis in GSD-III patients.
Moreover, these differences in glucose recycling correlated with significant dif-
ferences in the glucose C-1 13C nuclear magnetic resonance splitting pattern in
plasma, suggesting that such 13C nuclear magnetic resonance analysis of plasma
may be used to noninvasively diagnose defects in gluconeogenesis.
Another type of disorder to which MFA has been applied is hereditary fructose
intolerance (HFI), an inborn deficiency in the ability of aldolase B to split fruc-
tose-1-phosphate. Continuous exposure of these subjects to parental fructose dur-
ing infancy may result in liver cirrhosis, mental retardation, and death. In most
cases of HFI, final diagnosis of aldolase B deficiency is usually performed in
liver biopsy specimens. As an alternative, Gopher et al (27) proposed a method
for noninvasive in vivo diagnosis of HFI based on mass isotopomer analysis. In
control and HFI children, steps involved in fructose metabolism were quantified
by analyzing plasma glucose isotopomer populations after nasogastric infusion
of D-[U-13C]fructose. After administration of labeled fructose, the conversion of
fructose to glucose was significantly lower in HFI than in control children as
determined by this method, supporting its validity as a diagnostic test. Further-
more, it was found that the generally accepted pathway of fructose conversion to
glucose, by fructose-1-phosphate aldolase to triose phosphate, accounts for only
one-half of the total amount of fructose conversion in normal subjects. It is sug-
gested that a direct pathway from fructose to fructose-1,6-bisphosphate by 1-
phosphofructokinase exists, accounting for the remainder of fructose conversion.
Diabetes has also been the subject of many studies using stable isotopomer
methods. A series of extensive studies by Cohen (10–12) using streptozotocin-
diabetic rats has shown that the increase in relative flux through pyruvate car-
boxylase and the inhibition of flux through pyruvate kinase that prevents
548 KOFFAS ET AL
reconversion of phosphoenolpyruvate into pyruvate may be concerted actions

leading to the enhanced gluconeogenesis found in this model of diabetes. Tayek
& Katz (90) compared the relative contributions of gluconeogenesis and glyco-
genolysis with postabsorptive glucose production in normal and non–insulin-
dependent diabetes mellitus (NIDDM) humans. They found that total glucose
production was elevated in NIDDM patients compared with control subjects and
that fractional contribution of gluconeogenesis was comparable, raising doubts
about the widely held notion that synthesis of hepatic glycogen is seriously
impaired in NIDDM. On the other hand, Landau et al (54) estimated the contri-
bution of gluconeogenesis to glucose production in insulin-dependent diabetes
mellitus (IDDM) patients to be significantly less than in normal subjects, also
based on analyses of isotopomer distribution. More recently, Peroni et al (70)
measured gluconeogenic fluxes in postabsorptive and starved normal and strep-
tozotocin-diabetic rats, in which it was found that the increased gluconeogenic
contribution to glucose production in diabetic rats relative to control rats in the
postabsorptive state was abolished in the starved state.
Once inherited disorders of metabolism are diagnosed and the affected bio-
chemical pathways have been identified, these diseases may be treated by using
genetic-engineering tools. The therapy for many inherited liver enzyme deficien-
cies requires the removal of toxic intermediate metabolites from the blood of
affected individuals. Recent research tries to focus on the removal of circulating
toxins by expressing the missing enzymes in tissues other than the liver. This will
hopefully positively influence the disease phenotype. Harding et al (29) success-
fully expressed the phenylalanine hydroxylase (PAH) activity in skeletal and car-
diac muscle of mice under the control of the mouse muscle creatine kinase
promoter. When they bred the muscle PAH-expressing mice with liver PAH-
deficient mice, a progeny was created that lacked PAH activity in liver but
expressed PAH in muscle. These mice exhibited hyperphenylalaninemia at base-
line, but serum phenylalanine levels decreased significantly when the mice were
supplemented with tetrahydrobiopterin, a required cofactor for PAH. This result
suggests that gene therapy targeted to heterologous tissue, such as muscle, will
be an effective treatment of selected inborn errors of metabolism.
Cell and Organ Physiology

A powerful feature of metabolic engineering is that it allows systematic investi-
gation of metabolic control and regulation in intact tissues. Given that many
metabolic disorders, such as liver cirrhosis, post-traumatic hypermetabolism and
muscle wasting, cancer cachexia, etc, have no clearly identifiable genetic origins,
it is clear that to develop therapeutics or treatment strategies, quantitative char-
acterization has to be achieved at a biochemical level. In this regard, the results
of cell or organ physiological studies within the framework of MFA could be
very valuable. For example, an important focus of tumor biology has been on
understanding the ability of tumors to adapt to adverse growth environments such
as hypoxic or hypoglycemic conditions. It has been suggested that the energy

metabolism of tumors is altered in such a way as to accommodate high levels of
anaerobic metabolism and alternate substrate utilization. Using AS-30D hepatoma
cells, Holleran et al (31) investigated the quantitative importance of acetoacetate
and glucose as substrates of energy metabolism in tumors. It was found that
acetoacetate diverted pyruvate from pyruvate dehydrogenase (PDH) to pyruvate
carboxylation. In contrast, dichloroacetic acid, a metabolic analog of acetoacetate
that is an activator of PDH, increased the oxidation of glucose largely through
PDH, indicating that PDH is not maximally active in the presence of dichloroac-
etic acid. Isotopomer spectral analysis of lipid synthesis demonstrated that, in the
absence of acetoacetate, glucose supplied 65% of the acetyl-CoA used for de
novo lipogenesis. Isotopomer spectral analysis refers to a variant of 13C-isoto-
pomer analysis particularly suited to the study of lipid metabolism (50). In the
presence of high levels of acetoacetate, glucose was replaced as a lipogenic pre-
cursor, and acetoacetate supplied 85% of the acetyl-CoA for lipogenesis vs only
2% for glucose. Thus AS-30D cells may have a large capacity for acetoacetate
use for de novo lipogenesis, leading to greater capacity for fat storage, which
may help survival in cachexic conditions.

Portais et al (71) used a mathematical model of the TCA cycle in combination
with 1H-13C-nuclear magnetic resonance to calculate the flux distribution in rat
brain tumor cells. In their study, it was found that the pyruvate carboxylase activ-
ity and the efflux from the TCA cycle in C6 glioma cells are estimated to be very
low, suggesting a lack of glutamine production in these cells. In a subsequent
publication (72), they reported that glutamine and glucose are metabolized com-
plementarily in C6 cells in that glutamine is mainly used for anaplerosis but not
a substrate for energy metabolism, whereas glucose is poorly anaplerotic and is
essentially used as energetic fuel. Using a similar method, Bouzier et al (5) found
that, unlike normal astrocytes, C6 cells preferentially use lactate as a substrate
for oxidative metabolism. Such characterizations of the differences in interme-
diary metabolism between tumors and their normal counterparts could lead to
better understanding of tumor proliferation and may be exploited to control tumor
growth in vivo.
MFA has also been used to characterize the effects of acute metabolic stresses,
such as hypoxia or reperfusion injury. Malloy et al (62) developed a model based
on isotopomer distribution of glutamate in heart tissue extracts to show that, in
perfused hearts exposed to a combination of substrates (lactate, acetate, glucose),
ischemia-reperfusion leads to an increase in the contribution of acetate and a
decrease in the contribution of lactate as sources of acetyl CoA. Ischemia-reper-
fusion injury also causes an increase in anaplerotic flux. However, exogenously
added aspartate or glutamate are not significantly metabolized. This is a finding
that establishes a protective role for aspartate and glutamate on myocardial ische-
mia that does not result from a direct mechanism involving the TCA cycle in the
heart tissue. Also in a perfused heart model, Laplante et al (55) established that
the cardioprotective effect of fumarate during ischemia or hypoxia occurs through
550 KOFFAS ET AL
its reduction to succinate. Another technique developed by Sherry et al (79, 88),

showed that lipoamide—an agent being considered to enhance recovery after
infarctus—prevented, in large part, the switch from lactate to acetate use induced
by ischemia.
Finally, over the past several years, protocols have been developed that allow
the extension of 13C-labeling–based flux analysis to noninvasive monitoring of
specific organs without biopsies. One such approach involves the use of xeno-
biotics such as phenylacetate, which in primates is excreted in urine as a glutamine
conjugate. Conjugation occurs specifically in the liver. Glutamine is synthesized
from a-ketoglutarate via glutamate without rearrangement of carbons. Thus, glu-
tamine carbons reflect the carbons of a-ketoglutarate. Consequently, by analyzing
the labeling pattern in the glutamine conjugate in urine, liver metabolic fluxes
may be estimated. This method was first validated by Magnusson et al (61), who
estimated the fluxes in and around the TCA cycle in human subjects by analyzing
the 14C distribution in excreted phenylacetate after infusion with [3-14C]lactate
and oral administeration of phenylacetate. Jones et al (40) improved the method
by identifying a stable isotope tracer, [U-13C]propionate, which is quantitatively

extracted into the liver from portal circulation. Coined as ‘‘chemical biopsy’’ by
DiDonato et al (17), these noninvasive organ-monitoring protocols used in con-
junction with 13C-labeling experiments could become an important research and
diagnostic tool, providing a more detailed assessment of the metabolic state of
liver in human metabolic disorders.
CONCLUDING REMARKS
The success of biotechnology and biomedical engineering applications of the type

addressed in this review—the production of primary and secondary metabolites,
the alteration and improvement of cellular properties, and the investigations of
the causes and potential treatments of diseases and injuries—are all subject to the
understanding of complex networks of metabolic pathways. Because its focus is
not on isolated reactions but on the interrelationships of reactions in networks,
the emerging field of metabolic engineering should be seen as particularly relevant
to these applications. The examples described above clearly demonstrate the
breadth of the range of advances realized through metabolic engineering.
Although metabolism and cell physiology provide the main context for ana-
lyzing reaction pathways, it should be pointed out that results of flux determi-
nation and control have still broader applicability. Thus, besides the analysis of
material and energy fluxes through metabolic pathways, the concepts of metabolic
engineering are equally useful in the analysis of information fluxes and of those
encountered in signal transduction pathways. Because the latter have not yet been
well defined, the main focus of this article has been on applications to metabolic
pathways. However, once the concepts of information pathways have crystallized,
we expect that many of the ideas and tools presented herein will find good use
in the study of the interactions of signal transduction pathways and the elucidation
of the complex mechanisms by which external stimuli control gene expression.
Metabolic engineering is the science aiming at a holistic understanding of
metabolic functions and cellular physiology. As such it provides a much needed
framework for analyzing measurements of differential gene expression obtained
through the application of emerging technologies such as DNA microarray
hybridization. These data, in combination with detailed flux measurements
obtained by available methods and others under development, offer the best prom-
ise for a systematic study and elucidation of metabolic networks. As accurate
measurements of gene expression, protein content, and in vivo fluxes become
readily available, intricate regulatory structures at the genetic and metabolic levels
will be gradually better understood using the principles of metabolic engineering.
This will have profound implications for the rational modification of metabolic
and signaling pathways both in a biotechnological context and in refining current
methods for target selection in drug development and gene therapy.
Visit the Annual Reviews home page at http://www.AnnualReviews.org.

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Annual Review of Bimedical Engineering
Volume 1, 1999
CONTENTS
A Dedication in Memoriam of Dr. Richard Skalak, Thomas C. Skalak 1
Tissue Engineering: Orthopaedic Applications, C. T. Laurencin, A. M. A.

19
Ambrosio, M. D. Borden, J. A. Cooper Jr.
Airway Wall Mechanics, Roger D. Kamm 47
Biomechanics of Microcirculatory Blood Perfusion, Geert W. Schmid-

73
Schönbein
Engineering and Material Considerations in Cell Transplantation, Elliot
103
L. Chaikof
Bioreactors for Haematopoietic Cell Culture, Lars Keld Nielsen 129

Implanted Electrochemical Glucose Sensors for the Management of

153
Diabetes, Adam Heller
Injectable Electronic Identification, Monitoring, and Stimulation Systems,
177
Philip R. Troyk
Robotics for Surgical Applications, Robert D. Howe, Yoky Matsuoka 211
Transport of Molecules, Particles, and Cells in Solid Tumors, Rakesh K.

241
Jain
Nucleic Acid Biotechnology, Charles M. Roth, Martin L. Yarmush 265
Fluid Mechanics of Vascular Systems, Diseases, and Thrombosis, David

299
M. Wootton, David N. Ku
Automatic Implantable Cardioverter-Defibrillators, William M. Smith,
331
Raymond E. Ideker
Engineering Aspects of Hyperthermia, Robert B. Roemer 347
3-D Visualization and Biomedical Applications, Richard A. Robb 377
Microfabrication in Biology and Medicine, Joel Voldman, Martha L.

401
Gray, Martin A. Schmidt
Engineering Design of Optimal Strategies for Blood Clot Dissolution,
427
Scott L. Diamond
Cellular Microtransport Processes: Intercellular, Intracellular and
463
Aggregate Behavior, Johannes M. Nitsche
New Strategies for Protein Crystal Growth, J. M. Wiencek 505
Metabolic Engineering, M. Koffas, C. Roberge, K. Lee, G.

535
Stephanopoulos
Ultrasound Processing and Computing: Review and Future Directions,
559
George York, Yongmin Kim
Telemedicine, Seong K. Mun, Jeanine W. Turner 589
Imaging Transgenic Animals, T. F. Budinger, D. A. Benaron, A. P.

611
Koretsky
Instrumentation for the Genome Project, J. M. Jaklevic, H. R. Garner, G.
649
A. Miller

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Annu. Rev. Biomed. Eng. 1999.

Key Words metabolism, flux, pathway

metabolic engineering is well suited as a framework for the analysis of genome-wide

intentional manipulation of metabolic pathways by humans to improve the prop-

well-defined genetic backgrounds. The redirection of cellular metabolism to cre-

in the form of new pathway construction. As such, differentiation from genetic

associated with metabolic engineering are the biosynthesis of secondary metab-

the fermentation characteristics of such strains differ, such differences may be

metabolites. A central goal of metabolic engineering is to achieve the production

of commodity chemicals by overexpressing key metabolic pathways that already

transducing each of several mutations one at a time, researchers combined, within

in the isoleucine pathway, allowed significant isoleucine accumulation by B. lac-

tofermentum strains (15).

pathways can be used to enhance the production of commercially desirable com-

pounds such as carotenoids (81).

the chromosome of E. coli K-12. Tryptophan producer derivatives of E. coli

metabolism. The main effect of overproducing the NADH oxidase was an

S. coelicitor, the only species with well-established genetics, to S. lividans,

duction of the cancer chemotherapy drugs epirubicin (48-epidoxorubicin) and 48-

epidaunorubicin. The medical and industrial significance of epirubicin lies in its

step in the synthesis of the daunorubin precursor, daunosamine. By incorporating

synthesis processes. Because of this, metabolic engineering is being called on to

provide the means of increasing the efficiencies and product concentrations of

engineering of polyketide synthases and at the same time to derive knowledge

CELL AND TISSUE ENGINEERING

to achieve previously inaccessible metabolic states.

The hemoglobin (VHb) of the microorganism Vitreoscilla species has been

a (which both stabilizes and induces p21), Fussenegger et al achieved effective

in metabolic engineering through a better analysis of experimental results and

Inborn Errors of Metabolism

I patients involving gluconeogenesis, suggesting a deficiency of a gluconeogenic

enzyme such as glucose 6-phosphatase. In contrast, gluconeogenesis was sug-

reconversion of phosphoenolpyruvate into pyruvate may be concerted actions

Cell and Organ Physiology

as hypoxic or hypoglycemic conditions. It has been suggested that the energy

may help survival in cachexic conditions.

its reduction to succinate. Another technique developed by Sherry et al (79, 88),

by identifying a stable isotope tracer, [U-13C]propionate, which is quantitatively

The success of biotechnology and biomedical engineering applications of the type

Visit the Annual Reviews home page at http://www.AnnualReviews.org.

T, Schoner BE. 1989. Production of a

hybrid macrolide antibiotic in Strepto-

cin-production gene cluster. Microbiol- synthesis of erythromycin analogs by an

muscle. Gene Ther. 5:677–83 Hegeman, PL Skatrud, p. 97. Washing-

cogen storage disease type I, studied contributions of gluconeogenesis to

engineering of modular polyketide syn- engineering in Lactococcus lactis by

duction: chromosomal integration of concentration and yield of L-lysine.

split pathway of lysine synthesis in 1,3-propanediol production by Esche-

Corynebacterium glutamicum–quantifi- richia coli expression genes from the

min C in higher plants. Nature 393:365– mobilis. Science 267:240–43

69 108. Zhao LS, Sherman DH, Liu HW. 1998.

Tissue Engineering: Orthopaedic Applications, C. T. Laurencin, A. M. A.

Airway Wall Mechanics, Roger D. Kamm 47

Biomechanics of Microcirculatory Blood Perfusion, Geert W. Schmid-

Bioreactors for Haematopoietic Cell Culture, Lars Keld Nielsen 129

Implanted Electrochemical Glucose Sensors for the Management of

Robotics for Surgical Applications, Robert D. Howe, Yoky Matsuoka 211

Transport of Molecules, Particles, and Cells in Solid Tumors, Rakesh K.

Nucleic Acid Biotechnology, Charles M. Roth, Martin L. Yarmush 265

Fluid Mechanics of Vascular Systems, Diseases, and Thrombosis, David