Inverse Metabolic Engineering:
A Strategy for Directed Genetic
Engineering of Useful Phenotypes
James E. Bailey,” Adriana Sburlati, Vassily Hatzimanikatis, Kelvin Lee,
Wolfgang A. Renner, and Philip S. Tsai
Institute of Biotechnology, ETH Zurich, CH-8093 Zurich, Switzerland
Received September 29, 1995/Accepted April 8, 1996
The classical method of metabolic engineering, identify-
ing a rate-determining step i n a pathway and alleviating
the bottleneck by enzyme overexpression, has motivated
much research but has enjoyed only limited practical
success. Intervention of other limiting steps, of counter-
balancing regulation, and of unknown coupled pathways
often confounds this direct approach. Here the concept
of inverse metabolic engineering is codified and its ap-
plication is illustrated with several examples. Inverse
metabolic engineering means the elucidation of a meta-
bolic engineering strategy by: first, identifying, con-
structing, or calculating a desired phenotype; second, de-
termining the genetic or the particular environmental
factors conferring that phenotype; and third, endowing
that phenotype on another strain or organism by di-
rected genetic or environmental manipulation. This para-
digm has been successfully applied in several contexts,
including elimination of growth factor requirements in
mammalian cell culture and increasing the energetic ef-
ficiency of microaerobic bacterial respiration. 0 1996 John
Wiley & Sons, Inc.
Key words: inverse metabolic engineering hemoglobin
cell cycle CHO cell culture culture fluorescence
INTRODUCTION
Metabolic engineering has been defined as “the improve-
ment of cellular activities by manipulation of enzymatic,
transport, and regulatory functions of the cell with the use of
recombinant DNA technology” (Bailey, 1991) (use of the
term “improvement” in this definition connotes an identi-
fied goal and, therefore, clearly refers to a directed manipu-
lation). The potential applications of metabolic engineering
span the entire spectrum of biotechnology, and encompass
creation of new processes and products as well as improve-
ment of existing processes. Considering the general feasi-
bility of introducing any heterologous genes (natural or syn-
thetic) and of making any change in the host genome, the set
of genetic possibilities available to the metabolic engineer is
almost infinite. Only a small number of these infinite pos-
sibilities will be effective in achieving the metabolic engi-
neer’s goals. This means that metabolic engineering is al-
most certain to fail unless powerful algorithms can be iden-
tified which greatly increase above random chance the
probability of identifying an effective genetic change.
* To whom all correspondence should be addressed. Fax: 411-633-10-5
Biotechnology and Bioengineering, Vol. 52, Pp. 109-121 (1996)
0 1996 John Wiley & Sons, Inc.
The classical problem in the early emergence of meta-
bolic engineering is identifying a flux-limiting step in a
specified metabolic pathway. This formulation of the objec-
tive embodies an implicit assumption of several layers of
knowledge about the pathway. Not only is the identity of the
pathway assumed-what steps occur-but also the identity
of the catalysts involved should be known. Furthermore, to
choose a possible flux-limiting step, other than one at ran-
dom, much more information must be available, whether in
terms of reaction kinetics, intermediate metabolite concen-
trations, or results from well-designed stimulus-response
experiments (Cornish-Bowden and Cardenas, 1990; Ga-
lazzo and Bailey, 1990; Schlosser et al., 1993).
Later developments of metabolic engineering have con-
sidered much more complicated metabolic networks and
objectives such as selectivity improvement or creation of a
pathway new to the original network. However, common to
most of these exercises is a rational, deductive approach
(Bailey, 1991). Typically, based on knowledge of the meta-
bolic system of interest, a genetic manipulation is proposed
which in some way has postulated potential benefit based on
the expected perturbation within the known biochemical
network. This classical way of posing the metabolic engi-
neering problem, and of formulating a solution strategy, will
here be termed constructive metabolic engineering.
Results from the constructive metabolic engineering ap-
proach have been mixed. Notable successes have been
achieved, for example, in bacterial processes for amino ac-
ids and a few other fine chemicals. However, in many cases,
the metabolic consequence of the genetic change based on
the constructive strategy differs substantially from that de-
sired (Bailey, 1991). Perhaps with the benefit of accumu-
lated hindsight, such failures are quite likely due to several
classes of limitations in knowledge of metabolic networks,
effects of which will here be called secondary responses to
metabolic engineering. First, any network contemplated in
undertaking a metabolic engineering constructive design,
now and in the foreseeable future, is always a subnetwork of
a much larger, much more complex global metabolic net-
work, at least at the level of a cell and usually extending to
an interacting multicellular population. The subnetwork of
interest is typically coupled with the global network through
common cofactors and metabolites and, possibly, more
CCC 0006-3592/96/010109-13
complicated interactions influencing levels of proteins ac-
tive in the subnetwork. Furthermore, the specific activities
of some proteins mediating critical subnetwork processes
are often regulated by the cells’ native control system
through inhibition, activation, phosphorylationldephos-
phorylation, association with other proteins or nucleic acids,
and other mechanisms. Typically, all such control connec-
tions to the subnetwork considered are not known or are not
considered in constructive metabolic engineering.
Another factor which has confounded some earlier con-
structive metabolic engineering projects is the capability of
enzymes to catalyze conversions of structural analogs of
their natural substrates. Combined with the presence of pre-
viously unobserved host cell enzyme activities, this cata-
lytic flexibility has resulted in several surprises, some posi-
tive, after introduction of heterologous activities to create a
new biosynthetic or biodegradative pathway (Bailey, 1991).
Further, a constructive metabolic engineering design often
must assume availability of required cosubstrates. A protein
activity added or amplified by metabolic engineering will
only succeed in some applications if that activity is suitably
localized in a particular subcellular compartment. Some het-
erologous proteins require cofactors or assembly into mul-
tiprotein complexes to be active. In addition, heterologous
proteins may misfold, aggregate, or be selectively degraded.
All of these factors imply that expression of a peptide does
not necessarily confer the desired corresponding activity.
While the underlying strategic concept has not yet been
discussed in a general sense, several earlier metabolic en-
gineering studies have been based on a different algorithm
for choosing potentially useful genetic changes. The ele-
ments and information flow in this alternative approach,
here termed inverse metabolic engineering, are illustrated
schematically in Figure 1. The starting point of an inverse
metabolic engineering route to an improved industrial or-
ganism is identification of a desired phenotype in a heter-
ologous organism or in a related model system. Then, based
on some inverse genetics strategy (this nomenclature is ex-
plained in a later section), the genetic bases for this desired
phenotype are defined or hypothesized. By transferring this
t -r-
-1 GENETICALLY MODIFIED
INDUSTRIAL ORGANISM
WITH DESIRED PHENOTYPE
Figure 1. Schematic diagram of information flow in inverse metabolic
engineering. The critical step is identifying the genetic basis for a desired
phenotypic characteristic. Genetic engineering then is applied to attempt to
confer that phenotype on an industrial organism.
110 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 52, NO. 1, OCTOBER 5, 1996
genetic basis to the chosen industrial organisms the goal is
achieved.
Even if the genetic manipulation(s) chosen based on an
inverse metabolic engineering strategy fail to achieve the
desired change in phenotype, the experiment provides in-
formation on the genetic stimulus-phenotype response char-
acteristics of the organism which may be useful in identi-
fying a subsequent, more effective metabolic engineering
strategy or for improving understanding of the organism’s
physiology. Such experience accumulated from results of
inverse metabolic engineering applications can provide a
foundation of information for evolution toward a more in-
formed, rational, constructive metabolic engineering ap-
proach to the system.
Before discussing the general obstacles and primary limi-
tations in realizing a desired outcome with an inverse meta-
bolic engineering strategy, the inverse metabolic engineer-
ing approach will be illustrated by two examples concerning
very different applications and also very different metabolic
systems.
V/TR€OSC/LfA HEMOGLOBIN: AN INVERSE
METABOLIC ENGINEERING STRATEGY TO
ALLEVIATE OXYGEN LIMITATION
Finding a way to deliver oxygen to the obligate aerobic
fungus, Penicillium, is perhaps the defining problem of bio-
chemical engineering. Despite advances in mixing and air
dispersion technology, in membrane systems, and in discov-
ery of various additives for media which can enhance oxy-
gen transfer, limitation of desired cellular activities by oxy-
gen supply remains an important concern in many situa-
tions. An inverse metabolic engineering approach to
alleviate adverse effects of inadequate oxygen availability
on bioprocess productivity was articulated and demon-
strated by Khosla and Bailey (1988).
The inspiration for this strategy was the report that an
obligate aerobic bacterium called Vitreoscilla synthesized
much greater quantities of a heme cofactor for a simple
hemoglobin when grown under oxygen-limited conditions.
The natural habitat of Vitreoscillu is an extremely poorly
oxygenated environment. This combination of phenotypic
properties suggested that synthesis of its hemoglobin could
be a genetic strategy employed by Vitreoscillu to improve
its metabolism and growth under extreme oxygen limita-
tion. Motivated by this hypothesis, efforts were initiated to
clone the gene for this hemoglobin and, subsequently, to
express this hemoglobin in a variety of aerobic industrial
microorganisms to see if this strategy would enhance cell
productivity, especially under oxygen-limited conditions.
The first such experiment showed that expression of VHb in
the bacteria Escherichia coli enabled growth to higher cell
densities in microaerobic cultivations (Khosla and Bailey,
1988).
Recent research has explored, in some detail, the rela-
tionship between the amount of VHb synthesized and the
corresponding growth phenotype under poorly oxygenated
conditions. The IPTG-inducible VHb expression vector il-
lustrated in Figure 2 was constructed and transformed into --)O.O05mMIPTG
E. coli W3110 (for details on materials and methods, see +0.OlmM IPTG
Tsai, 1995). The resulting metabolic engineered strain was *O.MmMIPTG

cultivated in parallel in different concentrations of IPTG.

Figure 3 illustrates the resulting time trajectories of cellular
VHb content and total cell protein during these experiments.
In the range of VHb concentrations observed in this experi-
ment, cellular protein accumulation increases with increas-
ing VHb expression, up to at least around a final level of 3.8
pmol of VHb per gram dry cell weight, at which point 10
saturation may be approached. This gradual and monotonic
response to induction of VHb expression is the most care-
fully controlled and convincing demonstration to date that
the enhanced microaerobic growth observed is in fact a
consequence of VHb expression and not possibly an artifact
due to some secondary response to metabolic engineering.
Moved by observations of improved oxygen-limited
growth of E. coli expressing VHb, extensive efforts were
undertaken to accomplish expression of VHb in a variety of
industrial organisms ranging from bacteria to yeasts to
higher fungi to cultured mammalian cells. A summary of the 1
10 20 30 40 50
results of these experiments is provided in Table I. In each
of the cases listed in Table I, there was a reason, a priori, to Time @)
expect that availability of oxygen, ATP, or proton motive
force might potentially limit the production metabolism of Figure 3. Time trajectories of intracellular VHb concentration and cor-
responding accumulation of cell dry weight in parallel, oxygen-limited
interest. It is also important to comment that, in several batch cultivations of E. coli W3110:pKTVl in different concentrations of
situations, the presence of VHb in the cell did not signifi- inducer IPTG (Tsai et al., 1995b).
cantly affect growth of the cells, but a significant response
of the product formation pathway and coupled processes to
VHb was nevertheless observed. In none of these experi- and there remains no, direct experimental proof that expres-
ments was the amount of VHb expressed altered to ascertain sion of VHb has any physiological consequence or meta-
whether higher expression might provide greater physi- bolic benefits in its original host Vitreoscillu. Therefore, all
ological response. of these earlier applications of VHb were motivated by a
These applications of VHb are perhaps the clearest, most hypothesis based on a natural phenotypic response. This
striking examples to date of the inverse metabolic engineer- strategy has successfully achieved significant metabolic im-
ing approach. When all of these experiments were designed provements in many different systems without any knowl-
and conducted, nothing was known about the mechanism of edge of the pathways involved or the steps in growth or

EcoR I ,
action of VHb within the cell. In particular, there was not,

~p~;dIII
product synthesis at which VHb exerts an influence.
As indicated above, pursuit of an inverse metabolic en-
gineering strategy at least provides stimulus-response in-
formation on how the physiology of the host organism is
affected (or not) by the genetic perturbation introduced.
Accumulation of this information, particularly in the case of
ssp I sal I many different types of substantial physiological responses
to a single gene product, as is the case with VHb, motivates
experiments designed to better understand the mode of ac-
tion and the detailed biochemical effects of effective genetic
modification.
Different avenues have been followed recently to char-
acterize in greater biochemical detail the response of E. coli
to expression of VHb. A starting point for all of these ex-
periments is the hypothesis that, as a reversible site of oxy-
ColEl
gen binding, VHb likely interacts with metabolism at the
Figure 2. The expression vector pKTVl employed for inducible expres- level of the respiratory chain. E. coli is a complicated sys-
sion of different levels of Virreoscilla hemoglobin (VHb) in E. coli W3110 tem in this respect because it possesses two different termi-
(Tsai et al., 1995b). nal oxidases, cytochrome 0, which is expressed under

BAILEY ET AL.: INVERSE METABOLIC ENGINEERING 111

Table I. Vitreoscilla hemoglobin (VHb) has been cloned and expressed in a variety of organisms used for production of primary and secondary
metabolites and cloned proteins.”

Organism Product or activity Effect of VHb Reference

Escherichia coli
JM 101:pRED2 Total cell protein 2.1-fold increase Khosla and Bailey (1988)
Gro21:pTCAT Total cell protein 30% increase Khosla et al. (1990)
W3110:pKTVI Total cell protein 2.2-fold increase Tsai et al. (1995)
Gro22:pTCAT Chloramphenicol acetyltransferase 80% increase Khosla et al. (1990)
(CAT) activity
Gr021 :pGE245 P-Galactosidase activity 40% increase Khoala et al. (1990)
JM103:pMK79 a-Amylase activity 3.3-fold incease Khosravi et al. (1990)
Psudomonas aeruginosa
B-77 1:pSC160 Viable cell number 11% increase Liu et al. (1995)
Xanthomonas maltophilia
XM:pSC I60 Viable cell number 15% increase Liu et al. (1995)
Bacillus subtilis
1012M15:pMKV6 Neutral protease activity 30% increase Kallio and Bailey (1996)
Corynebacterium glutamicum
13287:pFSl L-Lysine titer 30% increase Sander et al. (1993)
13287:pFSl L-Lysine yield on glucose 24% increase Sander et al. (1993)
Streptomyces lividans
TK64:pWLD5 Final cell density 50% increase Magnolo et al. (1991)
Streptomyces coelicolor
M145:pWLDlO Actinorhodin production 10-fold increase Magnolo et al. (1991)
Streptomyces rimosus Oxytetracycline production 2.2-fold increase Galazzo (personal communication)
Acremonium chrysogenum
C 1O:pULXTR1 Cephalosporin C production 3.2-fold increase DeModena et al. (1993)
Saccharomyces cerevisiae
SEY2101:pEX-2 Final cell density (growth on acetaldehyde) 3-fold increase Chen et al. (1994)
Chinese hamster ovary (CHO) cells
ATCC 9606:pMSG-VHb Tissue plasminogen activator production 40%-100% increase Pendse and Bailey (1994)

“Resultsrelative to controls not expressing VHb are indicated. Details on VHb-expression strategies, host strains and cell lines, and cultivation and assay
conditions are available in the indicated references.

well-aerated conditions and which extrudes four protons per
oxygen atom reduced, and a higher oxygen-affinity second
terminal oxidase, cytochrome d, which is expressed only
under microaerobic conditions and which extrudes only two
protons from the cytoplasm per oxygen atom reduced. The
efficiency of proton export from the cytoplasm is, of course,
the crucial energetic determinant for aerobic metabolism
given the many functions of the proton motive force, in-
cluding ADP phosphorylation, which are powered by reen-
try of those protons from the periplasm into the cytoplasm,
often through specific protein ports.
A series of different types of experiments have probed
this hypothetical locus for VHb action at increasing levels
of biochemical definition. First, the net proton efflux per
oxygen atom reduced was measured for a VHb-expressing
E. coli and a corresponding control. In a resting cell sus-
pension, E. coli-containing VHb exported three protons per
oxygen reduced, whereas VHb-free controls exported only
two (Kallio et al., 1994). In vivo nuclear magnetic reso-
nance (NMR) spectroscopy measurements, in a growing
fed-batch culture observed on-line, showed a twofold in-
crease in ATP accumulation due to the presence of VHb
(Chen and Bailey, 1994). Saturation transfer NMR experi-
ments on dense resting cell suspensions disclosed an in-
crease in ATP turnover of approximately 30% in VHb-
expressing cells (Chen and Bailey, 1994).
Consistency of all of these findings qualitatively with the
starting hypothesis then led to measurements of the levels of
the two terminal oxidases and estimation of their specific
activities in VHb-expressing E. coli and VHb-free controls.
Deconvolution of VHb, cytochrome 0, and cytochrome d
bands from in vivo absorption spectra showed that, under
microaerobic environments (dissolved oxygen less than 2%
air saturation), the presence of VHb increased cytochrome o
amount by more than fivefold, and increased cytochrome d
content by 1.5-fold. Based on oxygen-uptake kinetics mea-
surements of these mutants, the specific activity of cyto-
chrome o was enhanced in the presence of VHb, but no
difference was observed for the cytochrome d specific ac-
tivity (Tsai et al., 1995b).
Changes in respiratory chain activity and efficiency with
respect to proton pumping should alter both the rates of
NADH oxidation and ADP phosphorylation. These two
central cofactors are important links among central carbon
metabolism, respiration, and many other metabolic path-
ways. For this reason a metabolic flux analysis was con-
ducted of the central carbon pathways in E. coli in expo-
nential growth with and without VHb expression (these ex-
periments were done with the IPTG-inducible constructs
and so are based on identical genotypes) (Tsai et al.,1995a).
Figure 4 shows the estimated fluxes, which have been
scaled to a common glucose uptake rate of 2.6 mmol glu-

112 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 52, NO. 1, OCTOBER 5, 1996

 I
I
I 1.69
I 2H20 2
' 2.73
I

i
I
I NADHresp
- -.J 11.19
13.06
-338
-5*46
~ATP
overall

J. fm I 4.60 (NADH -> NADPH)

0.85 (NADH <- NADPH)
Biomass Biomass

Figure 4. Estimated fluxes (scaled to an equal glucose uptake rate) in E. coli central carbon metabolism and respiration for cultures with cloned VHb
(italic) and without (regular font) (Tsai et al., 1995b).

cose per gram dry cell weight per hour. A major effect of
VHb is evident at one of the first steps in central carbon
metabolism in which the flux of glucose into the cell is
partitioned from glucose-6-phosphate into either the pentose
phosphate pathway or the Embden-Meyerhof-Parnas
(EMP) pathway. Carbon flux into the cell is directed to a
much greater extent toward the pentose phosphate pathway
in VHb-expressing cells than in the VHb-free controls, en-
hancing synthesis of several biosynthetic intermediates.
Interestingly, fluxes to end-products of central carbon
metabolism are also redistributed in the presence of VHb.
Fluxes to lactate, succinate, ethanol, and formate are re-
duced, whereas, somewhat surprisingly, the flux to acetate
per glucose consumed is slightly increased when VHb is
present. (Note: These end-product fluxes are directly mea-
sured experimentally and used as inputs to the estimation of
intracellular metabolic fluxes; hence, these fluxes are pri-
mary, direct data and are completely independent of all
assumptions and approximations made in the metabolic flux
analysis.)

BAILEY ET AL.: INVERSE METABOLIC ENGINEERING 113

 Focusing now on respiration, the fluxes of NADH oxi-
dation and ADP phosphorylation, which here are estimated
from the metabolic flux analysis and not measured as in the
earlier studies, are significantly modified. The flux of oxi-
dation-linked ADP phosphorylation is estimated to be en-
hanced approximately 60% in VHb-expressing cells under
microaerobic conditions, a finding quantitatively consistent
with earlier direct NMR observations (Chen and Bailey,
1994).
The metabolic flux analysis also indicates a much greater
rate of NADH oxidation, suggesting that cells with VHb
may be in a significantly more oxidized state than VHb-free
controls. Culture fluorescence and culture redox potential
measurements were undertaken to examine this indication
more directly (Tsai et al., 199%). It was shown that VHb-
expressing E. coli cells are in fact in a more oxidized state.
Moreover, transients of VHb-expressing E. coli culture re-
dox potential and culture fluorescence following step
changes in aeration exhibit significant differences compared
with these same characteristics for VHb-free controls. A
scaledown experiment was conducted in which the air-flow
to the bioreactor was switched on and off with 5-s intervals.
The culture fluorescence of VHb-free E. coli exhibited sig-
nificant fluctuations in response to these externally imposed
variations in aeration. On the other hand, VHb-expressing
cells showed almost no transient changes in intracellular
NAD(P)H levels in response to the externally imposed fluc-
tuations in dissolved oxygen levels (Fig. 5).
These observations suggest an entirely new type of ap-
plication for VHb. It is now well established, both through
direct experimental measurements and through large-scale
5 CELL CULTURE
Do specific
(%I Fluorescence
Unit (SFV)
0 ditives cause complications in product purification; are of
20
40
0
I I I I I
I
I I I I I
0 6 0 m o 6 0 1 2 0
Time (sec)
Figure 5. Transients in culture fluorescence in response to imposed cy-
clic fluctuations in air sparging to the bioreactor (the resulting DO tran-
sients are also illustrated). The VHb expressing culture is much less sen-
sitive to external DO fluctuations than the control E. coli strain which has
no VHb (Tsai et al., 1995~).
114 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 52, NO. 1, OCTOBER 5, 1996
detailed computational simulations (Bajpai and Reuss,
1982; Oosterhuis and Kossen, 1984; Yegneswaran et al.,
1991), that the field of dissolved oxygen concentration as a
function of position within a bioreactor can be highly non-
uniform in large-scale equipment, with greatly reduced dis-
solved oxygen concentrations found far from the sparger-
impeller region. As a consequence of these spatial nonuni-
formities in dissolved oxygen concentration, cells
circulating in the bioreactor experience transient fluctua-
tions of dissolved oxygen concentration in their immediate
environment.
This situation contrasts sharply with those encountered in
smaller-scale bioreactors, in which internal dissolved oxy-
gen concentrations are relatively uniform throughout the
entire volume. Scaledown experiments in which fluctua-
tions in dissolved oxygen concentrations have been imposed
in small-scale equipment to simulate the large-scale situa-
tion have shown that production metabolism can be very
sensitive to these oxygen fluctuations. Based on the culture
fluorescence dynamics observed with and without VHb, it is
plausible that the presence of VHb may buffer the intracel-
lular biochemistry from external fluctuations in dissolved
oxygen concentrations which occur on a time-scale that is
interesting relative to mixing times in large-scale bioreac-
tors (typically in the range of 3 to 10 s). Thus, VHb-
expressing cells may well exhibit less sensitivity to scale,
and perform on a large scale more like the performance
observed in smaller, more spatially uniform, bioreactors.
Further investigation of this possibility remains a future
opportunity.
INVERSE METABOLIC ENGINEERING OF
CELL-CYCLE CONTROL TO ELIMINATE
EXOGENOUS MITOGENS IN MAMMALIAN
There are many motivations presently in different mamma-
lian cell culture applications to eliminate exogenously
added animal proteins from all stages of culture mainte-
nance, expansion, and application. These animal protein ad-
increasing concern as potential sources of adventitious
agents such as prion pathogens; and, in the case of animal
serum or its fractions, are typically highly variable, compli-
cated, uncharacterized mixtures.
To understand better how exogenous mitogens function
and to identify a target phenotype for an inverse metabolic
engineering approach for activating mammalian cell prolif-
eration in culture without mitogens, extensive experiments
have been conducted with wild-type Chinese hamster ovary
(CHO K12) cells (Renner et al., 1995) cultivated in a basal
medium, designated FMX-8, which contains all nutrients
required for growth of these cells (Zang et al., 1955). In the
absence of exogenous mitogens, CHO cells in this medium
remain viable for several days, but do not proliferate (see
Fig. 6A). Cell proliferation has been activated by the addi-
Figure 6. Microphotographs of (A) CHO K1 cells in FMX-8 basal medium, (B) CHO K1 cells in FMX-8 supplemented by 20 ng/mL bFGF, (C) CHO
K1 cells in FMX-8 supplemented by 1 p g insulidml, and (D) CHO K1:cyclin E cells in FMX-8 without any added exogenous protein (Renner et al., 1995).
tion of either basic fibroblast growth factor (bFGF) or in-
sulin, but the morphological phenotypes are clearly differ-
ent as seen in the microphotos in Figure 6A-D. Cells grown
with bFGF stimulation assume a rounded-up morphology
and tend to release from the surface of the cell culture dish,
while insulin stimulation gives rise to a strongly adherent
culture with extensive cell-cell contacts. The phenotype
evidenced by bFGF stimulation is of much greater potential
bioprocessing interest because of an increasing emphasis
industrially in stirred-tank, suspended-cell processes.
Although the entire molecular pathway is not yet defined
for any growth factor or mitogen, it is now generally clear
that growth factors influence cell function by binding to a
receptor, activating a signaling cascade, and ultimately af-
fecting the expression or biochemical state of one or more
molecules involved in regulation of the cell cycle. This
suggests the feasibility of eliminating a requirement for ex-
ogenous growth factors by genetic intervention to accom-
plish the same, or a functionally equivalent, change in levels
and activities of crucial cell-cycle regulating proteins. The
possibility of pursuing such a strategy has only recently
emerged due to rapidly increasing understanding of the mo-
lecular bases of cell-cycle regulation and also because of the
availability of genes and antibodies for many of these cm-
BAILEY ET AL.: INVERSE METABOLIC ENGINEERING
cia1 proteins. Figure 7 shows a summary of some of the
molecular species now understood to influence transitions
through certain critical cell-cycle checkpoints. Of greatest
interest for CHO K1 cells in culture is the GUS transition,
because quiescent cells tend to accumulate in the G,-phase.
Hence, genetic engineering to activate movement from G,-
into S-phase is the most probable and opportune target for a
metabolic engineering approach.
Based on the possible critical importance of cyclin E as a
modulator of the G,/S transition, and also on the availability
of genetic and analytical reagents, cyclin E expression in
CHO K1 cells cultured in protein-free, basal medium, in
this medium supplemented with bFGF, and in basal me-
dium plus insulin, cultures were determined (Renner et al.,
1995). Under the assay conditions employed, no cyclin E
was detected in the protein-free or insulin-supplemented
cultures, but a significant quantity of cyclin E appeared in
the bFGF-augmented culture. Motivated, then, by the pos-
sibility that the phenotype arising from bFGF stimulation
might be somehow mediated or driven through enhanced
cyclin E expression, an inverse metabolic engineering strat-
egy emerged.
Could enhanced expression of cyclin E give the desired
115

Figure 7. Schematic diagram showing many of the molecular species and
complexes involved in regulating transitions through checkpoints in the
cell cycle, which is depicted here as a circle running clockwise (adapted
from Hunter and Pines, 1994).
the cultivation there was no evidence of any cellkell ag-
gregation or clumping; the cells remained highly dispersed,
and grew to a density of 2.7 x lo6 cells/mL. Future studies
will examine whether CHO cells engineered to overexpress
cyclin E perform well for heterologous protein production
in addition to possessing the excellent culture growth char-
acteristics demonstrated here.
Given the complexity of the regulatory network control-
ling the G,/S transition, it is surprising that overexpression
of a single component involved in this network would suf-
fice to shift so drastically the function of the network; that
is, to activate cell proliferation. To gain better understand-
ing of this phenomenon, and to serve as a basis for further
development of metabolic engineering approaches to cell-
cycle manipulation, a mathematical model of the presently
articulated molecular system controlling the G,/S transition
has been formulated and analyzed (Hatzimanikatis et al.,
1995). This model consists of unsteady-state mass balances
for cyclin E, cdk2, cyclin E-phosphorylated cdk2 complex,
Rb-E2F complex, Rb, hyperphosphorylated Rb, and E2F,
written for a homogeneous collection of growing cells (and
therefore with time-increasing total volume). After assum-
ing equilibrium for association of Rb and E2F, the model
can be reduced to three nonlinear ordinary differential equa-

phenotype, namely the proliferating, surface-detached cul- 2.2- r 2.8 rlOO

ture? CHO K1 cells transfected with a cyclin E expression -

2.0
vector, after a short interval of selection in protein-free basal
medium, displayed this desired phenotype (see Fig. 6D) 1.8' -80
(Renner et al., 1995). The cells grew as rapidly as the bFGF-
1.6'
Glucose
2-2 T
stimulated culture and could be transferred into spinner- b8 .70
flask cultivations for fully suspended culture without further
adaptation. Subsequent analysis of the durations of the G,-, xb 1A.
'
2o
1.8

1.6
-60 -
5
S-, and G, + M-phases revealed that the CHO K1 cells 8 1.2'
1.4 3 -50 8
u$
Y
genetically engineered to overexpress cyclin E had essen- 1.0- 4
g
tially identical cell-cycle time distributions as the bFGF- -40
stimulated wild-type culture, and both of these had cell- t
8 .30
cycle subintervals which differed significantly from those
observed in an insulin-augmented culture. d . 0.8
. 0.6 s .20
The technological potential of a CHO cell line converted . 0.4
to protein-independent growth by this inverse metabolic en- - 10
' 0.2
gineering strategy has been investigated in a simple fed-
batch, compact loop bioreactor (COLOR) experiment in . 0.0 LO
0 24 48 72 96 120 144 168
which a single pulse of additional glucose was added when
glucose concentration in the medium fell below about 0.7 Time ($1
g/L. This cultivation was undertaken in an improved pro-
Figure 8. Growth trajectory of a bioreactor cultivation of CHO K1:cyclin
tein-free medium formulation. The seeding density em- E cells in a serum- and protein-free improved FMX-8 medium. The cul-
ployed ( lo4 cells/mL) was intentionally extremely low to tivation was carried out in a 2.3-L compact loop reactor (Bioengineering,
minimize any possible effects on the experiment of auto- CH). The cells from one late exponential T-150 flask were used as inocu-
crine factors. The cells entered exponential phase with no lum. The initial cell density was 10,000 cells/mL; a final cell density of 2.7
million cells/mL was reached after one single glucose refeed after 108 h.
discernible lag time, increased in numbers exponentially The specific growth rate, p, was 1.05 d-', which corresponded to a dou-
with a doubling time of 15.8 h, and retained a viability of bling time of 15.8 h. The viability exceeded 95% throughout the growth
over 95% until late in the culture when glucose again fell to phase of the cultivation. The cultivation conditions were controlled at the
low levels and culture growth stopped (Fig. 8). Throughout following values: T = 37°C. pH 7.25, PO, 50% air saturation, 580 rpm.

116 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 52, NO. 1, OCTOBER 5, 1996

tions which are coupled with four simultaneous algebraic
relations. A quiescent state is manifested mathematically in
this framework by a time-invariant solution of these equa-
tions, with the specific growth rate parameter correspond-
ingly zero. A limit-cycle, time-oscillating solution, with pe-
riod equal to the generation time, denotes a proliferating
state in this context.
This model is intended to provide qualitative guidance for
interpreting and undertaking metabolic engineering. For
such an application, the qualitative features of the model
and its solution set are the properties of greatest importance.
To display some of these general characteristics, the oper-
ating diagram displayed in Figure 9 has been computed.
According to these results, cells in certain quiescent states
can be converted to proliferation by increasing expression
of cyclin E, or by increasing expression of E2F. The first
indication has already been observed experimentally. Given
these results, subsequent effort was focused on cloning and
overexpression of human E2F-1 in CHO cells (an entire
family of E2F species has now been identified; E2F-1 de-
notes the particular member of this family used in these
experiments; Lee et al., 1995b).
The gene for human E2F- 1 was cloned into a mammalian
expression vector also containing a selection marker for
neomycin resistance and transfected into CHO K1 cells. A
number of neomycin-resistant clones were selected and
shown by Southem-blot analysis, immunoblots on SDS-
PAGE gels, immunoblots on two-dimensional protein elec-
Figure 10. Microphotographs showing cultures of (top) a clone of CHO
trophoresis gels, and immunofluorescent confocal micros- K1 transfected with an E2F-1 expression vector and (bottom) wild-type
copy to contain the E2F-1 gene and significantly enhanced CHO K l cells 3 days after transfer from 10% fetal calf serum (FCS)
levels of E2F protein. Clones transferred from FMX-8 supplementation to 0.5% FCS (Lee et al., 1996).
supplemented with 10% fetal calf serum (FCS) into 0.5%
FCS in FMX-8 grew well, while untransfected CHO Kl
cells were marginally viable (see Fig. 10); furthermore, adaptation, to grow in completely protein-free FMX-8 me-
clones overexpressing E2F- 1 were able, without extensive dium. Interestingly, these E2F- 1 overexpressing clones
grew tightly attached to the cell culture flask, in contrast to
the CHO cells genetically engineered to overexpress cyclin
E; this observation and other data indicate that the pheno-
typic responses to metabolic engineering of the cell cycle by
these different genes are significantly different, although
transfectants of both classes display the common character-
istic of proliferation without exogenous mitogenic stimula-
tion (Lee et al., 1995).

ANALYTICAL, CHEMICAL, AND INFORMATION

$ 102
--
10-3 10-2, 10-1 100 101 102
E2F total concentration
Figure 9. The rate of cyclin E synthesis (ordinate) and the concentration
of all forms of transcription factor E2F are two parameters in a mathemati-
cal model of regulation of the G,/S cell-cycle transition; these parameters
are also amenable to manipulation by metabolic engineering because the
genes for human cyclin E and E2F- 1 have been cloned. Here the nature of
the model solutions are indicated as a function of these two parameters. In
this bifurcation diagram, steady-state model solutions correspond to qui-
escence and certain limit-cycle solutions to proliferating cells (Hatzimani-
katis et al., 1995).
TECHNOLOGY FOR INVERSE GENETICS
The power of the technology for deciphering the genetic
basis for a given phenotype is a critical determinant of the
feasibility of inverse metabolic engineering. (The practice
of identifying this phenotype to gene mapping will here be
called “inverse genetics” to distinguish this terminology
from the already familiar ‘‘reverse genetics” language long
used to describe identification of the gene which encodes a
particular protein of interest.) Accordingly, a few comments
are offered here on the convergence of several different
ongoing, methodological advances and of rapidly expand-

BAILEY ET AL.: INVERSE METABOLIC ENGINEERING 117

ing database resources on the scope and speed of inverse
genetics. These remarks summarize a disparate body of in-
formation which is together known to many laboratories but
which may not be familiar to all biochemical engineers.
The multifactorial (epigenetic) nature of many desirable
phenotypes suggests that the manipulation of several differ-
ing genes can result in the same phenotype (to a first order).
However, the inverse metabolic engineering approach re-
quires the identification of only one or one set of genetic
targets that can yield a particular phenotype. To this end,
there currently exist several powerful techniques which pro-
vide information at the nucleic acid and/or protein level
which can be indispensable tools for distilling the essence of
particular phenotypes. A common first step useful in inverse
genetics is determining, relative to a control or parent strain,
which genes are expressed at higher or lower levels in the
strain exhibiting the phenotype of interest. This can be done
at the level of differences in mRNA (cDNA) populations of
proteins. Important determinants of the feasibility for elu-
cidating the genetic basis for a phenotype are the capabili-
ties (in terms of the variety and diversity) of the analytical
tools. Whole cDNA libraries for many cell types are com-
mercially available or can be easily generated using stan-
dard techniques.
For proteins, the advent of two-dimensional electropho-
resis (2DE) of proteins allows users the ability to screen the
entire soluble protein fraction of intra- and extracellular
gene products at the level of expression, synthesis, and deg-
radation. Recent methodological advances have addressed
previous limitations or reproducibility in this technique re-
sulting in an extremely powerful tool for inverse genetics
(see, e.g., Lee et al., 1995). As an example of the utility of
2DE in inverse genetics, Figure 11 depicts the intracellular
protein fraction from CHO Kl:E2F-1 cells with changes
noted relative to CHO K1:cyclin E cells grown in the ab-
sence of any external growth factors. Computer-aided
analyses of these gels reveal 257 spots which are upregu-
lated in CHO Kl:E2F-I cells, and 54 spots which are down-
regulated in CHO Kl:E2F-l cells relative to the cyclin E
cells. Highlighted are some of these gene products.
The bridge between these analytical tools and identifica-
tion of specific, individual genes of interest relies on ex-
periments performed at the micropreparative or preparative
levels. Here, one takes advantage of existing chemical tech-
nology, the limits of which are often determined by avail-
able instrumentation. The amplification of minute amounts
of DNA fragments by the polymerase chain reaction (PCR)
has revolutionized the ability to sequence DNA. mRNA
fragments can be similarly amplified by emerging tech-
niques in reverse transcript PCR (RT-PCR), which allows
simultaneous analysis of several transcripts from total RNA
and is particularly useful for relative or absolute quantifi-
cation of mRNAs (Montarras et al., 1994). Additionally,
this technique is suitable for distinguishing between closely
related mRNA transcripts independent of their relative
abundance. Nucleic acid sequencing has undergone another
118 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 52, NO. 1, OCTOBER 5, 1996
mild revolution with the automation of the electrophoresis
and the development of fluorescent markers to measure mi-
gration. These achievements have increased the sensitivity
and throughput for DNA sequencing. Further developments
which stem from the Human Genome Initiative should be
expected.
In contrast to the widespread use of DNA chemical tech-
nology, the technology available for protein analysis re-
mains largely in the hands of specialists. Automated gas-
phase sequencers for determining amino acid sequence in-
formation are expensive and require particularly skilled
technical staff. However, such technology is particularly
effective for identifying the genetic origins of particular
gene products (e.g., from 2D gel to primary sequence to
cloned gene). Thus, many labs have access to in-house or
on-site services for N-terminal sequencing. Mass spectrom-
etry applied to protein analysis is even more specialized,
expensive, and difficult than N-terminal sequencing. This
trade-off is offset by the thousandfold improvement in sen-
sitivity which addresses a critical rate-limiting step. It
should be noted that cDNA scanning and DNA sequencing
are very fast compared to 2D gel electrophoresis of proteins
and protein sequencing.
With sequence information in hand, one is inclined to
take advantage of the recent advent of the worldwide web of
computer information. The metabolic engineer can move
virtually effortlessly through all known genes and gene
products to determine the potential source of the phenotype
of interest via nucleic acid and protein database information
which is available to nearly every lab. Improved sequence
alignment algorithms have improved computational speed
and decreased rigidity in sequence matching.
Although all these technologies have become standard
fare in many labs they do have inherent limitations such as
sensitivity, speed, and, perhaps most importantly, cost. The
near-term future should bring dramatic (e.g., thousandfold)
improvements. One particularly interesting trend is the de-
velopment of nanotechnology. All-in-one instruments built
onto silicon chips can provide increased sensitivity of pro-
tein detection and offer improved throughput and speed for
nucleic acid analysis. Furthermore, existing manufacturing
techniques for computer chips will potentially provide the
end-user with cheap and simple technologies for inverse
genetics.
CONCLUSIONS: CHALLENGES AND
OPPORTUNITIES IN INVERSE
METABOLIC ENGINEERING
Analogies between current themes in mammalian biology
and molecular medicine, on the one hand, and constructive
and inverse metabolic engineering, on the other, may be
instructive. Metabolic engineering is essentially gene
therapy for industrial organisms. The critical step of iden-
tifying the genes primarily responsible for a given pheno-
type in inverse metabolic engineering is identical to discov-
 Basic

LOW
Mw
Figure 11. Results of two-dimensional electrophoresis of proteins from CHO Kl:E2F-1 cells. The positions of individual CHO cell proteins mapped in
this format are labeled. This protein pattern has been compared with that for CHO K1:cyclin E cells cultured under identical conditions. Circles indicate
some of the 257 different proteins present at significantly higher levels in the CHO KI:E2F-1 cells relative to the CHO K1:cyclin E cells. Squares indicate
positions of some of the 54 proteins relatively downregulated in the CHO Kl:E2F-l cells.

ering the genetic basis for disease. Many investigators have
commented on the great difficulty of the latter, particularly
for phenotypes which depend on changes in multiple genes
(Lander and Schork, 1994).
Microbial and cell culture systems offer data on genetic
stimulus-phenotypic response over a much broader range of
types of changes and responses, and on a time scale orders
of magnitude shorter, than feasible for humans or even so-
phisticated transgenic animal technology. Furthermore, ex-
tensive background on the genetic, physiological, and bio-
chemical properties of standard microbial systems, com-
bined with availability of increasingly automated analytical
instruments which rapidly provide rich datasets concerning
many cell parameters, provide a reference frame and a sen-
sitive response readout essential to establishing systematic
correspondences between genetic and phenotypic changes.
Clearly, defining the genome-phenotype mapping is an ex-
tremely difficult, long-term problem in general. Microbial
and cell culture systems provide excellent models for un-
dertaking this quest. The concepts, measurement technolo-
gies, algorithms, and information management methods de-
veloped in this relatively convenient framework should be
useful in establishing the genetic bases for disease and other
phenotypic properties of higher organisms.
As noted in the Introduction, constructive metabolic en-
gineering often fails because the designer is ignorant of
important processes connected with the pathway of interest.
Besides experience in metabolic engineering of industrial
organisms, the frequent experience of finding no phenotypic
change after gene knockouts in transgenic mice further il-
lustrates this class of difficulties. A potential advantage of
inverse metabolic engineering is initiation of the design pro-

BAILEY ET AL.: INVERSE METABOLIC ENGINEERING 119

cess with a known, different, desirable phenotype. Of course
the fundamental hurdle of discerning or discovering the cor-
responding genetic basis looms immediately as a great chal-
lenge.
Available algorithms for metabolic design for bioprocess
applications span a spectrum from “pure” inverse meta-
bolic engineering, in which a gene with completely un-
known function is found to confer a useful phenotype (as
was the case in the first experiments with Vitreoscillu he-
moglobin), to ‘‘pure” constructive metabolic engineering in
which well-defined functions in a highly characterized
metabolic network are modified in a predicted fashion. Be-
tween these extremes lies a continuum in which, as more
information and analyses develop, a metabolic system, or a
particular group of genes, can be used in an increasingly
constructive way. Most metabolic engineering strategies
evident in the current research combine elements of both
approaches, using some mechanistic information or hypoth-
eses in combination with phenomenological observations on
genetic change-phenotype response relations.
Maximizing success in metabolic engineering, whether
approached from a constructive or an inverse direction, de-
pends on knowledge of the mapping from genome to phe-
notype. This knowledge is presently very murky, and bring-
ing this relationship into clearer perspective will require
extensive further research, hopefully accelerated by future
conceptual and methodological breakthroughs. In the mean-
time, the inverse metabolic engineering strategy offers a
sometimes useful way to penetrate this fog somewhat, in a
direction distinct from the relatively traditional constructive
metabolic engineering approach.
This research was sponsored by the Swiss Priority Program in
Biotechnology (SPP BioTech).
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