Background
Metabolic Engineering
An emerging
approach to the understanding and utilization of metabolic processes is
Metabolic (or pathway) Engineering (ME). As the name implies, ME is the
targeted and purposeful alteration of metabolic pathways found in an
organism in order to better understand and utilize cellular pathways
for chemical transformation, energy transduction, and supramolecular
assembly. ME typically involves the redirection of cellular activities
by the rearrangement of the enzymatic, transport, and regulatory
functions of the cell through the use of recombinant DNA and other
techniques. Much of this effort has focused on microbial organisms, but
important work is being done in cell cultures derived from plants,
insects, and animals. Since the success of ME hinges on the ability to
change host metabolism, its continued development will depend
critically on a far more sophisticated knowledge of metabolism than
currently exists.
This knowledge
includes conceptual and technical approaches necessary to understand
the integration and control of genetic, catalytic, and transport
processes. While this knowledge will be quite valuable as fundamental
research, per se, it will also provide the underpinning for many
applications of immediate value.
Scope
The Metabolic
Engineering Working Group is concerned with increasing the science and
engineering community's level of knowledge and understanding of ME. The
Working Group strives to encourage and coordinate research in ME within
academia, industry, and government in order to synergize the Federal
investment in ME.
Introduction
In November 1995,
Science Advisor John H. Gibbons of the Office of Science and Technology
Policy (OSTP) released the report, "Biotechnology for the 21st Century:
New Horizons." This report was a product of the Biotechnology
Research Subcommittee (BRS) under OSTP, and identifies priorities for
federal investment and specific research opportunities in
biotechnology. These priorities include agriculture, the
environment, manufacturing and bioprocessing, and marine biotechnology
and aquaculture. The BRS formed several working groups to facilitate
progress on some of these key priorities. The Metabolic
Engineering Working Group (MEWG) was created to foster research in
Metabolic Engineering, an endeavor that can contribute to all of the
key priorities in the aforementioned report. The Working Group is
composed of Federal scientists and engineers who participate as part of
the activities of OSTP, and represent all of the major agencies
involved in Metabolic Engineering research.
In its on-going
efforts to promote and enhance the use of Metabolic Engineering (ME),
the Working Group sponsored its second annual Interagency Grantee's
Conference. This Conferenc was held June 28, 2001 at the National
Science Foundation, in Arlington, VA. The purpose of the
Conference was to showcase the Grantees from the first and second the
Interagency Announcements of Opportunities in Metabolic Engineering
(NSF 98-49 and NSF 99-85), and review their progress on their Metabolic
Engineering Research Grants.
Abstracts of Expert Presentations
Glycolytic
Flux in Escherichia coli: A Gene Array Perspective Comparing
Glucose & Xylose
L.O. Ingram
University of Florida
The simplicity of
the fermentation process (anaerobic with pH, temperature, and agitation
control) in ethanologenic Escherichia coli KO11 and LY01 makes this an
attractive system to investigate the utility of gene arrays for
biotechnology applications. Using this system, gene expression,
glycolytic flux and growth rate have been compared in glucose-grown and
xylose-grown cells. Although the initial metabolic steps differ,
ethanol yields from both sugars were essentially identical on a weight
basis and little carbon was diverted to biosynthesis. A total of 27
genes changed by more than 2-fold in both strains. These included
induction of xylose-specific operons (xylE, xylFGHR, and xylAB)
regulated by XylR and the cyclic AMP-CRP system, and repression of
Mlc-regulated genes encoding glucose uptake (ptsHIcrr, ptsG) and
mannose uptake (manXYZ) during growth on xylose. However, expression of
genes encoding central carbon metabolism and biosynthesis differed by
less than 2-fold. Simple statistical methods were used to investigate
these more subtle changes. The reproducibility (coefficient of
variation of 12%) of expression measurements (mRNA as cDNA) was found
to be similar to that typically observed for in vitro measurements of
enzyme activities. Using a student t-test, many smaller but significant
sugar-dependent changes were identified (p<0.05 in both strains). A
total of 276 genes were more highly expressed during growth on
xylose; 307 genes were more highly expressed with glucose. Slower
growth (lower ATP yield) on xylose was accompanied by decreased
expression of 62 genes encoding the biosynthesis of small molecules
(amino acids, nucleotides, cofactors, and lipids), transcription, and
translation; 5 genes were expressed at a higher level. In
xylose-grown cells, 90 genes associated with the transport, catabolism
and regulation of pathways for alternative carbon sources were
expressed at higher levels than in glucose-grown cells, consistent with
a relaxation of control by the cyclic AMP-CRP regulatory system.
Changes in expression ratios for genes encoding the
Embden-Meyerhof-Parnas (EMP) pathway were in excellent agreement with
calculated changes in flux for individual metabolites. Flux through all
but one step was predicted to be higher during glucose fermentation,
pyruvate kinase. Expression levels (glucose/xylose) were higher in
glucose-grown cells for all EMP genes except the isoenzymes encoding
pyruvate kinase (pykA and pykF). Expression of both isoenzymes was
generally higher during xylose fermentation but statistically higher in
both strains only for pykF encoding the fructose-6-phosphate activated
isoenzyme, a key metabolite connecting pentose metabolism to the EMP
pathway. The coordinated changes in expression of genes encoding the
EMP pathway suggest the presence a common regulatory system, and that
flux control within the EMP pathway may be broadly distributed. In
contrast, expression levels for genes encoding the Pentose-Phosphate
pathway were statistically similar regardless of sugar.
Maximizing
Ethanol Production by Engineered Pentose-Fermenting Zymomonas mobilis
Dhinakar Kompala
University of Colorado, Boulder
Zymomonas mobilis
has been metabolically engineered to broaden their substrate
utilization range to include D-xylose and L-arabinose at the National
Renewable Energy Laboratory in Golden, CO. Both
chromosomally-integrated and plasmid-bearing Z. mobilis strains that
are capable of fermenting the pentose D-xylose have been created by
incorporating 4 genes: 2 genes xylA and xylB encoding xylose
utilization metabolic enzymes, xylose isomerase and xylulokinase and 2
genes talB andtktA encoding pentose phosphate pathway
enzymes, transaldolase and transketolase. While the proof-of
principle that the metabolically engineered Z. mobilis strains are able
to ferment bother glucose and xylose to ethanol has been previously
established, our current research undertakes detailed quantitiative
investigations on the enhanced metabolic network to maximize the
ethanol production from glucose and xylose by these strains.
Two different
xylose-fermenting Z. mobilis strains were grown on glucose-xylose
mixtures in computer-controlled fermentors to analyze the extracellular
metabolite concentrations as well as the activities of several
intracellular enzymes from the xylose and glucose consumption
pathways. Dynamic profiles of these enzymes show dramatic
increases in the activities of the two xylose utilizing enzymes
immediately after the depletion of the preferred sugar, glucose.
We are now addressing the regulatory mechanisms underlying these
reproducible increases. First, the issues of regulation at the
protein synthesis level versus the enzyme activity level is being
resolved through quantification of the key intracellular protein
concentrations through proteomic analysis using 2 D gel electrophoresis
techniques. In parallel, we are characterizing the
intracellular concentrations of the key metabolites along the network,
namely the phosphorylated carbohydrates through NMR spectroscopy.
Subsequently, the issues of transporter limitations as well as the gene
expression regulation and dosage effects will be addressed in the next
year.
Metabolic
Engineering of Solvent Tolerance in Anaerobic Bacteria
E. Terry Papoutsakis
Northwestern University
Understanding
solvent (and other toxic chemical) tolerance of microorganisms is
crucial for the production of chemicals, bioremediation, and whole-cell
biocatalysis. It is also very important basic knowledge. Past efforts
to produce tolerant strains have relied on selection under applied
pressure and chemical mutagenesis, with some good results, but not
consistently so. We desire to examine if Metabolic Engineering
(ME) and genomic approaches can be used to construct more tolerant
strains for bioprocessing. The accepted dogma is that toxicity is due
to the chaotropic effects of solvents on the cell membrane. Impaired
membrane fluidity and function inhibit cell metabolism, and result in
cell death. We have found that in C. acetobutylicum, several
well-defined genetic modifications not related to membrane function
impart solvent tolerance (by 40-70%) without strain selection. This
suggests that we need to re-examine the accepted dogma. The objective
of this research is to identify genes that contribute to solvent
tolerance and to use genetic modifications (involving these genes) to
generate solvent tolerant strains. In view of the large number of
possible genes that may be involved in determining solvent tolerance,
we use DNA microarrays based on the genome sequence of C.
acetobutylicum. DNA microarrays were designed and constructed in our
laboratory in order to examine the large-scale transcriptional program
of the cells in response to various levels of butanol and other solvent
challenges. Many genes belonging to several classes (molecular pumps,
chaperonins (HSPs), primary metabolism, ATPases, sporulation,
transcriptional regulators, carbohydrate metabolism) were identified as
changing gene expression under solvent stress. Several of these genes
will be explored in ME studies.
Metabolic
Engineering of Methylobacterium extorquens AM1
Steven Van Dien
University of Washington
A stoichiometric
model of central metabolism was developed based on new information
regarding metabolism in this bacterium to evaluate the steady-state
growth capabilities of the serine cycle facultative methylotroph
Methylobacterium extorquens AM1 during growth on methanol, succinate,
and pyruvate. The model incorporates 20 reversible and 47
irreversible reactions, 65 intracellular metabolites, and
experimentally-determined biomass composition. The flux space for
this underdetermined system of equations was defined by finding the
elementary modes, and constraints based on experimental observations
were applied to determine which of these elementary modes give a
reasonable description of the flux distribution for each growth
substrate. The predicted biomass yield, on a carbon atom basis,
is 49.8%, which agrees well with the range of published experimental
yield measurements (37-50%). The model predicts the cell to be
limited by reduced pyridine nucleotide availability during
methylotrophic growth, but energy-limited when growing on multicarbon
substrates.
Mutation and
phenotypic analysis was used to test model predictions regarding key
enzymes for growth on C3 and C4 compounds. Three enzymes involved
in C3-C4 interconversion pathways were predicted to be mutually
redundant: malic enzyme, phosphoenolpyruvate carboxykinase, and
phosphoenolpyruvate synthase. Insertion mutations in the genes from the
genome sequence that are predicted to encode these enzymes were made,
and these mutants were capable of growing on all substrates tested,
confirming the model predictions. Likewise, citrate synthase and
succinate dehydrogenase were predicted by the simulations to be
essential for all growth substrates. In keeping with these
predictions, null mutants could not be obtained in these genes.
In addition, a random approach using transposon mutagenesis was used to
generate mutants with impaired growth on succinate or pyruvate. A
mutant in a gene predicted to encode a subunit of the NADH-quinone
oxidoreductase was obtained, and was unable to grow on succinate or
pyruvate but grew normally on methanol. Since this function is
necessary for the entry of NADH into the electron transport chain, this
finding supports the model prediction that NADH must be oxidized to
ultimately yield ATP during multicarbon growth, but not with methanol
as the carbon source. A transposon mutant in a putative
a-ketoglutarate dehydrogenase gene was also unable to grow on succinate
or pyruvate. However, the model does not predict this enzyme
activity to be required for growth on any substrate. In
situations such as this in which the phenotype does not agree with
predictions, the model has helped to identify errors in the current
understanding of Methylobacterium extorquens AM1 central metabolism.
Engineering
Plant One-Carbon (1-C) Metabolism
David Rhodes
Purdue University
Primary and
secondary metabolism intersect in the one-carbon (C1) area, with
primary metabolism supplying most of the C1 units and competing with
secondary metabolism for their use. This competition is
potentially severe because secondary products such as lignin, alkaloids
and glycine betaine require massive amounts of C1 units. Many
current metabolic engineering projects aim to change levels of these
products, or entail reducing the supply of C1 units. It is
therefore essential to understand how C1 metabolism is regulated at the
metabolic and gene levels so as to successfully engineer C1 supply to
match demand. Our project aims to acquire this
understanding. Specific objectives are: (1) to clone complete
suites of C1 genes from maize and tobacco, and to incorporate them into
DNA arrays; (2) to use sense and antisense approaches as well as
mutants to engineer alterations in C1 unit supply and demand; and
(3) to quantify the impacts of these alterations on gene expression
(using DNA arrays), and on metabolic fluxes (by combining radio- and
stable isotope labeling, MS, NMR and computer modeling).
Four findings from
Year 1 are summarized. All were unexpected and have implications
for engineering C1 metabolism: (1) Unlike other eukaryotes,
plants have methylenetetrahydrofolate reductases that use NADH rather
than NADPH as reductant, and are not allosterically inhibited by
AdoMet. (2) DNA arrays show that formate dehydrogenase and a
cluster of enzymes for methyl group synthesis and transfer are more
highly expressed in roots than leaves. (3) Metabolic flux
analysis and modeling of tobacco engineered to convert choline to
glycine betaine suggests a crucial role for a chloroplast choline
transporter. (4) Plants have an unsuspected source of formate ö
the irreversible hydrolysis 10-formyltetrahydrofolate, via an enzyme
previously known only in prokaryotes. The first and last of these
findings depended on genomics-based approaches, and illustrate the
value of bioinformatics in metabolic engineering.
Significant
findings from Year 2 include: (1) Confirmation of the crucial role for
a chloroplast choline transporter in conversion of choline to glycine
betaine by metabolic flux analysis and modeling of transgenic tobacco
expressing choline monooxygenase and betaine aldehyde dehydrogenase in
the chloroplast, or choline oxidase and betaine aldehyde dehydrogenase
in the cystosol. (2) Analysis of 14C-formaldehyde and
14C-serine metabolism in leaves of near-isogenic maize lines differing
for alternative alleles of a single locus conferring glycine betaine
accumulation (Bet1/Bet1) or lack thereof (bet1/bet1), show markedly
different fluxes of radiolabel into choline moieties under salinity
stress. Despite these large differences in flow of C1 units into
choline moieties, no significant differences between near-isogenic
maize lines were found in the mRNA transcript abundances of any of the
C1 enzymes, with the single exception of
phosphoethanolamine-N-methyltransferase, which shows a modest 2-fold
down-regulation in the glycine betaine-deficient (bet1/bet1)
line. The latter result suggests control of C1 flux into choline
moieties primarily by post-transcriptional mechanisms.
Tobacco lines
expressing antisense methylenetetrahydrofolate reductase, antisense
S-formylglutathione hydrolase, and formate dehydrogenase have been
derived and are currently being characterized for C1 gene expression
and metabolic fluxes. A dynamic kinetic model of the intersecting
transmethylation, methionine salvage and S-methylmethionine cycles has
been developed, and is being used to explore the effects of altering
one or more enzyme levels on metabolism of U-13C5-methionine.
Carbohydrate
Engineering for Generating Sialylated Glycoproteins in Insect Cells
Michael J. Betenbaugh
Johns Hopkins University
Insect cells are
used to generate of a variety of biotechnology products. Many of the
most valuable biotechnology products are glycoproteins that include
oligosaccharides attached to the protein at particular amino acids.
These oligosaccharides can be extremely important to the therapeutic
activity of biopharmaceuticals in humans. Unfortunately,
processing in insect cells yields glycoproteins with different
oligosaccharides from those generated by human and other mammalian
hosts. While mammalian cells produce complex oligosaccharides often
terminating in the sugar, sialic acid, insect cells typically generate
simplistic oligosaccharides terminating in mannose or
N-acetylglucosamine. Since these covalently-attached
carbohydrates can significantly affect a protein's structure,
stability, biological activity, and in vivo circulatory
half-life, the objective of this project is to manipulate
carbohydrate-processing pathways in insect cells to generate complex
sialylated glycoproteins. The sialylation reaction involves the
addition of a donor substrate, cytidine monophosphate-sialic acid
(CMP-SA) onto a specific acceptor carbohydrate via an enzymatic
reaction in the Golgi apparatus. Evaluation of the nucleotide-sugars in
Sf-9 and High Five insect cells grown in serum-free medium revealed
negligible levels of CMP-SA to suggest a limitation in the donor
substrate levels. Consequently, the genes responsible for
generating CMP-SA must be engineered into insect cells using metabolic
engineering strategies. Unfortunately, the mammalian genes were
unknown so bioinformatics approaches were implemented to identify
putative human genes based on known bacterial sequences. When the
enzymes encoded by these genes are expressed with baculovirus vectors,
sialic acids and the donor substrate (CMP-SA) can be generated in
insect cells at levels exceeding those typically observed in mammalian
cell lines. Furthermore, the enzymes have broad substrate
specificities which may allow for the generation of glycoproteins with
different sialic acid termini. In addition to producing the donor
substrate, CMP-SA, the correct acceptor carbohydrate acceptors must be
generated in insect cells. Collaborating scientists are
generating correct carbohydrate acceptors by expressing favorable
glycosyltransferase enzymes such as galactose transferase and by
evaluating methods to inhibit unfavorable cleavage reactions. The
completion of the sialylation reaction will be obtained by expressing
the catalyzing sialyltransferase enzyme in the presence of these
correct acceptor and donor substrates. Engineering the
sialylation reaction into insect cells may increase the value of insect
cell-derived products as vaccines, therapeutics, and
diagnostics. Humanizing insect cells and other recombinant
DNA hosts will make expression systems more versatile and may
ultimately lower biotechnology production costs. In the future a
particular host may be chosen based on its efficiency of production
rather than its capacity to generate particular oligosaccharide
profiles.
Modeling
Metabolic Pathways: A Bioinformatics Approach
Imran Shah
University of Colorado
The overall goal of this project is to develop novel bioinformatics
tools to aid metabolic engineering (ME). The final final product this
project is a predictive computational system for metabolic pathway
elucidation utilizing high-throughput biomolecular data (mostly genomic
sequence and expression), background biological knowledge and novel
inference techniques. To achieve this goal we are developing
bioinformatics software to address the following challenges: (i)
biochemical data representation and integration from public domain
sources, which is necessary to effectively compute with biomolecular
information; (ii) the accurate assignment of biocatalytic function to
protein sequences using machine learning methods, which is necessary to
place putative proteins in a biochemical context, and (iii) the
elucidation of pathways by heuristic search, which is necessary to
automatically relate sets of putative enzymes in a broader metabolic
context. When implemented the system will be made available to the ME
community through interactive web-accessible software. We are
approaching the problem in a general manner so that the system will be
useful in annotating whole microbial genomes, in finding alternative
routes in a partially complete pathways, or even elucidating pathways
that have not been observed before.
In
Silico Analysis of the Escherichia Coli Metabolic Genotype and the
Construction of Selected Isogenic Strains
Bernhard O. Palsson
University of California-San Diego
Small genome
sequencing and annotation are leading to the definition of metabolic
genotypes in an increasing number of organisms. We show how in silico
metabolic genotypes are formulated based on genomic, biochemical, and
strain-specific data. Such metabolic genotypes have been
formulated for E. coli, H. influenzae, and H. pylori. The in
silico models are based on the philosophy of using applicable
physico-chemical (such as stoichiometric structure) and capacity
(maximum fluxes) constraints on the integrated functioning of the
metabolic networks. Given these constraints, optimal phenotypes
can be computed and compared to experimental data. They are found
on the edge of the allowable solution spaces ö a space that basically
represents the reaction norm of the defined genotype ö where the
governing constraint on cellular functions can be identified. For
E. coli, this process leads to quantitative prediction of growth and
metabolic by-product secretion data in batch, fed-batch, and continuous
cultures, and to the accurate prediction of the metabolic capabilities
of 73 of 80 mutants examined. Furthermore, we present
mathematical methods that allow for the analysis, interpretation,
prediction, and engineering of the metabolic genotype-phenotype
relationship, and for the interpretation of expression array data.
Key refs:
J.S. Edwards and B.O. Palsson, "The Escherichia coli MG1655 in
silico metabolic genotype; Its definition, characteristics, and
capabilities," Proc. Natl Acad Sci (USA), 97: 5528-5523 (2000).
J.S. Edwards, R.U.
Ibarra, and B.O. Palsson, "In silico predictions of Escherichi coli
metabolic capabilities are consistent with experimental data," Nature
Biotechnology, 19:125, 2001
Metabolic
Engineering of Microorganisms
Jay D. Keasling
University of California
The goal of this
work is to develop the experimental and theoretical methods to
introduce multiple, heterologous, biodegradation pathways into a single
organism and to optimize the flux through those pathways for the
remediation of toxic or recalcitrant organic contaminants. The
objectives of this work are: (1) to find and clone a gene that encodes
an enzyme capable of degrading diethylphosphate, (2) to clone and
express a pathway for complete mineralization of p-nitrophenol
phosphate, (3) to clone and express a phosphotriesterase capable of
hydrolyzing parathion, (4) to develop a co-culture biofilm capable of
degrading parathion (as a proof-of-concept), and (5) to combine all of
the genes in a single organism for complete mineralization of parathion
or paraoxon.
Metabolic
engineering offers the opportunity to expand the role of
bioremediation. Traditional metabolic engineering involves
overexpression of a desired protein and leads to a high metabolic
burden on the cell. The purpose of this work is to develop
strategies to help reduce this burden and make an engineered organism
more environmentally effective.
Parathion
(O,O-diethyl-O-p-nitrophenyl phosphorothioate), an organophosphate
pesticide which has been widely used and is highly toxic, was chosen as
the model compound for this project. Parathion is also
structurally and functionally similar to many chemical warfare agents
(including VX and soman).
Metabolic
Engineering of Isoprenoid Production
Jay D. Keasling
University of California
The objectives of
this work are (i) to maximize the production of the isoprenoid
precursor isopentenyl diphosphate in E. coli by expressing the genes
for either the mevalonate-dependent or the mevalonate-independent
synthesis pathway using the metabolic engineering tools developed in
this laboratory; (ii) to maximize production of the primary precursors
to the terpenoids: geranyl diphosphate, farnesyl diphosphate, and
geranylgeranyl diphosphate; (iii) to introduce into E. coli the genes
for specific classes of terpenoids and optimize production of these
ãnaturalä terpenoids; and (iv) to use laboratory evolution of terpene
cyclases to produce novel terpenoids or to change the distribution of
products made by terpenoid biosynthetic enzymes.
To accomplish this
work, we are (i) cloning the genes encoding the enzymes in the
non-mevalonate IPP biosynthetic pathway and express these genes under
the control of inducible promoters on high, medium, and low-copy
plasmids; (ii) cloning the genes for synthesis of DMAPP, GPP, FPP, and
GGPP and express these genes under the control of inducible and
constitutive promoters on high, medium, and low-copy plasmids; (iii)
cloning the genes for various plant and fungal terpenes and express
these genes under the control of inducible and constitutive promoters
on high, medium, and low-copy plasmids; and (iv) mutating the terpene
cyclases genes using mutagenic PCR and gene shuffling. For the
maximization of IPP, DMAPP, and GGPP production, we will express the
genes for lycopene synthesis and look for deep red colonies (containing
large quantities of lycopene).
Metabolic
Engineering to Study the Regulation/Plasticity of, and to Modify
Diterpene Metabolism in Trichome Gland Cells
George J. Wagner
University of Kentucky
Plant trichome
glands represent potential "green-factories" for the biosynthesis of
useful chemicals (molecular farming). These factories require
only energy from the sun, carbon dioxide from the air, water, and
minerals as feedstocks. Before this potential can be realized, however,
the regulation and plasticity of carbon flow in trichome glands must be
better understood, and protocols for engineering glands to produce
desired chemicals must be developed. The specific objectives of
this project are 1) to investigate the regulation/plasticity of carbon
flow in the biosynthesis of trichome-exudated diterpenes of glands, and
2) to study the feasibility of introducing heterologous genes into
glands to facilitate molecular farming. Exudating plant trichome
glands are specialized tissues that occur on the aerial surfaces of
about 30% of higher plants. They produce exudates that serve the
plant in pest/insect resistance, temperature control, etc. We
isolated a gland-specific c-DNA library, which yielded a P450 gene
involved in the conversion of cembratriene-ol (CBT-ol) to
cembratriene-diol (CBT-diol), the major diterpene of the experimental
tobacco, T.I. 1068. This plant can accumulate up to 17% of leaf
dry weight as trichome exudate, and CBT-diol accounts for 60% of
exudate weight. Knockdown of the P450 gene activity (using
antisense and co-suppression strategies) resulted in a 20-fold increase
in CBT-ol and a corresponding decrease in CBT-diol. Exudate from
high CBT-ol plants was more toxic to aphids, and high CBT-ol plants had
greatly reduced aphid colonization. Thus, we have metabolically
engineered the last step in the biosynthesis of the major exudate
diterpene and significantly altered natural-product-based aphid
resistance in this plant. Knockdown strategies (antisense,
co-suppression, and RNA interference) are being applied to determine
the function of additional trichome-specific genes, and to determine
the impact of altering their activities on exudate chemistry.
Full-length genes of known function will be introduced into host
plants, trichome-specifically, to determine the ability glands in these
plants to accommodate heterologous diterpene biosynthetic
genes. A trichome-specific promoter has been isolated that
can serve in planned transformation experiments designed to
metabolically engineer glands.
Aromatic
Amino Acid Biosynthesis in Archaeoglobus fulgidus
H.G. Monbouquette
University of California Los Angeles
The aromatic amino
acid synthesis pathway has been engineered successfully for the
synthesis of natural and unnatural chiral amino acids, which are
important drug intermediates, as well as other industrially important
aromatics, such as indigo. Production of aromatics via engineered
microbes offers both environmental and economic advantages including
exclusive use of aqueous solvent and non-toxic intermediates, and lower
raw material cost. Intense interest therefore has developed in
the enzymes of these metabolic pathways. A. fulgidus is
representative of the third, most primitive domain of life, and the
aromatic amino acid synthesis pathways have not been explored in these
microorganisms despite the fact that they may offer a far more robust
set of biosynthetic enzymes well suited both for in vivo and in vitro
synthesis applications. Recently, the entire genome of A.
fulgidus was sequenced and a thorough study of open reading frames for
sequences homologous to known enzymes was conducted. It is
noteworthy that a number of enzymes involved in common aromatic amino
acid synthesis routes were not identified on the genome. Our goal
is to identify these new enzymes/pathways by a functional proteomics
approach made possible by our demonstrated ability to culture A.
fulgidus to the 100-liter scale, and to identify, isolate, sequence,
clone and express (in E. coli) new enzymes from this microbe.
This project will establish a functional proteomics approach involving
coordinated use of high-throughput LC/MS-based enzyme assays, DNA
microarrays, and gene cloning and expression for fast screening of
enzyme activities and for identification of genes in hypothesized
metabolic pathways.
The following was
accomplished in the first year of this project: (1) the 15 A. fulgidus
open reading frames (ORFs) homologous to known genes in the aromatic
amino acid synthesis pathways were cloned in E. coli and were
sequenced, (2) a putative gene for a novel bifunctional phosphoribosyl
(PRA) anthranilate transferase/indoleglycerol phosphate (IGP) synthase
was found to be two separate genes, (3) prephenate dehdrogenase
activity was confirmed for the over-expressed product of a putative
trifunctional chorismate mutase/prephenate dehydratase/prephenate
dehydrogenase gene, (4) over-expressed shikimate dehydrogenase was
purified and partially characterized, and (5) a method for determining
95% confidence intervals for DNA microarray data was developed.
Of the 15 cloned ORFs, nine were over-expressed as soluble
products. An effort to obtain soluble products of the remaining
genes and to characterize the recombinant enzymes is continuing.
A preliminary characterization of the recombinant shikimate
dehydrogenase was conducted. The enzyme exhibits similar kinetics
to the E. coli enzyme, albeit at a temperature optimum of ~90 °C.
The prephenate dehydrogenase activity of the putative trifunctional
enzyme suggests that this may indeed be a novel fusion of catalytic
functions, although chorismate mutase and prephenate dehydratase
activity has not been confirmed. Work is ongoing to develop LC/MS
as a tool for high throughput enzyme assays and to refine the DNA
microarray technique such that LC/MS and DNA microarrays may be used in
complementary fashion to identify new enzymes and metabolic
pathways. This approach will be used in the second year of the
grant to identify the novel enzyme(s) catalyzing the first two steps in
the shikimate pathway as well as the phosphorylation of shikimate.
Conference Agenda
June 28, 2001
-- Room 110
8:15
Welcoming and Introductory Remarks
MARYANNA
HENKART, Chair, Biotechnology Research Working Group
FRED HEINEKEN, Chair, Metabolic Engineering Working Group -- Introduction
8:30 Glycolytic
Flux in Escherichia coli: A Gene Array Perspective Comparing Glucose
& Xylose by LONNIE INGRAM
8:50 Maximizing
Ethanol Production by Engineered Pentose-Fermenting Zymomonas mobilis
by DHINAKAR KOMPALA
9:10 Metabolic
Engineering of Solvent Tolerance in Anaerobic Bacteria by TERRY
PAPOUTSAKIS
9:30 Break
9:45 Welcoming Remarks by MARY CLUTTER, Chair,
Subcommittee on Biotechnology
10:00 Metabolic
Engineering of Methylobacterium extorquens AM1 by STEVEN VAN DIEN
10:20 Engineering
Plant One-Carbon (1-C) Metabolism by DAVID RHODES
10:40 Carbohydrate
Engineering for Generating Sialylated Glycoproteins in Insect Cells
by MICHAEL BETENBAUGH
11:00 Break
11:15 Modeling
Metabolic Pathways: A Bioinformatics Approach by IMRAN SHAH
11:35 In
Silico Analysis of the Escherichia Coli Metabolic Genotype and the
Construction of Selected Isogenic Strains by BERNHARD PALSSON
11:55 Lunch
1:00 Discussion: in
vitro Metabolic Engineering
2:00 Break
2:15 Metabolic
Engineering of Microorganisms by JAY KEASLING
2:55 Metabolic
Engineering to Study the Regulation/Plasticity of, and to Modify
Diterpene Metabolism in Trichome Gland Cells by GEORGE WAGNER
3:15 Aromatic
Amino Acid Biosynthesis in Archaeoglobus fulgidus by HAROLD
MONBOUQUETTE
3:35 Open Discussion
4:25 Closing Remarks
4:30 Adjourn
Agency
Activities in Metabolic Engineering
U.S Department of Agriculture
The Agricultural
Research Service (ARS) and the Forest Service (FS) conduct metabolic
engineering research through the Federal laboratory system while the
Cooperative State Research, Education, and Extension Service (CSREES)
supports metabolic engineering research through competitive research
grants and through formula-based programs in cooperation with the
states.
USDA research
activities encompass animal sciences, plant sciences, commodity
conversion and delivery, environmental sciences (air, soil, water),
human nutrition, and integration of agricultural systems.
Metabolic
engineering technologies are being developed and applied across the
above research areas and include the following goals:
 |
To modify microbial metabolism for the
production of commercially useful products, chemicals, biofuels, and
biomolecules from agricultural commodities and resources. |
|
To develop genetic and other techniques
for altering metabolic pathways to understand basic processes
associated with microbial based natural or newly developed biocontrol
agents resulting in elimination, decreased use, or increased
environmental bioremediation of both agricultural wastes and
agricultural chemicals such as herbicides, insecticides, fungicides, or
biocides. |
|
To improve efficiency of production and
decrease losses due to environmental stresses, diseases, pathogens,
parasites, or pests by altering host metabolism using genetic or other
techniques to apply metabolic engineering at the tissue, organ, or
whole organism level of animals or plants, alone or in combination with
the microorganisms associated with these hosts. |
Ongoing research
includes:
|
Metabolic engineering for the
development of superior fuel ethanol producing microorganisms.
Microorganisms that normally use multiple substrates are being
engineered for enhanced ethanol production, and microorganisms that
normally make ethanol are being engineered to use multiple substrates.
|
|
Metabolic engineering for the
development of superior solvent producing anaerobic bacteria.
Specifically, the fermentative enzymes involved in butanol production
are being analyzed in order to manipulate metabolic fluxes from
acidogensis to solventogenesis. |
|
Metabolic engineering of anaerobic
bacteria for improved animal performance. The specific approach is to
enhance xylan degradation of feed material by introducing into the
rumen a genetically modified bacterium that overproduces xylanase.
|
|
Metabolic engineering of toxigenic fungi
and host plants. Specifically, the genes involved in aflatoxin
biosynthesis have been identified and a master switch gene discovered.
By engineering plants to favor production of a metabolite that
interferes with this master gene, aflatoxin production may be prevented
in the host plant. |
|
Modify metabolite distribution in
plants. One specific approach is to transfer the liquid wax producing
capability of jojoba into a metabolic pathway for commercially viable
oilseed rape and soybeans. |
National Institute of Standards and Technology
NIST has internal
research programs in the Biotechnology Division, and extramural
collaboratively funded research and development programs through the
Advanced Technology Program that are related to the scientific field
known as Metabolic Engineering. Each of these programs have different
foci and management structures, but share the overall goal of fostering
the commercialization of recent scientific advances in areas related to
biotechnology, such as biocatalysis and metabolic engineering.
Biotechnology
Division (Intramural)
In the intramural
programs of the Biotechnology Division ( http://www.cstl.nist.gov/biotech
) , which is one of five Divisions of the Chemical Sciences and
Technology Laboratory, the mission is to advance the commercialization
of biotechnology by developing the scientific/engineering technical
base, reliable measurement techniques and data to enable U.S. industry
to quickly and economically produce biochemical products with
appropriate quality control. The mission is carried out in
collaboration with industry, other government agencies and the
scientific community. The primary research efforts that relate to
Metabolic Engineering are in Bioprocess Engineering, Structural
Biology, DNA Technologies, and Biomolecular Materials groups.
The Bioprocess
Engineering ( http://cstl.nist.gov/div831/bioprocess
) activity includes biophysical property evaluation where
thermophysical and thermochemical properties are being obtained,
evaluated, codified and modeled for biochemicals, proteins and
biosolutions of interest in metabolic pathway engineering. A research
program in biocatalysis is underway to solve technical roadblocks in
the commercial development of enzymes that build new complex molecules
used in advanced drug or food product design. Other investigations
include developing DNA-based reference standards for detecting and
quantifying biotech crops, and fluorescence standards for interpreting
DNA microarrays.
The Structural
Biology activity includes x-ray and NMR measurements of atomic
structures of prototypical proteins, enzymes, enzyme-substrate
complexes and model DNA systems. A research program in
biothermodynamics uses state-of-the-art calorimetric methods to study
protein-protein and protein-substrate interactions, and computational
models are developed that relate structure to function. Physical and
biochemical methods are used to characterize protein behavior,
including the study of membrane-embedded proteins to understand signal
transduction. Computational chemistry and modeling develops methods to
model the energetics and dynamics of interactions between substrates
and active sites of enzymes. Modeling techniques to understand the
relationship between protein sequence and structure are being developed.
The DNA
Technologies activity includes development of methods and standards for
DNA profiling for forensic and other uses. Research is being conducted
to develop the next generation of DNA profiling based on polymerase
chain reaction (PCR) technology including new methods development for
rapid DNA extraction, amplification, separation, and computer imaging.
DNA sequencing develops specific reference materials and technical
expertise that are essential for DNA Genomic research in the public and
private sector. This activity also provides quality assurance expertise
to the developers of technology that proposes to use DNA recognition
sites on silicon chips for the diagnosis of human genetic diseases.
Research on DNA damage and repair is developing methods to characterize
DNA damage on a molecular scale using GC/MS techniques. Studies of both
in-vivo and in-vitro systems are underway to understand both damage (as
low as one base per million) and repair mechanisms.
The Biomolecular
Materials activity develops generic measurement technologies utilizing
both optical and electrochemical approaches for applications in
clinical diagnostics, bioprocessing, and environmental monitoring.
Research on lipid membranes and membrane proteins is being performed to
provide an understanding of materials and methods that will enhance the
development of this important class of molecules in sensor and other
applications. The light-sensitive protein, bacteriorhodopsin is being
studied as a potential source for the storage and retrieval of
information. Studies are underway to understand and control the
mechanism of this optical transition, and to develop methods of
immobilizing this protein to increase its stability.
Advanced
Technology Program (Extramural)
The Advanced
Technology Program within NIST provides funding to support innovative
research and development, which are likely to lead to inventive new
technologies and products that will have positive economic benefits for
the United States. ATP has in the past, and continues to fund projects
in Metabolic Engineering. These projects include the modification of
enzymatic pathways in microorganisms and improved bioprocessing
technologies to produce, in a cost-effective way, monomers used in the
synthesis of thermoplastics, essential cofactors for human health,
disease-targeted therapeutics and desulfurized crude oil. Support also
has been provided to companies seeking to engineer the synthesis of
isoprenoids in yeast and biopolymers in the fibers of cotton plants.
The production of better goods at lower costs and the utilization of
renewable biosystems are potential benefits to be derived from these
projects. As documented in more than a dozen White Papers submitted to
ATP, industries' future commitments for applications of metabolic
engineering are expansive and cover wide areas including immobilized
biocatalysis, novel bioreactors, value-added crops, better nutrition
and an improved environment
Department
of Defense
The Department of
Defense (DoD) currently supports a broad range of research in the area
of metabolic engineering through the Army Research Office (ARO) and
other Army research activities, the Air Force Office of Scientific
Research (AFOSR), the Office of Naval Research (ONR), and the Defense
Advanced Research Projects Agency (DARPA). The specific focus of the
ARO, AFOSR, ONR, and DARPA efforts will be summarized and future
directions in metabolic engineering research and technology development
will be addressed.
The broad needs
for the DoD that can be served through research efforts in metabolic
engineering are summarized below. These science and technology targets
will provide enhanced and expanded capabilities for the missions of the
services and provide greatly expanded capabilities for the civilian
sector.
|
Materials |
|
Processes |
|
Devices |
|
Fabrication Schemes |
|
Information Processing |
Current interests
in metabolic engineering at ARO are focused on two related topics: the
characterization of biochemical pathways and enzymatic mechanisms and
the genetic manipulation of protein structure and function. The goal is
to develop a detailed understanding of how macromolecules have been
tailored to execute their designated functions and how they interact
with other macromolecules. With this information, it will be possible
to engineer enzymes and metabolic pathways to exhibit a set of specific
functions and properties, according to Army needs. ARO currently
supports research in several areas, including: how molecular transport,
subcellular compartmentalization, and reaction sequences are involved
in enzymatic regulation and superstructural formation; understanding
and manipulating aminoacylation of tRNAs to produce, using cellular
translation machinery, new polymeric peptide materials containing
non-natural amino acids; the role and regulation of "stress" proteins
differentially expressed in response to environmental or external
stimuli; and the design and implementation of unique enzymatic
strategies for the biodegradation of environmental pollutants.
For the AFOSR,
space and aerospace materials are often produced by complex sequences
of reactions involving toxic solvents and expensive catalysts. Some
materials are derived from structures that are difficult to synthesize
with traditional chemistry. Because of their remarkable specificity and
efficiency, biocatalysts can enable the synthesis of a wide range of
materials. They can catalyze de novo synthesis from renewable
feedstocks, specific reactions in synthesis of monomers that are
difficult to accomplish with conventional chemistry, and modification
of polymers or composites at several stages of synthesis and
assembly. Biocatalysts have substantial potential for deposition
of thin films of organic or inorganic material including silicates.
Development of biocatalytic approaches to synthesis will enable the
development of materials with novel properties, reduce the cost of the
material and eliminate the environmental impact of toxic chemical
reagents.
AFOSR-supported
work at the Air Force Research Laboratory has also led to the discovery
of new catabolic pathways used by bacteria for the biodegradation of
synthetic organic compounds. A variety of novel enzymes catalyze key
steps in the pathways. The objective of the current work is to
characterize the enzymes to determine the reaction mechanisms and then
to explore the potential for use of the enzymes as biocatalysts for the
synthesis of chemical feedstocks used in the production of space and
aerospace polymers. Strategies are also being developed for the
biological destruction of chemicals by bacterial enzymes.
One of the
metabolic engineering foci at ONR, currently, is the microbial
synthesis of energetic materials (EM) and EM precursors for the
purposes of cost and environmental impact. Practically all such
materials are non-natural products and their biosynthesis therefore
requires the re-engineering of existing pathways and/or the assembly of
new or hybrid pathways in one or more host organisms. An example of a
simple EM precursor now under study is 1,2,4-butanetriol, which as its
energetic trinitrate is used as a plasticizer in propellant and
explosives formulations. More advanced EM targets, such as RDX, HMX and
Cl20, involve high density fused ring cores with multiple nitramino
(C-N(NO2)) substituents. While these are very difficult targets, they
suggest worthwhile research goals such as the biosynthesis of highly
electron withdrawing substituents on carbon (as in C-nitramino) or the
assembly of strained heterocyclic rings. Clearly, a
theoretical/experimental approach to the prediction of the true scope
of enzyme reaction specificity, with energetic boundaries, would be
particularly valuable in the design of pathways for EM biosynthesis.
Other non-polymeric targets, besides EM, would include novel
photonic/electronic/optical materials. Persons interested
in metabolic engineering opportunities at ONR are strongly advised to
communicate with Dr. Harold J. Bright (703-696-4054, brighth@onr.navy.mil) before
submitting a proposal.
DARPA's metabolic
engineering programs are driven by an interest in protecting human
assets against biological threats and using biology to enhance both
human and system performance. The general concept of this thrust is to
understand how nature controls the metabolic rate of cells and
organisms (e.g., extremophiles, hibernation) and apply this
understanding to problems of interest to DoD. Examples of current
investments in metabolic engineering include efforts to develop
technologies for engineering cells, tissues and organisms to survive in
the battlefield environment so they can be used as sensors. DARPA
is also developing technologies that permit the long-term storage of
cells including human blood. More complete descriptions of
current DARPA programs and solicitations in these areas can be viewed
at http://www.darpa.mil/dso.
U.S. Department of Energy
The Department of
Energy is supporting over $25 million in metabolic engineering
research, largely through the Offices of Science (SC), Energy
Efficiency and Renewable Energy (EE), and Environmental Management
(EM). The research falls in two main categories: 1) basic research,
which involves the advancement of metabolic engineering fundamental
knowledge and capabilities, and 2) applied research, which employs
metabolic engineering techniques in development of target products. The
basic research efforts of the Department reside within SC, whereas most
of the applied research in this area is conducted within EE. In
general, these research efforts are conducted by universities, national
laboratories, and industry.
The Department's
goals related to metabolic engineering research are to:
|
To expand the level of knowledge and
understanding of metabolic pathways and metabolic regulatory mechanisms
related to the development of novel bio-based systems for the
production, conservation, and conversion of energy. |
|
Apply metabolic engineering techniques
to enhance and develop plants and microorganisms for use in the
production of chemicals and fuels or for environmental remediation of
waste sites. |
Metabolic
engineering research within SC is supported predominantly through the
Office of Basic Energy Sciences (BES) and Biological and Environmental
Research (OBER). Most of BES's metabolic engineering research resides
within the Energy Biosciences program which has the mission to generate
the fundamental knowledge required for the development of novel
bio-based systems for the production, conservation, and conversion of
energy. A significant part of the program has been and continues to be
aimed at the development of metabolic engineering capabilities related
to plants and fermentative microbes. These activities include defining
metabolic pathways, characterization of the catalytic properties of
enzymes, determining metabolic regulation mechanisms, development of
gene transfer capabilities, kinetic analysis of the flow through a
pathway, and in a few instances the actual metabolic engineering of
specific pathways. The program focuses on the development of basic
scientific knowledge as opposed to the development of specific
processes.
The metabolic
engineering research within OBER resides in three divisions: Health
Effects and Life Sciences Research, Medical Applications and
Biophysical Research, and Environmental Sciences. Most of the research
is conducted in association with the human genome, microbial genome,
structural biology, and environmental remediation programs. OBER's
research in this area is directed toward enhancing fundamental
knowledge of metabolic pathways and addresses the development of tools
and capabilities to elucidate the kinetics and mechanisms of microbial
metabolic pathways; to create useful pathways for biotransformation of
metals for biodegradation of toxic organics; and to understand complex
relationships between genes, the proteins they encode, and the
biological functions of these proteins in the whole organism.
In complement with
its core research efforts, SC is conducting joint research with EM in
support of their environmental restoration efforts and with EE in
support of their fuels and chemicals production efforts. These newly
formed partnerships demonstrate the spirit of collaboration and
coordination within the Department, which combines science with
technology to fulfill DOE's research missions.
Metabolic
engineering research within EE is supported through the offices of
Transportation Technologies (OTT), Industrial Technologies (OIT), and
Utility Technologies (OUT). As applied R&D efforts, the focus is on
specific research and market issues within the purview of the
respective office. For example, research in OTT focuses on ethanol
production using bacteria and yeast that feed on sugars derived from
non-agricultural feedstocks. In OIT, the focus is on the development of
bioprocesses and new chemical synthesis routes using whole organisms or
enzymes in the production of chemicals and materials. Finally, the
research in OUT focuses on the use of photosynthetic microorganisms,
such as cyanobacterium or alga blue or green algae in the production of
hydrogen. In each of these program efforts, the R&D activities
address metabolic engineering to increase the production of the
product(s) desired by either enhancing existing pathways, constructing
new pathways, or designing alternative pathways.
Environmental
Management (EM) has a modest biotechnology research effort in support
of its mission in waste management related to the clean-up and
restoration of the U.S. national laboratory sites. The focus of this
research involves bioremediation, including intrinsic, chemical
bioaugmentation, and phytological approaches to clean-up chlorinated
compounds, heavy metals, and other hazardous organics. Metabolic
engineering approaches are being used to improve the effectiveness and
efficiency of their environmental clean-up efforts by enhancing,
augmenting, or creating new metabolic pathways within target organisms
or plants. More recently, EM has teamed with SC to pursue basic
research needs in various areas of national laboratory clean-up issues
and waste management.
Environmental Protection Agency
Developing
Metabolic Engineering Strategies
The mission of the
Environmental Protection Agency is to protect human health and the
environment from adverse effects of anthropogenic activity. Included in
this mission are various elements for which metabolic engineering can
play a useful role.
One prominent
concern is the introduction of chemicals to the environment which may
have detrimental effects on humans and other biota. As mandated
by statute and implemented by rule, the Agency routinely conducts
evaluation of chemicals intended for use, currently in use, or
determined to exist at significant levels in the environment. From
these evaluations, the Agency may decide to implement management
strategies designed to limit the potential for adverse effects.
The application of
novel technologies such as the use of biotechnology as a substitute to
conventional manufacturing and processing of raw materials into final
products is consistent with the mission of the Agency. EPA
implements this by supporting development of technologies which 1) use
chemical substitutes that are less toxic; 2) produce more efficient
activity resulting in decreased requirement for the chemical or; 3)
develop engineering procedures which produce little or no toxic end
products. Finally, consistent with the pollution prevention ethic is
the reevaluation of chemical stewardship from one of "cradle to grave"
to a more multigenerational philosophy in which a chemical may be
utilized successively in different forms prior to final disposal.
Metabolic engineering has a role to play by enabling the development of
biological mechanisms for production or use that meet one or more of
these criteria.
While it is
generally accepted that chemical-based technologies have evolved to
provide a higher standard of living for the general population, it is
also recognized that the use of some chemicals, either through the
chemical characteristics or the handling, synthesis or disposal, have
produced negative effects on human health and/or the environment.
Advances in technology allow scientists to better predict the potential
for adverse effects from exposure to chemicals as well as mechanisms to
diminish the negative effects of chemical production such as production
of toxic byproducts and disposal of the chemical. The approach, which
strives to identify synthetic pathways that are less polluting than
existing pathways and that encourages the development of nontoxic
chemical products, is referred to as "Green Chemistry". The use
of metabolic engineering to evaluate the potential for increased risk
from chemicals, by allowing the study of responsible metabolic pathways
and by permitting modification of such pathways to reduce risk, is
another way in which metabolic engineering firs within the EPA mission.
Finally, basic
research, which utilizes methods of metabolic engineering, can provide
longer range approaches to assist EPA in its overall mission of
protecting human health and the environment. The EPA supports
extramural metabolic engineering research through the Technology for a
Sustainable Environment (TSE) program, which awards grants in the area
of pollution prevention. Since 1995, the TSE program has funded
metabolic engineering research related to methanol conversion, solvent
tolerance, biopolymer production and pesticide production-all focused
on the elimination of pollution at the source
National Institutes of Health
National
Institute of General Medical Sciences
The National
Institute of General Medical Sciences (NIGMS) supports metabolic
engineering research, usually in the form of grants to investigators in
universities (R01s) or in small businesses (SBIRs). These grants
support basic research in two general areas: (1) the development of
microbial or plant-based metabolic routes to useful quantities of
ãsmallä molecules such as polyketides; (2) the development of a much
better understanding of the control architecture that integrates the
genetic and catalytic processes in normal and aberrant cells.
During fiscal 2002, the NIGMS is providing $13.6 million (47 grants)
for the support of research directly involving metabolic
engineering. Examples of funded projects include (1) a study of
the pikromycin biosynthetic pathway, and (2) an in silico studies of E.
coli growth.
National Science Foundation
The Directorate
of Engineering supports several investigators in the area of
metabolic engineering. One common feature of these research projects
involves purposeful changes in organism behavior for increased product
yields and levels for both wild type and recombinant systems. In
addition, the improved biodegradation of toxic compounds is also being
approached through metabolic engineering. Biological processes of this
type have significant industrial potential, but in many cases still
require the necessary biochemical engineering to translate them into a
scalable process. In order to obtain the highest yields of metabolite
products, restructuring of the central pathways for carbon catabolism
and dispersal of incoming carbon into synthetic pathways will be
necessary. Because of the tight integration among these pathways and
the energy-producing pathways, restructuring of this central core of
metabolism will require a systems approach, which considers the
interactions of the pathways concerned with the other metabolic
subsystems in the cell. The system is complicated by regulation at both
genetic and enzyme levels of all of these interacting metabolic
subsystems. Therefore, an important aspect of the engineering research
is the development of the mathematical systems, and control theory
needed for a quantitative analysis and understanding of the metabolic
changes which are initiated by the manipulation of the enzymatic,
transport and regulatory functions of the cell. Examples of metabolic
engineering research supported in this Directorate include: (1) the use
of linear optimization theory for the network analysis of intermediary
metabolism, (2) the development of methods to select the internal
fluxes for experimental measurement based on their sensitivity to
experimental error, (3) the development of a method to determine flux
control coefficients using transient metabolite concentrations, and (4)
a study of network rigidity to help overcome the cell control
mechanisms that resist flux alterations at branch points in metabolic
pathways.
The Directorate
for Biological Sciences (BIO) supports a broad range of research
activities directed at increasing the knowledge base required for
metabolic engineering. Examples of several BIO activities with
implications for Metabolic Engineering include the following: (1) the
ãArabidopsis Genome Research Initiative:ä a multinational research
cooperation to sequence the entire genome of the model plant,
Arabidopsis thaliana, in order to establish baseline genomic data for
plants, and to develop microarrays and other technology that can be
used for further applications; (2) the ãPlant Genome Research Programä
which supports research on plant genome structure and function.
Research supported by this program is characterized by a systems
approach to plant genome research that builds upon recent advances in
genomics, bioinformatics, and plant biology. This program has already
funded over 70 groups of investigators, often consortia of several
universities and industries, to carry out sequencing and functional
genomics projects. Supported efforts range from sequencing
agriculturally important genomes (maize, soybean, tomato), to
technology development, to focused applications (stress tolerance,
pathogen responses, cotton fibers). (3) The "Microbial Observatories
Initiative includes the study of novel microorganisms in soils, marine
sediments, and aquatic environments. The tremendous diversity of
currently undescribed microorganisms offers potential metabolic
engineering spin-offs such as new pathways for biodegradation of
environmental toxins and novel pharmaceuticals. (4) The BIO
Directorate is in the second year of the ã2010 Projectä that supports
research to determine the function of all genes in Arabidopsis thaliana
by the year 2010. In the first year, 26 awards were made in support of
creative and innovative research designed to determine the function of
networks of genes and to develop new tools for functional genomic
approaches.
The Directorate
for Geosciences supports research related to ME in marine
ecological systems. Examples of research areas include: (1)
determination of the physico-chemical requirements for the maintenance,
growth, and regulation of marine microbes; (2) identification,
isolation, and determination of the function of enzymes responsible for
useful degradation processes; (3) exploration of marine viruses and how
they can be used in genetic engineering; (4) development of molecular
assays for harmful species of marine microbes; (5) determination of
cellular and biochemical control of trace metal limitation; (6)
characterization of enzymes and genes associated with nitrogen fixation
in cyanobacteria; and (7) identification and characterization of marine
microbes and consortia that degrade, detoxify, or metabolize marine
pollutants.
The Directorate
for Mathematical and Physical Sciences supports a number of
projects involving metabolic engineering. Of particular interest
is the use of new enzymes to facilitate catalytic processes such as the
desymmetrization of achiral molecules and the development of new
bacterial strains that will be useful for the conversion of
petrochemical and other industrial byproducts into useful or benign
derivatives. Theoretical work continues to explore the basis of
information encoding which is the foundation of molecular genetics and
its associated properties of self-replication and the nonrandom
organization of genetic material into specific shapes. Bridges to
the experimental realm provide ever more elegant examples of synthetic
structures that mimic genetic principles. These experiments are
expanding our understanding of the underlying chemistry of genetic and
biochemical processes and provide the basis for such functional
examples of chemical systems patterned after living systems as enzyme
mimics. Additionally, the increasing understanding of the
specific ways that drug molecules interact with gene-derived entities
is the basis for a new era of chemotherapy.
