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.