|
|
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 fourth annual
interagency workshop of federal scientists and engineers
associated with ME research. This workshop was held May 31, 2000
at the National Science Foundation, in Arlington, VA. The
purpose of the workshop was to showcase the grantees from the
first the Interagency Announcement of Opportunities in Metabolic
Engineering, which was issued in January 1998. In
addition, an afternoon session was devoted to a better
understanding of bioinformatics and metabolic engineering,
chaired by topic experts. The workshop was designed to be
a tutorial for agency representatives and decision makers that
participate in reviewing and funding proposals in related
disciplines. The Metabolic Engineering Working Group has
since issued a second Announcement of Opportunities in Metabolic
Engineering and selected research grantees for 1999 - 2000.
Purpose
of Workshop
The purpose of the fourth
Interagency Workshop on Metabolic Engineering was to:
Educate Federal agency personnel
on emerging metabolic engineering issues through presentations
by experts in the field, and highlight the progress of the
grantees from the first Interagency Announcement.
Discuss possible topic areas of
interest amongst the agencies for future Interagency
Announcements on Metabolic Engineering.
The Scope of
the 1998 Interagency Announcement
Three topic areas were specified
in the FY 1998 Interagency Announcement and awards were made in
each area. The topics were:
Instrumentation, sensors, new
analytical tools, and new cell and molecular biology methods
which facilitate the study of metabolic pathways, especially
those technologies that allow the examination of individual
cells.
Quantitative and conceptual
models integrated with experimental studies that better
characterize the regulation and integration of complex,
interacting metabolic pathways.
The use of bioinformatics to
deduce the structure, function, and regulation of major
metabolic pathways from the genomic sequence data bases.
1998
Interagency Metabolic Engineering Grants
| Principal
Investigators |
Institute |
Title |
Award
Amount |
Award Time
Period
|
| L.O. Ingram,
J.F. Preston, & K.T. Shanmugam, |
University of
Florida |
Advanced
Ethanologenic Biocatalysts for Lignocellulosic
Fermentations |
$498935 |
FY1998-FY2000 |
| Michael J.
Betenbaugh |
Johns Hopkins
University |
Carbohydrate
Engineering for Generating Sialylated Glycoproteins in
Insect Cells |
$814706 |
FY1998-FY2000 |
| Andrew D.
Hanson |
University of
Florida |
Engineering
Plant One-Carbon (1-C) Metabolism |
$268000 |
FY1999-FY2001 |
| Bernhard
Palsson |
University of
California-San Diego |
In Silico
Analysis of the Escherichia Coli Metabolic Genotype and
the Construction of Selected Isogenic Strains |
$274593 |
FY1999-FY2001 |
| Bernhard
Palsson |
University of
California-San Diego |
Computational
Infrastructure for Engineered Microorganisms |
$850000 |
FY1998-FY2000 |
| Bernhard
Palsson |
University of
California-San Diego |
Genomically
Based Models for Antimicrobial Development |
$680957 |
FY1998-FY2000 |
| Mary E.
Lidstrom, Steven Van Dien |
University of
Washington |
Metabolic
Engineering of Methylotrophic Bacteria for Conversion of
Methanol to Higher Value Added Products |
$380000 |
FY1998-FY1999 |
| Jay D.
Keasling |
University of
California-Berkeley |
Strategies for
Metabolic Engineering of Environmental Microorganisms -
Application to Degradation of Organophosphate
Contaminants |
$397749 |
FY1999-FY2001 |
Abstracts
of Expert Presentations
ENGINEERING PLANT ONE-CARBON
METABOLISM
Andrew Hanson
University of Florida
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 will be
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 B 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.
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. Unfortunately, processing in insect cells yields
glycoproteins with different carbohydrate structures 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 simpler
oligosaccharide structures. 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 humanized glycoproteins terminating in sialic acid. The
sialylation reaction involves the addition of a donor substrate,
cytidine monophosphate-sialic acid (CMP-SA or CMP-NeuAc), onto a
specific acceptor carbohydrate via an enzymatic reaction in the
Golgi apparatus. Therefore, each of the three reaction
components, donor substrate, acceptor substrate, and enzyme,
must be engineered into insect cells using metabolic engineering
strategies. Production of the donor substrate, CMP-SA, will be
achieved by adding key metabolic precursors such as N-acetylmannosamine
(ManNAc) to the growth media and by genetically manipulating
insect cells to express limiting enzymes in the CMP-SA
production pathway. These genes have been obtained from known
mammalian sequences or identified using homology searches of
known bacterial sequences. The generation of correct
carbohydrate acceptors is achieved by suppressing unfavorable
cleavage reactions and by enhancing the expression of favorable
glycosyltransferase enzymes such as galactose transferase. 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.
APPLICATION OF FUNTIONAL GENOMICS
TO THE DEVELOPMENT
AND OPTIMIZATION OF BIOCATALYSTS FOR RENEWABLE FUELS AND
CHEMICALS
Lonnie O. Ingram
University of Florida
Today, the genetic piping of
metabolic pathways to fuel ethanol and higher value products
such as aromatics or plastics, and the hyper-expression of
recombinant proteins in cells as factories for
biotransformations offer the potential to replace imported
petroleum with renewable biomass. Previous USDA and DOE awards
have supported the development of recombinant Escherichia
coli K011, an organism capable of producing ethanol
efficiently from monomers of all carbohydrate constituents in
lignocellulose. Subsequent awards developed recombinant
Klebsiella oxytoca P2 for cellulose bioconversion,
eliminating the need to externally supply _-glucosidase. Current
funding has extended cellobiose utilization to recombinant E.
coli, and engineered the expression and secretion of high
levels of Erwinia endoglucanases in both organisms.
Studies funded by the Metabolic Engineering Working Group (USDA-NRI
& DOE-BES) have improved our removal of toxins generated
during dilute acid hydrolysis pretreatments of biomass, moving
toward a simplified process. With the completion of the E.
coli genome, the most widely used microbial platform for the
new biotechnology, it is now time to apply Functional Genomics
to these problems. Continuing research will focus on the
molecular tuning of biocatalysts to maximize resistance to
toxins and ethanol, to increase rates of glycolytic flux, and to
reduce the time required for the completion of bioconversions.
Initial studies have investigated the expression of the entire E.
coli genome during model fermentations of 100 g/L xylose to
50 g/L ethanol. Initial examination of these data have provided
a wealth of new information concerning the isoenzymes used in
central pathways, changes in gene expression responsible for
increased flux, clues to genes involved in ethanol tolerance,
evidence for unexpected co-regulation of central metabolism
genes, etc. Our initial Transcriptome data has facilitated the
development of many hypotheses that can be readily tested using
expression vectors and chromosomal mutations. Although we have
none as yet, the development of companion information concerning
the Proteome by 2-D gel analysis or other methods would further
enhance the uitility of the data.
Transcriptome and Proteome data
should be used in the near term to guide the molecular tuning of
recombinant biocatalysts for renewable fuels and chemicals. This
Functional Genomic data should be shared with the research
community in publications and on the WWW. Results from this type
of work could serve as a base for a variety of biotransformation
processes, and for the improved utilization of genomic
information from other organisms in biotechnology applications.
METABOLIC ENGINEERING OF
METHYLOTROPHIC BACTERIA FOR CONVERSION OF METHANOL TO HIGHER
VALUE-ADDED PRODUCTS
Mary Lidstrom
University of Washington
Methanol is an attractive
possibility as an alternative to petroleum as a chemical
feedstock. It is relatively inexpensive, soluble in water, and
since it is produced from methane, it is a renewable resource.
Methylotrophic bacteria are capable of growth on one-carbon
compounds, such as methanol. As such, they represent the
potential to convert methanol to a variety of potential
products. In order to manipulate methylotrophic metabolism,
metabolic engineering will be required, both to understand
methylotrophy in more depth and to alter the flow of carbon from
methanol to desired products. Methylotrophs are actually growing
on formaldehyde, their key intermediate for both carbon and
energy metabolism. Therefore, the key to manipulating
methylotrophic metabolism in methylotrophs is to understand
formaldehyde handling. The organism of choice for this project
is Methylobacterium extorquens AM1, an _-proteobacterium
that grows on methanol, methylamine, and a variety of
multi-carbon compounds. It is already known that about 75 gene
products are involved in methylotrophic metabolism in this
organism, a substantial toolkit of genetic capabilities are
available, and in collaboration with the UW Genome Sequencing
Center, an unfinished genome sequencing project is in progress.
We have initiated metabolic engineering studies by examining the
pathway of polybetahydroxybutyrate (PHB) synthesis. Not only is
this polymer a potential target product, it is a major part of
the biomass generated during regular growth on methanol.
Therefore it is important to understand it=s role in overall
metabolism during growth on methanol. We have cloned the genes
for PHB synthesis and degradation using information from the
genome sequencing project, and have generated mutants in these
genes. Surprisingly, mutants in PHB synthesis are unable to grow
on methanol. Further analysis has suggested that D-betahydroxybutyrate,
the precursor to PHB synthesis, is an intermediate in a part of
the serine cycle that involves the conversion of acetylCoA to
glyoxylate. We are currently examining the role of NADPH/NADH
ratios in methylotrophic metabolism, using a combination of
metabolic modeling and metabolic engineering.
STRATEGIES FOR METABOLIC
ENGINEERING OF ENVIRONMENTAL MICROORGANISMS: APPLICATION TO
DEGRADATION OF ORGANOPHOSPHATE CONTAMINANTS
Jay D. Keasling
University of California, Berkeley
Biodegradation of
readily-degradable contaminants has proven to be an effective
treatment strategy for environmental restoration. Although
bacteria have the capacity to transform a number of chemicals,
many compounds have novel structures or substituents rarely
found in nature and are recalcitrant to biodegradation or are
extremely toxic. To expand the range of compounds that can be
degraded by biological systems, we must assemble the appropriate
enzymatic reactions to catalyze the transformations, either by
introducing the genes for the enzyme-catalyzed reactions into a
single bacterium or by assembling a consortium of bacteria
containing one or more of the necessary enzymes to undertake the
transformation. Analogous to the design of chemical
manufacturing facilities, the flow of chemicals through
enzymatic reactions within a cell must be optimized within the
context of other cellular processes to ensure that the toxic
compound is fully degraded, to minimize the generation of
undesirable products, and to ensure that the engineered organism
can compete in the environment.
Advances in molecular biology
have given rise to a number of tools to manipulate gene
expression. However, most of these tools have been developed for
overproduction of pharmaceutical proteins and, as such, have not
been optimized for metabolic engineering of environmental
organisms. Furthermore, there are few technologies available to
coordinately regulate multiple, heterologous, biodegradation
pathways in a single organism, particularly for degradation of a
contaminant.
The goal of this work has been 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. This
work focuses on the biodegradation of the organophosphate
contaminants by an engineered strain of Pseudomonas putida.
We have chosen parathion as a model compound because (i)
it has been widely used as a pesticide, (ii) it could
potentially serve as a source for carbon, phosphorus, and sulfur
for cell growth, (iii) no single organism has been
isolated that can use it as a sole carbon and phosphorus source,
and (iv) it is similar in structure to a number of other
important environmental contaminants, such as nerve agents and
other pesticides, but is relatively safe for use in the
laboratory. In addition, the initial enzyme in parathion
degradation (parathion hydrolase), which hydrolyzes parathion to
p-nitrophenol (PNP) and diethyl thiophosphate (DETP), has
been shown to hydrolyze many organophosphate contaminants. We
have chosen a well-studied species of the common soil bacterium Pseudomonas,
Pseudomonas putida, as its genetics and metabolism have
been well characterized.
The specific aims of this work
are as follows: (i) to develop a flux-based, metabolic
model for Pseudomonas putida to predict necessary fluxes
through the native and heterologous pathways for optimal growth
and biodegradation; (ii) to clone the genes for DETP
degradation from Comomonas acidovorans; (iii)
to place the genes for the enzymes involved in DETP degradation
in an operon; (iv) to introduce into P. putida the
gene encoding parathion hydrolase and the operons of genes
responsible for PNP and DETP degradation; (v) to
coordinate expression of the opd gene and the PNP and
DETP operons at the optimal levels for maximal growth and
biodegradation rates.
Although this work has focused on
the degradation of parathion, we anticipate that the
technologies developed here will be applicable to the
degradation of other organophosphate contaminants, such as nerve
agents, and recalcitrant organic contaminants. The development
of rational metabolic engineering technologies for environmental
restoration will lead to improved degradation rates, more
complete degradation of the contaminant, and bacteria that can
compete better in the environment. The application of
biodegradation to treat extremely toxic contaminants, such as
organophosphate nerve agents, may necessitate such strategies.
IN SILICO ANALYSIS OF THE ESCHERICHIA
COLI METABOLIC GENOTYPE AND THE CONSTRUCTION OF SELECTED
ISOGENIC STRAINS
Bernhard Palsson
University of California, San Diego
During the first year of this
program we have made significant progress towards the stated
goals.
Goal #1: Automate the
generation of metabolic genotypes
Task 1: We have constructed a new
in silico metabolic genotype for Haemophilus
influenzae Rd (strain KW20) and have nearly completed a
model for Helicobacter pylori (strain 26695). The
genes present in each genome were determined using genomic
microbial databases accessible on the Internet, as well as
published biochemical data. A list of the genes present is
compared to our database of known metabolic reactions (initiated
with our in silico E. coli K-12 strain) for the purpose
of (1) associating each gene in the list with one or more
reactions catalyzed by the gene as found in our database; and
(2) expanding our database to accommodate more reactions. The
corresponding stoichiometric matrices are being formed for each
new strain and used in flux-balance and pathway analysis.
Task 2:
Information for the construction of a physiological database
has been gathered in a literature survey focusing on E. coli
K-12, H. influenzae Rd KW20 and H. pylori 26695.
We have also performed experiments with E. coli K-12 in
our laboratory, using glucose, succinate and acetate as carbon
sources for the purpose of determining more strain-specific
parameters.
Task 3:
The FBA program was developed to give both quantitative and
visual results to the linear optimization of a given objective
function, most commonly growth. The program calculates
traditional linear programming values such as the objective
function and shadow prices. The results are then graphically
displayed in pathway form with the corresponding fluxes labeled
for each reaction and the utilized pathways highlighted.
Task 4:
This task has not yet been addressed.
Goal #2: Develop methods to
determine genotype properties and capabilities
Module 1:
The FBA program that we developed allows for the deletion of any
number of genes. The corresponding phenotype can than be
analyzed in silico. A metabolic model of Haemophilus
influenzae has been developed by our group and will be used
to do deletion analyses similar to those done in E. coli.
In addition, the FBA program has been used to do robustness
analyses in E. coli. Such an analysis examines the
sensitivity of the growth function to altered flux levels of
essential genes that have been identified in the central
metabolic pathways.
Module 2:
The FBA program is designed to plot Phenotypic Phase Planes (PhPP),
which are a phenotypic mapping for biomass generation as a
function of a primary carbon source and oxygen uptake rate.
These PhPPs show the metabolic shifts that occur with various
oxygenation and substrate levels. Experiments have begun in the
lab to verify the metabolic shifts predicted using the in
silico metabolic model for E. coli.
Module 3:
This module is in progress.
Panel
Discussion
The afternoon session consisted
of a panel discussion lead by George Church and Bernhard Palsson.
The workshop focused on the
Metabolic Engineering program and desirable changes in its
direction. It has become clear that the emphasis of this program
will become the use of microbial genetics and systems analysis
methods to attempt to synthesize mechanistic description of the
genotype-pheotype relationship; or in other words to go from
genomics to phenomics.
This grand challenge involves the
development of instrumentation, data basing, algorithm
development, and model formulation. These issues were discussed
on a wide basis and all opinions were heard.
Issues raised and focused
recommendations:
A. Data generation (to drive
computation work):
A.1) More quantitative data is
needed. Where the costs of this could benefit greatly from new
instrumentation encourage clear communication with the
appropriate engineering groups.
A.2) Generate ways to
estimate/catalog physico-chemical properties of protein (enzymes
in particular).
A.3) Make funding for arrays
available once sequences are established especially since this
is a small fraction of the sequencing cost.
A.4) Instrumentation for
high-throughput phenotyping is needed. Desired phenotypic data
include: growth rates of cells and organisms, RNA, protein and
metabolite assays on single cells and populations.
B. Databases and data sharing.
B.1) It was observed that purely
database creation grants have not received high marks during
peer review. Applicants should be encouraged to couple such
databases with creative goals, methods, and/or models.
B.2) Standardization is needed
for both software and data and both syntax, semantics.
B.3) Encourage deposit of
computationally parseable versions of data generated under
program where it is retrievable (e.g. www). As needed, design
databases to connect new data types with new applications.
C. Models, Software and
Algorithms:
C.1.) Algorithm and model
development is good, but more software is needed. Models need to
be (at a minimum) reproducible by experts by a simple download
and run.
C.2) Point-and-click web
accessible software is needed for more general use.
C.3) Standardization/portability
is needed for math models.
C.4) Biology is stated by many to
be too complex for math analysis, but the converse would be
stated by systems scientist, namely that it is hard to
understand such complex processes without a model.
C.5) What is the accuracy
available (false negative and positive rates) for pathways
generated de novo in silico? Encourage estimates of the costs of
improving the accuracy.
C.6) Encourage determination of
the level/accuracy of kinetic constants that are needed for good
models.
D. Administrative:
D.1) Broaden announcement to
include 'functional genomics'.
D.2) Microbial genomics needs to
be merged into metabolic engineering.
D.3) Be careful in the use of
language in announcement
 | bioinformatics vs.
functional genomics,
| math model vs.
reconstruction,
| deduction vs. engineering,
| Physico-chemical-properties
vs functional genomics. |
| | |
Conclusions
and Discussions of the Panel Discussion
The attendees at The Panel
Discussion focused on the future needs for augmenting the
bioinformatic content in the Metabolic Engineering Joint
Research activity. Attendees included all of the Year 1
grantees, several of the Year 2 awardees, and about 15-20
representatives from the agencies participating in the MEWG. The
conclusions and recommendations from this
Discussion represent the collective opinions of the attendee
group as reflected in comments made at the Discussion as well as
several e-mail responses sent to one or more of the MEWG
representatives.
The consensus was that the
program needs to foster the development of tools to facilitate
the translation of genomic information into real biological
processes -- e.g directed protein synthesis and metabolism.
Tools that were specifically discussed included databases,
functional genomics, and nucleic acid and protein
high-throughput screening methodologies. On the subject of
databases, the observation was made that good metabolic models
are data starved --- there is a need for genomic, proteomic and
metabolomic data that is validated, widely distributed (e.g. the
web-based Biology Workbench), and curated. It was also noted
that data generation, accumulation and genomic-to-phenomic
modeling should be done in the context of the new processes and
products that can be realized by recombinant DNA technology.
DNA, RNA and protein array technology should be fostered in the
program, and it was noted that many of the Years 1 and 2
grantees are already using these methodologies and in several
cases making important contributions to the advancement of these
tools. Finally, opinions were widely voiced that modeling work
needs to be tightly coupled to experimental effort, and that the
program should be kept appropriately broad so that opportunities
for investigator-driven research are maximized.
AGENDA
8:00 am
Welcoming
and Opening Remarks
MARYANNA HENKART, Chair,
Biotechnology Research Working Group
FRED HEINEKEN, Chair,
Metabolic Engineering Working Group
8:15 am Engineering
Plant C1 Metabolism
ANDREW HANSON, University of
Florida
8:45 am Carbohydrate
Engineering for Generating Sialyated Glycoprotiens in Insect
Cells
MICHAEL BETENBAUGH, Johns
Hopkins University
9:15 am Metabolic
Designs to Maximize Ethanol Production from Lignocellulose
LONNIE INGRAM, University of
Florida
10:15 am
Welcoming Remarks
MARY CLUTTER, Chair,
Subcommittee on Biotechnology
10:30 am Metabolic
Engineering of Methylobacterium extorquens AM1 for Conversion of
Methanol to Higher Value Added Products
MARY LIDSTROM, University of
Washington
11:00 am Strategies
for Metabolic Engineering of Environmental Organisms:
Application to Degradation of Organophosphate Contaminants
JAY KEASLING, University of
California - Berkeley
11:30 am Progress
Report Grant BES 98-14092
BERNARD PALSSON, University of
California - San Diego
1:15 pm
Introduction to Afternoon Session
Convener BERNARD PALSSON
1:20 pm Measuring
& Modeling Cellular Metabolic & Regulatory Networks
GEORGE CHURCH, Harvard
University
1:35 pm
The Needed Information Technology Infrastructure
JOHN WOOLEY, University of
California - San Diego
1:50 pm Predicting
Physico-Chemical Properties of Gene Products
MICHAEL GILSON, National
Institute of Standards and Technology
2:20 pm Numerics
and Modeling Philosophies
LESLIE LOEW, University of
Connecticut Health Center
2:50 pm
Introduction to general discussion on Bioinformatics
MARK SEGAL, Environmental
Protection Agency
VINCE VILKER, National Institute of Standards and Technology
3:05 pm
General Discussion and Future Directions
BERNARD PALSSON, University of
California - San Diego
GEORGE CHURCH, Harvard University
4:15 pm
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 modelled 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. Investigations are currently focused on finding
generic routes for meeting the energy requirements of these
enzymes, and on developing synthetic methods of carrying out
cell functions like electron transfer between proteins.
The Structural Biology activity
includes x-ray amd 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 modelling develops methods to model the
energetics and dynamics of interactions between substrates and
active sites of enzymes. Modelling 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
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, bioprocessessing, 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) and the Office of Naval Research (ONR).
The specific focus of the ARO, ONR and AFOSR 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 manipulationof 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.
The AFORS focus on environmental
biotechnology for cleanup and detoxification of hazardous
chemicals requires considerable emphasis on the use of metabolic
engineering. Many strains of microorganisms can readily use
natural organic compounds such as jet fuel and gasoline as their
source of carbon and energy. Such microorganisms provide the
basis for the extensive recent successes in bioremediation.
Metabolic engineering offers the potential for development of
strains able to degrade such pollutants under adverse conditions
such as in the presence of heavy metals, at elevated
temperatures, or at extremes of pH.
AFOSR researchers are also using
metabolic engineering in the development of microorganisms able
to use synthetic organic compounds including nitro- and
chloro-substituted compounds as growth substrates. Finally, a
variety of reactions capable of detoxifying hazardous chemicals
are known to be catalyzed by microorganisms that are unable to
use the chemicals as growth substrates. Such processes can be
effective for treatment of contaminants if the appropriate
microbes can be stimulated to produce the necessary enzymes and
confactors to sustain the reactions. Metabolic engineering
offers the potential for uncoupling the production of the
enzymes from the growth of the organisms. Researchers are
currently developing constitutive strains with altered surface
properties to allow transport in the subsurface or adherence to
substrates in bioreactors. Future applications of metabolic
engineering will involve the construction of strains able to
degrade or synthesize a wide variety of materials relevant to
not only the military, but also civilian applications.
The current ONR program provides
a broad base in funding of research that addresses fundamental
issues associated with metabolic engineering and, at the same
time, targets a niche which is under represented in the other
DoD services and in other agencies. A significant portion of the
ONR program targets marine organisms as cellular factories for
metabolic engineering and exploits many of the novel and unique
features of marine bacteria and algae to fabricate
nanostructures in which composition and shaped are defined
simultaneously. Current program activities address the use of
combinatorial approaches for the development of (1) biosensor
devices and whole-cell biosensors, (2) new macromolecular
materials, (3) novel processes and catalysts, (4) molecular
composites and, (5) designer fabrication schemes. ONR is
addressing the role of extracellular enzymes as catalysts for
bioremediation, fabrication and for immobilized synthetic
activities. In addition enzymes are being engineered to perform
in non-aqueous environments which will be critical to materials
synthesis, biosensor technologies, bioremediation and other
critical Navy and DoD applications. Future directions include
the production of proteins that serve in non-metabolic
transformations such as those proteins functioning in cellular
information processing, including signaling cascades,
protein-based circuits and metabolic switching in which multiple
metabolic pathways are coupled. Lastly, future activities will
also target multi-enzyme complexes that are coupled to generate
novel structures and capabilities like those involved
in polyketide synthesis.
U.S.
Department of Energy
The Department of Energy is
supporting over $25 million in metabolic engineering research,
largely through the offices of Energy Research (ER), 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 ER, 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 ER is supported predominantly through the Office of Basic
Energy Sciences (BES) and Health and Environmental Research
(OHER). Most of BES's metabolic engineering research resides
within the Division of Energy Biosciences. The mission of the
Division is 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 OHER 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. OHER'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, ER 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 OER to pursue basic research needs
in various areas of national laboratory clean-up issues and
waste management.
The biological research
activities of the Department are monitored and coordinated
through the BioEnergy Coordinating Committee (BECC). BECC is an
interagency committee open to all organizations involved in
bio-energy research and development. The committee is comprised
of about 45 representatives from seven agencies and meets on a
quarterly basis. The objectives of BECC are to 1) achieve
effective coordination of DOE's bio-energy R&D; 2) assure
optimum use of DOE's existing expertise in bio-energy R&D;
3) provide a resource for industry and others to access
information rapidly in DOE bio-energy programs; and 4) achieve
rapid communication within DOE of new developments,
opportunities, and problems in bio-energy research and
technology development.
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. A prominent
concern is the introduction of chemicals to the environment
which may have detrimental effects on humans and other biota. As
mandated by Congress, 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.
To fulfill its congressionally mandated responsibilities, the
Agency dedicates a significant portion of its resources to the
development of risk assessment tools.
Coordinately, the Agency, through
its stated mission as well as the implementation of
congressional initiatives, such as the Pollution Prevention Act,
has initiated 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
utilitized successively in different forms prior to final
disposal.
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".
Because the EPA functions in both
regulatory and scientific modes, there is a need to foster
greater understanding of risks associated with both conventional
as well as novel technologies. Failure to do this can result in
expensive and needless regulatory oversight, while prolonging
the process of bringing "environmentally friendly"
products online. This talk will provide a discussion of both
risk assessment tools and some recent developments which have
been brought to EPA's attention either through Agency funding or
regulatory review. Among the topics that will be discussed are
the following:
- development of a biotechnology
risk assessment program with a focus on addressing technical
issues that are growing more complex;
- substitution of microorganisms
to manufacture adipic acid starting with microbial nutrients
rather than benzene;
- construction of a microorganism
which uses biological fluorescence to detect the presence of
biologically available toxic materials;
- generation of a biomass
conversion process which produces alcohol from biomass products.
The presentation will discuss
recent developments in these fields as well as opportunity to
better coordinate work in these areas between federal
departments and agencies. The objective will be to leverage
increasingly smaller resources to maximize benefits across the
government, as well as presenting a more consistent approach to
developmental and regulatory activities to nongovernmental
agencies.
National
Institutes of Health
NIGMS/NIDDK
The National Institute of General
Medical Sciences (NIGMS), in conjunction with the National
Institute of Diabetes, Digestive and Kidney Diseases (NIDDK),
issued a program announcement in September 1995, in an effort to
stimulate research in metabolic engineering. This announcement
is part of a long-term, ongoing effort, and application for
support of research in metabolic engineering are still being
encouraged. Through this initiative, the NIH hopes to encourage
basic research that will facilitate both the development of
microbial or plant-base production routes for useful quantities
of "small" molecules (such as antibiotics and other
drugs) AND a substantially heightened understanding of the
control architecture that integrates the genetic and catalytic
processes in normal and aberrant cells. During fiscal 1998, the
NIGMS and NIDDK provided over $2.3 million for the support of
research directly involving metabolic engineering. Examples of
work funded through this initiative include (1) a study of the
genes and enzymes which represent rate-limiting steps in the
biosynthesis of beta-lactam antibiotics; (2) a study of the
origin of bioactive marine natural products at the cellular
level within selected deep water sponges; and (3) a study of the
feasibility of genetically engineering fungi to produce novel
polyketides with pharmacological potential.
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 division 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 activities: (1) the “Arabidopsis
Genome Research Initiative:” 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 “Plant Genome
Research Program” 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 BIO activity has already funded 23
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" a new initiative
announced in November 1998, which 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 ongoing
“Life in Extreme Environments (LEXEN) Initiative” also
offers a strong link to Metabolic Engineering because of the
wealth of metabolic pathways evolved by organisms that have
adapted to environmental extremes. Also included in the LEXEN
program are proposals to investigate the potential for habitable
environments on other planets. (5) Finally, the BIO Directorate
has recently announced the “2010 Project” that will support
research to determine the function of all genes in Arabidopsis
thaliana by the year 2010. The program will focus on
supporting creative and innovative research designed to
determine the function of a network of genes and to develop new
tools for functional genomic approaches.
The Directorate
for Geosciences supports research related to ME in marine
systems. 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 generation 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 of a new era of chemotherapy.
Workshop
Participants
| No. |
Name |
Institution |
E-Mail |
| 1. |
James J.
Anderson |
NIH |
andersoj@nigms.nih.gov |
| 2. |
Michael J.
Betenbaugh |
Johns Hopkins
University |
beten@jhu.edu |
| 3. |
David J. Boron |
DOE |
David.Boron@hp.doe.gov |
| 4. |
Harold J.
Bright |
ONR |
BrightH@onr.navy.mil |
| 5. |
Michael W.
Broder |
EPA |
broder.michael@epamail.epa.gov |
| 6. |
George M.
Church |
Harvard
University |
church@arep.med.harvard.edu |
| 7. |
Mary Clutter |
NSF |
mclutter@nsf.gov |
| 8. |
Jeffrey L.
Conrad |
USDA |
JConrad@reeusda.gov |
| 9. |
Joanne D.
Culbertson |
NSF |
jculbert@nsf.gov |
| 10. |
Michael M.
Domach |
Carnegie
Mellon University |
md0q@andrew.cmu.edu |
| 11. |
Marvin Frasier |
DOE |
marvin.frazier@science.doe.gov |
| 12. |
Michael Gilson |
NIST |
gilson@indigo14.carb.nist.gov |
| 13. |
Bruce Hamilton |
NSF |
bhamilto@nsf.gov |
| 14. |
Andrew D.
Hanson |
Univeristy of
Florida |
adha@gnv.ifas.ufl.edu |
| 15. |
Philip
Harriman |
NSF |
pharrima@nsf.gov |
| 16. |
Maryanna
Henkart |
NSF |
MHenkart@nsf.gov |
| 17. |
Fred G.
Heineken |
NSF |
FHeineke@nsf.gov |
| 18. |
Geoffrey
Holdridge |
WTEC, Inc. |
gholdrid@itri.loyola.edu |
| 19. |
Lonnie O.
Ingram |
University of
Florida |
lingram@micro.ifas.ufl.edu |
| 20. |
Warren Jones |
NIH |
JonesW@GM1.NIGMS.NIH.gov |
| 21. |
Arthur Katz |
DOE |
Arthur.Katz@oer.doe.gov |
| 22. |
Jay D.
Keasling |
University of
California - Berkeley |
keasling@socrates.berkeley.edu |
| 23. |
Ashok Kolaskar |
American Type
Culture College |
akolaskar@atcc.org |
| 24. |
Dhinakar S.
Kompala |
University of
Colorado |
kompala@colorado.edu |
| 25. |
Mary Lidstrom |
University of
Washington |
lidstrom@u.washington.edu |
| 26. |
Liang-Shiou
Lin |
USDA |
llin@reeusda.gov |
| 27. |
Leslie Loew |
University of
Connecticut Health Center |
les@volt.uchc.edu |
| 28. |
Elbert L.
Marsh |
NSF |
emarsh@nsf.gov |
| 29. |
Gail McLean |
USDA |
gmclean@reeusda.gov |
| 30. |
Robert Menzer |
EPA |
Menzer.Robert@epamail.epa.gov |
| 31. |
Bernhard O.
Palsson |
University of
California - San Diego |
bpalsson@bioeng.ucsd.edu |
| 32. |
E. Terry
Papoutsakis |
Northwestern
University |
e-paps@northwestern.edu |
| 33. |
Dewey D.Y. Ryu |
University of
California - Davis |
ddyryu@ucdavis.edu |
| 34. |
Mark Segal |
EPA |
Segal.Mark@EPAMail.EPA.Gov |
| 35. |
Imran Shah |
University of
Colorado |
Imran.shah@uchsc.edu |
| 36. |
Alison Sipe |
NSF |
asipe@nsf.gov |
| 37. |
Marvin
Stodolsky |
DOE |
Marvin.Stodolsky@science.doe.gov |
| 38. |
George B.
Vermont |
NSF |
gvermont@nsf.gov |
| 39. |
Vincent L.
Vilker |
NIST |
vilker@nist.gov |
| 40. |
George J.
Wagner |
University of
Kentucky |
gwagner@ca.uky.edu |
| 41. |
Sharlene
Weatherwax |
DOE |
sharlene.weatherwax@science.doe.gov |
| 42. |
William
Weigand |
NSF |
wweigand@nsf.gov |
| 43. |
Eugene Wong |
NSF |
ewong@nsf.gov |
| 44. |
John Wooley |
University of
California - San Diego |
jwooley@ucsd.edu |
|
|
