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.
