The Genomic Science program within the U.S. Department of Energy’s (DOE) Office of Biological and Environmental Research (BER) supports basic interdisciplinary research aimed at achieving a predictive, systems-level understanding of plants, microbes, and microbial communities to advance BER energy and environmental missions. Building on an ever-growing body of genomic sequence information, the program integrates large-scale systems biology (transcriptomics, proteomics, metabolomics, and other “-omics” approaches) with computational modeling to comprehensively understand the foundational principles that rule biological systems. Achieving this knowledge enables the design and engineering of DOE-relevant plants and microbes for defined purposes and reveals insights into their responses to environmental inputs. Within the Genomic Science program, Biosystems Design research has contributed to the advancement of bioengineering, often referred to as synthetic biology.
New genome-scale engineering and synthetic biology tools are opening new avenues for systems biology research. These tools not only enable the design of tailored plants and microbes by genome editing or introduction of megabase-size synthetic constructs with orthogonal pathways, they also advance fundamental understanding of biological function and inspire novel approaches for its manipulation on a large scale. Critical components of these tools are computational modeling and computer-aided design methods that integrate large -omics datasets to solve grand challenges in biological engineering.
In fiscal year 2017, BER solicited integrative, multidisciplinary applications to conduct fundamental genomics and systems biology research and technology development to design novel biosystems within the following research areas:
Building on prior funding from BER for systems biology work on Rhodococcus opacus, this project will apply innovative combinations of -omics and mass spectrometry data analysis, flux analysis, and machine learning to develop improved genome-scale models that will guide multiplex genome engineering of R. opacus strains with branchedchain fatty acid esters (BCFAE) biosynthetic capacity and improved tolerance to lignin degradation products. The project will take advantage of this organism’s capability to use both polysaccharides and lignin as carbon sources as well as its natural tolerance to phenolic compounds such as those produced by lignin breakdown.
The strength of this project is that it will advance biological
engineering to a new level of throughput and complexity.
Guided by computer-aided design platforms, this research
will develop new finely tuned microbial strains containing
orthogonal regulatory networks. The redesigned strains will
be tailored to grow on a variety of feedstocks, tolerate toxic
metabolites and other stresses, and produce valuable molecules.
Building on technologies and knowledge developed
with prior DOE funding from the Biosystems Design program,
the project will further develop the necessary technical
and computational infrastructure to achieve its goals,
initially working with Escherichia coli and Saccharomyces
cerevisiae and later transferring the technologies to other
non-model bacteria and yeast species.
The goal of this research is to integrate genome-scale
modeling efforts of the diatom Phaeodactylum
tricornutum to
inform experimental
research for
the production of
high-value and fuel-related
metabolites.
Leveraging prior
work on this organism
under DOE
Biosystems Design
funding, this project
will apply genome-scale
engineering
to optimize carbon,
energy, photosynthesis,
and metabolite
flux to shift the
native P. tricornutum metabolism from
carbohydrate storage
into lipid and/
or branched chain
amino acid (BCAA)
accumulation. The team will focus on compartmentalizing
relevant biosynthetic pathways on artificial
chromosomes to obtain modified diatom strains with
high productivity of triacylglycerols or BCAAs.
This project will focus on Rhodosporidium toruloides and Issatchenkia orientalis, two industrial yeast species, to
develop the tools needed to turn these organisms into
model systems for biological design. R. toruloides will be
engineered to produce fatty alcohols from lignocellulose-derived
sugars, and the target products for I. orientalis will be organic acids such as muconic acid and methyl
methacrylate. The project will leverage genome-scale
engineering tools previously developed for Saccharomyces
cerevisiae by the PI’s laboratory that apply CRISPR-Cas
methods for multi-gene disruptions (homology integrated
CRISPR-Cas or HI-CRISPR) and CRISPR-based
gene activation, interference, and deletion (CRISPR-AID).
A highly automated robotic pipeline already
operational at the University of Illinois will be used for
high-throughput genetic manipulation and engineering
of the two yeast species.
This project will develop an integrated set of technologies
for high-throughput metabolic pathway design and
engineering of Clostridia, called the Clostridia Foundry
for Biosystems Design (cBioFAB), to produce fuels and
chemicals from lignocellulose-derived syngas. Using
multi-omics, computational modeling, genome engineering,
and cell-free and in vivo technologies, the Foundry
will focus on the production of acetone, butanol, butanediol,
and mono-ethylene glycol as proof of concept. The
project will leverage DOE Joint Genome Institute (JGI) efforts to sequence the genomes of multiple Clostridium strains and use a computer-aided design tool called Biochemical
Network Integrated Computational Explorer
(BNICE) developed by team members to identify relevant
metabolic genes and pathways that will be subsequently
tested in cell-free systems. New CRISPR-based genome editing
techniques will be developed for Clostridium, and
metabolic ensemble modeling will be used to predict in
vivo pathway performance from cell-free assays.
The model system on which this project will focus is the
oleaginous green alga Chromochloris zofingiensis because
it is fast-growing, can be cultured at high densities, and
accumulates triacylglcyerols
(TAG)
at the highest
levels observed
among microalgae. C. zofingiensis metabolic
regulation
is unique because
in the presence of
glucose, photosynthesis
is switched
off by degrading
the thylakoid
membranes and
photosynthetic
machinery, while
lipid production
is substantially
increased; the
process is reversed when the glucose supply is exhausted.
The project will leverage a high-quality genome assembly
of the C. zofingiensis genome recently generated by the
research team and will enhance it with multi-omics and
physiological analyses to build a model to guide metabolic
engineering of this alga for the production of biofuel
precursors.
To identify the plant genes involved in the development
of symbiotic nodules containing nitrogen-fixing
microbes (diazotrophs), this project will analyze the
evolution of those symbioses. Prior studies by members
of the research team showed that microbe-mediated
nitrogen fixation was originated and lost multiple times
during the evolution of the nodulating plant clade.
Therefore, investigators intend to identify the genetic
machinery used by nodulating plants and introduce it
into non-nodulating bioenergy crops such as poplar,
thereby reducing this crop’s need of nitrogen fertilization.
To achieve this ambitious goal, the team will
conduct extensive phylogenetic analysis of over 20,000
species using available genomic sequence data as well
as samples from multiple plant species, including preserved
museum specimens from around the world.
Selected engineered poplar lines will be tested for their
ability to establish symbiotic relationships with diazotrophs,
as well as for their biomass productivity and
composition.
To engineer bioenergy crops that can grow in dry, marginal
lands, this project will introduce into poplar trees tolerance
to different environmental stresses such as water deficit,
high soil salinity, and heat. As plants are often exposed
to combinations of these different types of abiotic stress,
poplar lines will be engineered to express multiple stressrelated
genes, each one controlled by a synthetic promoter
that is only induced under specific stress conditions and
within the tissues where its function is needed, improving
biomass production under unfavorable conditions while
avoiding negative effects of unwanted transgene expression.
The project will focus on a poplar hybrid clone for which
a genome sequence is available and will design tissue- and
stress-specific synthetic promoters using plant promoter
databases and computational tools to identify cis-regulatory
elements.
Building on prior work on the model grass Setaria,
funded by DOE as part of the Biosystems Design program,
this project aims at increasing water use and photosynthetic
efficiencies in the bioenergy crop sorghum.
Leveraging the resources previously developed by the
research team for Setaria, investigators will accelerate the
generation of engineered high-biomass sorghum lines that grow in marginal lands using innovative genomeediting
tools, genome-scale modeling, -omics approaches,
genome-wide association studies, and high-throughput
phenotyping in both laboratory and field settings. The
work will exploit the efficient transformation and screening
pipelines available for Setaria to facilitate gene discovery
and pathway design that can be transferred to
sorghum. Because biomass productivity strongly depends
on photosynthesis and water availability, the engineering
work will focus on three main topics: enhanced carbon
assimilation, increased root water uptake, and reduced
water loss through stomata.
This project will pursue an innovative approach to
engineer oil accumulation in vegetative tissues of
bioenergy crops. Leveraging previous efforts toward
producing
oil in sugar
cane leaf
tissue, the
researchers
will attempt
to redirect
and increase
TAG accumulation
in
the stems of
energy cane
and Miscanthus.
As
these species
are close
relatives of
sugar cane, CRISPR/Cas9-based genome-editing technologies
previously developed in sugar cane will likely
be transferable to these new plant systems along with
novel genome-engineering methods. The ultimate goal
of the project is to reach a TAG accumulation of up to
20% of dry stem weight, which represents a productivity
increase of more than one order of magnitude
relative to oilseed crops. Computational modeling will
be used to design improved photosynthetic capacity
that they expect to reach 50%, along with increased
nitrogen and water use efficiency, to account for the
required redirection of carbon flow from lignocellulosic
biomass to oil in the redesigned plant lines.
The goal of this project is to divert carbon flow from
starch accumulation to oil production in resting fronds
of Lemnaceae (duckweed) species, thereby redesigning
existing
commercial
strains
towards
biofuel
production.
Lemnaceae’s rapid
growth,
high proportion
of photosynthetic
tissue
due to its
lack of lignified supportive tissue, and its ability to grow
in marginal wastewater environments are among the
advantages of these small aquatic plants. An available
genome sequence and other -omics resources will be
leveraged, along with other products from prior DOE
funding (ARPA‐E) to significantly increase oil content
in Lemnaceae. Metabolic pathways will be re‐engineered
to maximize TAG yields while preserving rapid
biomass accumulation, and tools for genetic manipulation,
metabolic flux models, and a catalog of key transcription
factors and promoters will be developed to
elevate this emerging plant model system to a potential
bioenergy crop.
This project will establish Camelina sativa as a commercially
viable, dedicated biofuel and bioproduct
feedstock. Its low agronomic input requirements, natural
tolerance to different stresses, available molecular
and genomics tools and resources, and suitability of its
oil for transportation as liquid fuel blends are some of Camelina’s advantages over other oilseed crops. However,
oil yield from Camelina seeds is relatively low. To
improve oil production, an integrative approach including
metabolic flux modeling, proteomics, regulatory
network analysis, and multi‐gene stacking and genomeediting
techniques will be employed to optimize photosynthetic
carbon fixation in leaves, its transport to
seeds, and the allocation of fixed carbon to TAG. With
this approach, the team aims at increasing oil production
to 300% per hectare, thus greatly enhancing the
feasibility of developing Camelina as a cost‐competitive
bioenergy crop.
Dr. Pablo Rabinowicz
U.S. Department of Energy
Office of Biological and Environmental Research
Phone: 301-903-0379
E-Mail: pablo.rabinowicz@science.doe.gov
