March 2021 - JBEI Research Highlight Slides

Performance-Based Payments for Soil Carbon
Sequestration Can Enable a Low-Carbon Bioeconomy
Background
• Incentivizing bioenergy crop production in locations with marginal soils,
where low-input perennial crops can provide net carbon sequestration
and economic benefits, will be crucial to building a successful
bioeconomy.
Approach
• We developed an integrated assessment framework to compare
switchgrass cultivation with corn-soybean rotations on the basis of
production costs, revenues, and soil organic carbon (SOC) sequestration
at a 100 m spatial resolution.
• We calculated profits (or losses) when marginal lands are converted from
a corn-soy rotation to switchgrass across a range of farm gate biomass
prices and payments for SOC sequestration in the State of Illinois,
United States.
Outcomes and Impacts
• The annual net SOC sequestration and switchgrass yields are estimated
to range from 0.1 to 0.4 Mg ha–1
and 7.3 to 15.5 Mg dry matter ha–1
,
respectively, across the state.
• Without payments for SOC sequestration, only a small fraction of
marginal corn-soybean land would achieve a 20% profit margin if
converted to switchgrass
• Including $40–80 Mg–1
CO2e compensation could increase the
economically viable area by 140–414%.
• With the compensation, switchgrass cultivation for 10 years on 1.6
million ha of marginal land in Illinois will produce biomass worth $1.6–2.9
billion (0.95–1.8 million Mg dry biomass) and mitigate 5–22 million Mg
CO2e.
Mishra et al. (2021) Environmental Science & Technology, doi: 10.1021/acs.est.0c06452
Figure 1. Integrated assessment framework for the
systematic analysis of land use change.
Figure 2. Switchgrass biomass yield under rain-fed
conditions simulated by DAYCENT for the State of Illinois (a).
Projected change in annual soil organic carbon due to
decade-long bioenergy-based land use conversion (corn-
soybeans to switchgrass) on marginal lands (b).

Regional environmental controllers influence
continental scale soil carbon stocks and future
carbon dynamics
• Understanding the influence of environmental factors on soil
organic carbon (SOC) is critical for quantifying and reducing the
uncertainty in carbon climate feedback projections under
changing land use and climatic conditions.
Goncalves et al. (2021) Scientific Reports,
DOI: 10.1038/s41598-021-85992-y.
• Higher precipitation and lower temperatures were associated
with higher levels of SOC stocks in majority of ecoregions.
• Change in vegetation properties was important controller of
SOC stocks in drier ecoregion such as North American
deserts, whereas soil types and topography were more
important predictors of SOC stocks in American prairies.
• The SOC stocks did not change substantially under SSP126
until 2100, however SOC stocks decreased up to 21% under
SSP585.
• We explored the effect of climatic variables, land cover types,
topographic attributes, soil types and bedrock geology on SOC
stocks of top 1m depth across conterminous United States
ecoregions.
• Coupling 4559 soil profile observations with high-resolution
data of environmental and climatic factors, we identified (1)
dominant environmental controllers of SOC stocks in 21 US
ecoregions, and (2) projected change in SOC stocks under
SSP126 and SSP585 climate change scenarios.
Approach
Background
Figure: Baseline soil organic carbon stocks in 0-1m depth
interval of continental US (a) and projected change (b) by
2100 under high emission scenario (SSP585).
(a)
(b)

Omics-driven biotechnology for industrial applications
Background
• The use of biological systems to manufacture bioproducts of
commercial relevance (i.e., biomanufacturing) is a key component
of the biotechnology industry
• Knowledge gained from “omics” experiments aid the development
and advancement of biotechnology research toward establishing
robust industrial biomanufacturing platforms
Approach
• This review focuses on recent advances in “omics” technologies
and their influence on the production of biofuels and bioproducts,
agricultural and food biotechnology, and COVID-19 research
• “Omics” technologies can accelerate the metabolic engineering of
biofuels and bioproducts via the DBTL cycle framework
• Proteomics and metabolomics are now routinely applied to the
pathway engineering studies in microbial hosts
• Over the last 20 years, there is a growing trend in the use of
genomics and proteomics in bioenergy research as evidenced by
the number of publications during that time
• Additionally, there is a growing trend in the use of genomics,
proteomics, and metabolomics in natural products and materials
research
• Multi-omics data integration and interpretation tools can derive
new insights from data including biomarker predictions
• Genomics sequencing appears to be key to the development of
next generation vaccines against viruses such as SARS-CoV-2,
whereas transcriptomics, proteomics, and metabolomics can aid
the development of therapeutic drugs for COVID-19
Amer et al. (2021) Frontiers in Bioengineering and Biotechnology, doi: 10.3389/fbioe.2021.613307
In the last 20 years, genomics has shown a steady growth in
biotechnology research. However, the emergence of transcriptomics,
proteomics, and metabolomics will be key to development of next
generation biofuels and bioproducts

Nanotechnology to advance CRISPR–Cas genetic
engineering of plants
Background
• CRISPR–Cas technology holds much potential for transforming
plant functional genomics research, improving crop resilience to
abiotic and biotic stresses, and rapidly introducing new
desirable traits into crops
• Widespread application of CRISPR technologies in plants faces
several barriers, such as limitations of plant tissue and cell
culture, lack of methods that work across species, and efficient
CRISPR cargo delivery
• There is great potential for nanomaterials to address many
central bottlenecks of CRISPR-based genome editing, but
several nanotechnology-specific advances are needed to
realize the potential of these tools in plant biology
Demirer et al. (2021) Nature Nanotechnology, doi: 10.1038/s41565-021-00854-y
Fig. 1 CRISPR–Cas reagent
delivery to diverse plant
species, cells and
organelles. a, Examples of
plant species, cell types
and organelles that can be
targeted by the CRISPR–
Cas system. b, The CRISPR–
Cas genome-editing
system can be delivered in
into the plant cell in form
of RNPs or plasmids
containing genes that
encode Cas and the sgRNA.
Perspectives
Moving the field forward
we need to:
• Establish an upper limit
for the nanoparticle-
loaded cargo size and
amount for CRISPR
DNA (dependent on the
nanoparticle type and
surface chemistry)
• Determine if
nanoparticles can carry
CRISPR reagents to
plant plastids and
mitochondria
• Establish the
compatibility of
nanoparticles with plant
tissue culture and
regeneration protocols
(when germline
transformation is not
plausible)
• Determine the
persistence of
nanoparticles in the
offspring of edited
plants to facilitate
regulation

Association of gene expression with syringyl to
guaiacyl ratio in sugarcane lignin
Background
• Sugarcane bagasse is a highly abundant resource that may be used as
a renewable feedstock for the production of biofuels and bioproducts.
• Altering the ratio of the lignin units, syringyl (S) and guaiacyl (G), which
comprise the native lignin polymer in sugarcane, may facilitate the
processing of bagasse.
• Identifying genes and markers associated with S/G ratio is key for
expediting the development of bioenergy varieties of sugarcane.
Approach
• This study aimed to identify genes and markers associated with S/G
ratio in order to accelerate the development of sugarcane bioenergy
varieties with modified lignin composition.
• The transcriptome sequences of 12 sugarcane genotypes that
contrasted for S/G ratio were compared and there were 2019
transcripts identified as differentially expressed (DE) between the high
and low S/G ratio groups.
• The frequencies of SNPs in expressed transcripts were compared
between the two groups to identify specific alleles linked with the
phenotype in terms of S/G ratio.
• There were 2019 transcripts identified as differentially expressed
between the genotypes contrasting for S/G ratio.
• 2063 SNP loci across 787 unique transcripts showed group specific
expression that impacted S/G ratio.
• The DE transcripts and SNP alleles identified in this study may be
valuable for breeding sugarcane varieties with altered S/G ratio that is
tailored for efficient deconstruction and conversion into biofuels and
bioproducts.
Hodgson-Kratky et al. (2021) Plant Mol Biol. doi: 10.1007/s11103-021-01136-w
Expression of transcripts in sugarcane (Saccharum spp. hybrids)
genotypes with contrasting syringyl to guaiacyl (S/G) ratios mapped
against the SUGarcane Isoform-sequencing Transcriptome
reference and mean transcript expression levels.
Multidimensional scaling plot of sugarcane (Saccharum spp. hybrids)
genotypes in triplicate with contrasting syringyl to guaiacyl (S/G)
ratios. Distances represent average log2(fold changes) (log2FC)
computed from trimmed mean of M-values-normalized log2(counts
per million) values for the 500 transcripts.

The Arabidopsis thaliana nucleotide sugar transporter
GONST2 is a functional homolog of GONST1
Background
• Nucleotide sugars are the substrate for the synthesis of plant cell
wall polysaccharides, and are synthesized primarily in the
cytosol.
• Transporters are required to move nucleotide sugars to the Golgi
lumen, when much of the polysaccharide synthesis occurs
• By controlling substrate availability, these transporters are key
control points in cell wall biosynthesis
Approach
• We determined the in planta function of the final member of the
GDP-sugar binding clade of nucleotide sugar transporters
(GONST2) by characterizing a plant lacking a functional
GONST2 gene (gonst2 mutant).
• We have shown that GONST2, like its homolog GONST1,
provides substrate specifically for GIPC (glycosylated
sphingolipid biosynthesis), and that disruption of this affects
cellulose biosynthesis
• Our work indicates the importance of GIPC glycosylation in
susceptibility to some pathogens, with gonst2 plants twice as
resistant to powdery mildew.
• We have also failed to identify a nucleotide sugar transporter for
GDP-Mannose for mannan biosynthesis, despite characterizing
all available candidates. This implies that either (a) a new class
of transporter may be involved or (b) the mannan synthases use
a mechanism similar to the cellulose synthases, where the
enzyme also translocates the substrate. This is important when
considering efforts to engineer cell walls with increased mannan.
Jing et al. (2021) Plant Direct, doi: 10.1002/pld3.309
Figure 1: Crystalline cellulose content in gonst1 and gonst2 mutants.
Figure 2: Susceptibility of gonst2-1 plants to biotrophic and necrotrophic pathogens. (A)
Golovinomyces orontii (B) Botrytis cinerea.

Biophysical analysis of the plant-specific GIPC sphingolipids
reveals multiple modes of membrane regulation
Background
• Glycosyl Inositol Phosphoryl Ceramides (GIPCs) were
discovered in plants and fungi during the 1950’s. They are
estimated to make up ~40% of the plant plasma membrane.
• They have been poorly studied due to challenges in isolation,
caused by their polar glycan headgroup.
• Loss of GIPC glycosylation is lethal, and misglycosylation affects
diverse abiotic and biotic stress processes, as well cellulose
biosynthesis
Approach
• In this multi-national project, led by CNRS France, we developed
an improved method for isolating GIPCs, and characterized their
biophysical properties. JBEI was able to contribute our glycan
analysis expertise, using methods we’d previously developed.
• We showed that GIPCs increase the thickness and
electronegativity of model membranes
• We also showed that GIPCs interact different with different
phytosterols and regulate the gel-to-fluid phase transition during
temperature variations
• These data are important for developing a revised model of the
plant plasma membrane in which the GIPCs play a central role.
• This is critical to understanding how processes such as cellulose
biosynthesis, which is synthesized by transmembrane enzyme
complexes, are regulated.
Mamode Cassism et al. (2021) JBC, doi: 10.1016/j.jbc.2021.100602
Figure 1: Structure of a GIPC
Figure 2: GIPC extraction protocol

Can Multiple Ions in an Ionic Liquid Improve the
Biomass Pretreatment Efficacy?
Background
• Pretreatment with ionic liquids (ILs) is an attractive approach to
enable efficient biomass deconstruction into intermediates for
bioconversion to fuels and chemicals
• Imidazolium-based ILs are effective pretreatment solvents but have
high cost and limited compatibility with enzymes and
microorganisms, whereas more biocompatible and economical Ils
offer lower deconstruction efficiency
• This study focuses on the development of new versatile IL-based
biomass processing approaches for both chemicals and energy
Approach
• Develop unique “double salt” ILs (DSILs, systems containing three or
more ions) by incorporating multiple ions with known distinct
pretreatment mechanisms and integrate synergistic advantages of
the individual components
• For sorghum, DSIL composed of cholinium, lysinate, and palmitate
ions not only enabled a 98% glucose yield but was also found to be
biocompatible in a one-pot configuration, producing the biofuel
precursor bisabolene using an engineered strain of the yeast
Rhodosporidium toruloides.
• For pine, pretreatment with the combination of imidazolium,
cholinium, acetate, and lysinate ions achieved 80% glucose and 70%
xylose yields at 20wt% solids loading.
• This work demonstrates a single example of the large number of
DSIL combinations that one can design to enable efficient
fractionation of biomass and downstream processing, which provides
a positive environmental and economic impact
Yao et al. (2021) ACS Sustainable Chemistry & Engineering, doi: 10.1021/acssuschemeng.0c09330
Sorghum Pretreatment
PT of Pine
One-pot on Sorghum

Background
• In California, woody biomass comprises the largest fraction of
underutilized biomass available for biofuel production
• Conversion to biofuels is challenged both by recalcitrance to
deconstruction and by organic acids and phenolic compounds
generated during pretreatment
• JBEI developed a one-pot technology that addresses these
challenges, and it has been shown to be very effective at the lab
scale
Approach
• In a collaboration with Aemetis and the ABPDU, JBEI scientists
have scaled-up an integrated one-pot process for deconstruction
and conversion of California woody biomass using the ionic
liquid (IL) cholinium lysinate [Ch][Lys] as the pretreatment
solvent
• Evaluated the impact of solid loading, solid removal, yeast
acclimatization, fermentation temperature, fermentation pH, and
nutrient supplementation on final ethanol yields and titers
• Scaled one-pot process to 680L pilot-scale fermentation,
achieving >80% deconstruction efficiency, >90% fermentation
efficiency, 27.7 g/L ethanol titer, and >80% ethanol distillation
efficiency from the IL-containing hydrolysate post fermentation
• This study is the largest scale demonstration of IL pretreatment
and biofuel conversion known to date, and the overall biomass-
to-ethanol efficiencies are the highest reported to date for any
IL-based biomass-to-biofuel conversion process
Barcelos et al. (2021) ACS Sustainable Chemistry & Engineering,
DOI: 10.1021/acssuschemeng.0c07920
High-Efficiency Conversion of Ionic Liquid Pretreated
Woody Biomass to Biofuel at the Pilot Scale

Deconstruction of Woody Biomass via Protic and
Aprotic Ionic Liquid Pretreatment
Background
• Woody biomass derived from forestry residues has the potential to be
used as a feedstock in the production of biofuels and bioproducts, and its
removal from forests can help mitigate disastrous wildfires in fire-prone
states like California
• JBEI has developed a one-pot ionic liquid (IL) technology that can
effectively liberate fermentable sugars from woody biomass and convert
them to biofuels, but needs further optimization before it is suitable for
scale-up
Approach
• This study evaluated woody biomass types (pine, almond, walnut, and fir)
from California as potential biofuel feedstocks
• The feedstocks were pretreated with the ILs cholinium lysinate ([Ch][Lys])
and ethanolamine acetate ([EOA][OAc]), followed by enzymatic
hydrolysis and fermentation of lignocellulosic sugars to produce ethanol
• Evaluated the impact of solid loading, solid removal, yeast
acclimatization, fermentation temperature, fermentation pH, and nutrient
supplementation on final ethanol yields and titers
• Under optimal conditions, [EOA][OAc] pretreatment and enzymatic
hydrolysis generated glucose and xylose yields in the range of 24−82 and
14−80%, respectively, while glucose and xylose yields for the [Ch][Lys]
ranged between 28−83 and 23−80%, respectively
• Blends of feedstocks were also explored, and a mixed blend (MB) with a
mass ratio of 2/2/1 (almond/walnut/pine) resulted in maximum glucose
and xylose (>90%) yields using [Ch][Lys]
Das et al. (2021) ACS Sustainable Chemistry & Engineering, DOI:10.1021/acssuschemeng.0c07925
Glucose and xylose yields from enzymatic hydrolysis
of MB and pine after [Ch][Lys] pretreatment
Results from batch ethanol fermentation of pine
and pine/almond/ walnut hydrolysates
pretreated with [Ch][Lys]

Functional genetics of human gut commensal
Bacteroides thetaiotaomicron reveals metabolic requirements for
growth across environments
Background
• The rapid development in metagenomics and microbiome
research have brought in promises to use microbes to
solve environmental and human health issues.
• Harnessing the beneficial outcomes of microbes,
especially newly identified ones, is limited by our lack of
understanding of the physiology of those organisms, as
the functions of most of their genes are unknown.
• RB-Tnseq is a powerful approach to annotate gene
functions in a high throughput way.
Approach
• Generate a random-barcoded transposon mutant library in
the human gut microbe, B. thetaiotaomicron.
• Large scale phenotyping of the mutant library across over
500 experiments, including different carbon sources,
stress conditions, and during mono-colonization of
gnotobiotic mice.
Outcomes and impacts
• Identification of specific phenotypes for over 500 genes in
100 different conditions.
• A 3-keto-glucoside hydrolase is important for disaccharide
utilization.
• B. thetaiotaomicron uses a tripartite multidrug resistance
system to cope with bile salts stress.
• B. thetaiotaomicron alternate biosynthetic enzymes based
on ammonium availability in the gut.
Figure 1. High-throughput functional genomic study of B.
thetaiotaomicron by RB-Tnseq approach. Phenotyping a
random-barcoded transponson mutant library of B.
thetaiotaomicron resulted in discovering novel genes related to the
function of bile salt stress resistance, disaccharide metabolism and
alternative biosynthetic pathways.
Liu, H., Shiver, A.L., Price, M.N., et al. 2021. Cell reports 34(9), p. 108789. doi: https://doi.org/10.1016/j.celrep.2021.108789

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