Summaries of the research published by the Joint BioEnergy Institute (JBEI) in September 2022

1. Office of Biological and Environmental Research
Multifunctional Landscapes for Dedicated Bioenergy Crops Lead to
Low-Carbon Market-Competitive Biofuels
Background/Objective
• Ecosystem services generated by cultivating bioenergy crops could provide
cost and carbon footprint reduction benefits to biorefineries, yet there is no
systematic studies to quantify such benefits.
• This study aimed to determine the threshold values of ecosystem services
required to reach cost parity with gasoline and a net zero-carbon footprint.
Approach
This study developed an integrated modeling approach that combines
biophysical models, ecosystem services valuation, TEA, and LCA models to
perform rigorous cost and carbon footprint analyses.
Results
Ecosystem services benefits reduced ethanol selling price and carbon footprint,
respectively, by 20-55% and 44-81%.
Significance/Impacts
The study provides generalizable insights into how ecosystem services generated
from bioenergy crops impact on the production cost and carbon footprint of
biofuels. The overall benefits are largely dependent on nitrate reduction, carbon
sequestration, and pollination services benefits.
Baral, N.R., Mishra, S.K., George, A., Gautam, S., Mishra, U., and Scown, C.D., 2022. Multifunctional Landscapes for Dedicated
Bioenergy Crops Lead to Low-Carbon Market-Competitive Biofuels . Renewable and Sustainable Energy Reviews, 169,
10.1016/j.rser.2022.112857.
In this figure, the dashed lines represent targeted cost of $0.79/L-gasoline
equivalent ($3.gge) and the dotted line represents last 10-year (2012-
2021) average gasoline price at the refinery gate of $0.54/L.
(a) Selling price of ethanol (b) Carbon footprint of ethanol

2. Office of Biological and Environmental Research
Expression of Dehydroshikimate Dehydratase in Sorghum Improves Biomass
Yield, Accumulation of Protocatechuate, and Biorefinery Economics.
Background/Objective
• Engineering bioenergy crops to accumulate coproducts in planta is
an attractive approach to increasing the value of biomass and
enabling a sustainable bioeconomy.
• Our goal was to assess the impact of accumulating protocatechuate
(DHBA) in sorghum.
Approach
We introduced a bacterial 3-dehydroshikimate dehydratase (QsuB) in
sorghum to overproduce DHBA in biomass.
We conducted techno-economic analysis (TEA) to evaluate the potential
impact on biofuel production based on the DHBA titers achieved.
Results
Field-grown engineered sorghum accumulated DHBA at 0.3 dw% and
showed increased biomass yields (up to +29%). TEA indicates that such
titer and yield increase positively impact the economics of biofuels.
Significance/Impacts
Engineered sorghum accumulates economically relevant amounts of
DHBA, which can be purified from biomass to generate extra revenue.
Tian et al., 2022. ACS Sustain. Chem. Eng. DOI: 10.1021/acssuschemeng.2c01160
Controls QsuB transgenics
Figure 1: (a) Strategy to produce DHBA from the shikimate pathway
and (b) DHBA titers obtained in field-grown engineered sorghum.
b
Figure 2: TEA results of
integrated cellulosic
biorefinery producing
DHBA as the coproduct.
MESP when producing
DHBA onsite is shown.

3. Office of Biological and Environmental Research
Heterologous Expression, Characterization, and Comparison of
Laccases from the White Rot Causing Basidiomycete Cerrena Unicolor
Background/Objective
Laccases are a crucial class of lignin-degrading enzymes but are difficult to
heterologously express and characterize. This publication describes heterologous
expression and characterization of two laccases from Cerrena unicolor.
Approach
• Selected laccases from C. unicolor with lowest calculated aggregation
propensities. Lc1 and Lc2 share ~65% sequence identity.
• Laccases were heterologously expressed in Komagataella pastoris (formerly
Pichia pastoris).
• Characterized for their stability and activity toward catalysis of breaking bonds
commonly found in lignin using JBEI’s LigNIMS assay platform (Figure 1).
Results
• Laccase activity was much higher at acidic pH and in the presence of
syringaldehyde as a mediator (Figure 2).
• Lc1 and Lc2 had half-lives of 16 min and 185 min at 60°C, respectively.
• Molecular dynamics simulations showed the longer half-life of Lc2 was due to
an increased number and persistence of salt bridges compared to Lc1.
Significance/Impacts
Cerrena unicolor is a promising source of laccases capable of catalyzing
cleavage of b-O-4 ether and C-C bonds in lignin and Lc1 and Lc2 are
currently being used in JBEI’s lignin depolymerization processes.
Mai Pham, L.T., Deng, Northen, T., Singer, S., Adams, P., Simmons, B., K., Sale, K., 2022.
Heterologous Expression, Characterization, and Comparison of Laccases from the White Rot Causing
Basidiomycete Cerrena Unicolor. Catal. Res. 02, 1–33. doi:10.21926/cr.2203028
Figure 1. Model lignin compound used to study laccase catalyzed bond
breaking using nanostructure-initiator mass spectrometry (NIMS)
Lc1
81.8%
conversion
58.0%
conversion
Lc2
pH pH
Figure 2. Profiles of Lc1 and Lc2 catalyzed bond breaking as
a function of pH in presence of the mediator syringaldehyde.

4. Office of Biological and Environmental Research
Transcriptome and metabolome integration in
sugarcane through culm development
Background/Objective
A better understanding of sugarcane growth with a finer definition of links between enzymes, metabolites, and metabolic
pathways through development is required to support selection in improved breeding programs for yield, fibre quality,
sugar, chemical bioproduct, and plant synthetic biology
Approach
Integration of two large datasets, metabolomics and
transcriptomics, from a sample collection of 1440 internodes
across 24 sugarcane cultivars through five different
developmental stages
Results
Study revealed a global view of essential metabolic pathways
identifying critical genes involved in carbon partitioning
during different stages of development
Significance/Impacts
Enables opportunities in synthetic biology and metabolic
engineering to facilitate the optimized production of biofuels
and a broad range of (high-value) biomaterials
Food Energy Security 2022, e421, doi: 10.1002/fes3.421

5. Office of Biological and Environmental Research
Renewable Schiff-Base Ionic Liquids for
Lignocellulosic Biomass Pretreatment
Background/Objective
The application of ionic liquids (ILs) technologies for sustainable processing of biomass to meet
the large quantities of biofuels, demands a reliable and renewable source rather than
rapidly depleting fossil resources for IL productions
Approach
Utilize underutilized lignin component to obtain
renewable ILs
Results
The fully protonated Schiff-Base ILs (green
boxes) were most effective in affording up to
87% glucose and 76% xylose release
Significance/Impacts
Demonstrate the ease of synthesis of renewable
ILs designed to be task specific including
biomass pretreatment, enabling an overall lower
environmental and economic impact.
Molecules 2022, 27, 6278, doi: 10.3390/molecules27196278

6. Office of Biological and Environmental Research
Using High Throughput ‘Omics Data to Guide
Strain Engineering in Pseudomonas putida
Background/Objective
For high performing engineered strains (>40% MTY), different
engineering strategies are required to realize additional product
titer, rate, or yield gains.
Approach
Using 13C metabolic flux analysis, metabolomic data, and
existing RNAseq datasets, further characterize the behavior
of P. putida strains that already produce 45% MTY of
indigoidine from glucose.
Results
This approach tested new strategies to further improve
indigoidine rate and titer by identifying competing
metabolic reactions, thus enabling greater flux to the
desired final molecule.
Significance/Impacts
This is the first characterization of a multiplex
CRISPRi/dCpf1 strain with 13C MFA and demonstrates
the distinct analysis required for other highly edited
strains.
Czajka et al, Metabolic Engineering Communications, September 13, 2022.
doi.org/10.1016/j.mec.2022.e00206

7. Office of Biological and Environmental Research
Engineering sorghum for higher 4-hydroxybenzoic
acid content
Background/Objective
• Engineering crops to accumulate coproducts in planta can increase the
value of biomass and enable a sustainable bioeconomy.
• A technoeconomic analysis previously indicated that accumulating the
valuable compound 4-hydroxybenzoic acid (4-HBA) in sorghum could
improves the economics of biofuels.
Approach
We introduced bacterial chorismate pyruvate-lyase (UbiC) and 3-deoxy-D-
arabino-heptulonate-7-phosphate synthase (AroG) in sorghum to
overproduce 4-HBA (Figure 1).
Results
Field-grown engineered sorghum accumulated 4-HBA in biomass (up to
1.2 dw%) without major impact on biomass yields (Figure 2).
Significance/Impacts
Engineered sorghum accumulates economically relevant amounts of 4-HBA,
which can be purified from biomass to generate extra revenue.
Lin et al., 2022, Metab. Eng. Commun. DOI: 10.1016/j.mec.2022.e00207
Figure 1: Strategy to produce 4-HBA from the
shikimate pathway in sorghum.
Figure 2: (a) 4-HBA titers and (b) biomass yields from two
field-grown engineered sorghum lines (Eng-1 and Eng-2)
compared to wild-type (WT) controls.

8. Office of Biological and Environmental Research
In Situ Synthesis of Protic Ionic Liquids (PILs) for Biomass Pretreatment
Background/Objective
• The cost of many ionic liquids remain prohibitively high for their use in biomass
deconstruction thus limiting process scalability and commercialization.
• This study presents a simple and effective method for producing PILs “in situ”, which
promotes improved economic and environmental benefits.
Approach
In situ synthesis of ionic liquids was demonstrated by mixing the individual components
of the PIL ethanolamine acetate ([Eth][OAc]) with lignocellulosic biomass immediately
prior to biomass pretreatment. Techno-economic and life-cycle analyses were employed
to assess the impact of deploying this technology.
Results
The pretreatment and enzymatic hydrolysis of biomass with the in situ-synthesized
[Eth][OAc] was found to have equal efficiency compared to using pre-synthesized
IL in terms of sugar yields.
Significance/Impacts
Techno-economic analysis demonstrated a cost reduction of up to $2.9/kg for the in situ
produced PIL. Life-cycle assessment also shows the in situ method generates up to 59%
less GHG emissions per kilogram of PIL.
https://doi.org/10.1021/acssuschemeng.2c01211
The in situ PIL synthesis method involving simultaneous PIL synthesis/biomass
pretreatment that eliminates the use of solvents, cooling water, and downstream
purification
Glucose, xylose, and arabinose yields recovered after enzymatic hydrolysis of
the pretreated biomass for varying pretreatment solvents.
Production cost ($/kg) of the protic ionic liquid, [Eth][OAc] (a) and carbon
footprint (kg CO2e/kg) of the protic ionic liquid, [Eth][OAc] (b) under different
synthesis processes

9. Office of Biological and Environmental Research
JBEI Enabled

10. Office of Biological and Environmental Research
Over-expression of the Global Regulator
LaeA Improves Itaconic Acid Yield
Pomraning K.R., Dai Z., Munoz N., Kim Y.M., Gao Y., Deng S., Lemmon T., Swita M.S., Zucker J.D., Kim J., Mondo S.J., Panisko
E., Burnet M.C., Webb-Robertson B.M., Hofstad B., Baker S.E., Burnum-Johnson K.E., Magnuson J.K. (2022) “Itaconic acid
production is regulated by LaeA in Aspergillus pseudoterreus.” Metab Eng Commun. doi: 10.1016/j.mec.2022.e00203
Background/Objective
• Itaconic acid is a secondary metabolite produced industrially by Aspergillus
pseudoterreus
• LaeA is a known global regulator of secondary metabolite clusters and other
metabolic activities
• Phosphate limitation is a trigger of itaconic acid production in the native strain
Approach
• LaeA was over-expressed and deleted in an itaconic acid producing strain of A.
pseudoterreus
• Multi-omics and substrate/product/cell mass data was collected and analyzed
Results
• Over-expression of LaeA increased itaconic acid yield due to less of the mock
lignocellulosic substrate (glucose/xylose) being diverted to cell mass
• Physiologically, this effect was due to increased expression of key biosynthetic
pathways and decreased phosphate scavenging and acquisition genes
Significance/Impacts
• Lignocellulosic feedstocks are often rich in nutrients, like PO4, that can lead to
biomass accumulation at the expense of productivity of the desired biofuel or
bioproduct global regulators of metabolism, like LaeA may be useful targets for
engineering strains that are robust to bioprocess heterogeneity

11. Office of Biological and Environmental Research
Whole-genome sequencing-based characteristics
of Escherichia coli Rize-53 isolate from Turkey
Background/Objective
Urinary tract infections (UTIs) are one of the most common infectious diseases
identifying genetic determinants of antibiotic resistance in a clinical isolate of UTI-causing Escherichia coli.
Approach
The clinical isolate was obtained from a urine sample of a UTI patient in Turkey and identified via 16S rDNA sequencing
Results
Ten antibiotic resistance genes were identified in the genome of the isolate: serotype of the isolate were identified
as ST2141 and O107/H39, respectively
Significance/Impacts
Genetic determinants of resistance to tetracycline, β-lactam and aminoglycoside antibiotics were identified using
WGS in a uropathogenic E. coli from ST2141 lineage and O107:H39 serotype, isolated in Turkey.
Turumtay (2022) doi: 10.17219/acem/152704

12. Office of Biological and Environmental Research
Background/Objective
• X-ray footprinting and mass spectrometry (XFMS) is a method that utilizes high flux
density X-ray beams for in situ hydroxyl radical labeling to map protein interactions
and confirmation.
• Conventional sample delivery systems face great challenges improving hydroxyl
radical labeling efficiency and data measurement workflows with restricted XFMS
sample analysis throughput.
Approach
• Implement a container-free liquid jet sample delivery system at high flux density X-
ray beam.
• Develop an automated inline fluorescence X-ray dose analysis system
Results
• Together, the technologies significantly advance the throughput and opens new
directions for XFMS experiments
Significance/Impacts
• This method greatly reduces the sample exposure times to as short as one microsecond.
• Enables the use of high hydroxyl radical scavenging buffers in XFMS analysis.
• Enables more accurate/rapid dosage response analysis after protein irradiation
Rosi, et al., Commun Biol . doi: 10.1038/s42003-022-03775-1
An automated liquid jet for fluorescence dosimetry
and microsecond radiolytic labeling of proteins
Fig 1. Instrument design for automated liquid-jet
fluorescence dosimetry and sample exposure.
Components–beam alignment module (BAM), liquid jet
module (LJM), fluorescence imaging module (FIM), laser
assisted pre-alignment unit (LAPU), photomultiplier tube
(PMT), and data acquisition (DAQ). Intact mass analysis
of exposed CytC protein sample using the developed
automation system shows a significant amount of
modification within 10 μs.