May 2023 - Summary slides of Joint BioEnergy Institute (JBEI) research on biofuels and bioproducts.

1. Office of Biological and Environmental Research
Field performance of switchgrass plants
engineered for reduced recalcitrance
Background
• Switchgrass is an important potential bioenergy crop
• Biomass is recalcitrant to deconstruction
Approach
Switchgrass was engineered to reduce recalcitrance by expressing
dehydroshikimate reductase (QsuB) to reduce lignin or AT10 acyltransferase to
reduce cell wall crosslinking. These plants had been described before in
greenhouse studies and were now tested in multi-year field trials.
Results
Reduced recalcitrance was observed in engineered plants, but the effect was
smaller than in greenhouse studies. The plants expressing QsuB showed an
increased yield of about 16% over the three years. A similar increase was
previously observed in sorghum plants expressing QsuB,.
Significance/Impacts
The study shows the importance of testing engineered plants in the field as
results from greenhouse studies often do not translate to the field. The increase
in biomass yield is important and together with reduced recalcitrance would
lead to a lower biofuel production cost.
Eudes A., et al. Front Plant Sci. DOI: 10.3389/fpls.2023.1181035
Figure 1: Biomass yield in the first two years of trial. Three QsuB and
two AT10 (FT) lines were tested. The error bars indicate standard
deviations for the total yearly yield. Total biomass yield in each year with
the same letter are not significantly different (Fisher’s least significance
test, p > 0.05). The experimental plot design was a randomized complete
block design with four replicates for each transgenic line and controls.

2. Office of Biological and Environmental Research
pGinger family of expression plasmids
Background/Objective
Metabolic engineering and synthetic biology are predicated on the
precise control of gene expression.
Approach
The pGinger family of plasmids is constitutes that will enable
both constitutive and inducible gene expression in a wide
range of non-model Proteobacteria.
Results
The pGinger suite of expression plasmids comprises 43 plasmids
that will enable precise constitutive and inducible gene expression
in a wide range of gram-negative bacterial species.
Significance/Impacts
As synthetic biology expands beyond model organisms more
tools will be required that function robustly in a wide range of
bacterial hosts.
Pearson A.N., et al. Microbiology Spectrum. DOI:10.1128/spectrum.00373-23

3. Office of Biological and Environmental Research
Background/Objective
• Biomanufacturing is important for sustainable development
• Lack of many reactions limits the power of biomanufacturing
• Bringing unnatural reactions into microbes opens new possibilities
Approach
Genes for substrates biosynthesis and engineered enzyme were combined
in the microbe to produce unnatural products through carbene reactions.
Results
• BGC for azaserine was identified and heterologously expressed
• Carbene reactions were completely integrated into biosynthesis
Significance/Impacts
This study established a microbial platform for introducing unnatural
carbene reactions and paved the way for application of carbene reactions
in potential industrial scale bioproduction.
Huang, J., et al. Nature. DOI: 10.1038/s41586-023-06027-2
(a) Some selected natural diazo compounds. (b) Biosynthesis of carbene
precursor azaserine in S. albus. (c) Biosynthesis of unnatural
cyclopropanes by an abiological carbene-transfer reaction
Complete integration of carbene-transfer
chemistry into biosynthesis
a
c
b
Fig. 2c
Retention Time (min)
3 3.5 4 4.5 5 5.5 6 6.5 7 7.5
Gene
cluster
TSB
TSB
1B
1B
Medium
+
-
+
-
Azaserine

4. Office of Biological and Environmental Research
Solvent-Assisted Poly(lactic acid) Upcycling under Mild
Conditions
Background/Objective
• A circular bioeconomy requires that bio-based chemicals and materials have
viable paths to being recovered and recycled.
• Ionic liquids can be used as a catalyst for recycling bio-based polymers.
Approach
The study combines experimental work with process design and simulation to
identify strategies for recycling poly(lactic acid) in mixed waste streams from
material recovery facilities (containing PLA).
The result is a novel process that produces lactides at mild conditions with a low
GHG footprint and high lactide yields.
Results
Significance/Impacts
The study suggests that ionic liquids can play an important role not only in
biorefineries to pretreat biomass, but also in the process of recovering and
recycling biomaterials to reduce plastic waste and improve circularity.
Hubble, D., et al. ACS Sustainable Chemistry & Engineering. DOI: 10.1021/acssuschemeng.2c06500

5. Office of Biological and Environmental Research
Engineering Rhodosporidium toruloides for production
of 3-hydroxypropionic acid from lignocellulosic
hydrolysate
Background/Objective
• 3-hydroxypropionic acid (3HP) is one of the top 12 US DOE’s platform chemicals, yet its bulk production
through chemical synthesis is limited by high costs and environmental impacts
• Objective of this study is to engineer R. toruloides for production of 3HP from lignocellulosic hydrolysate
Approach
• Metabolic engineering to introduce a 3HP producing pathway into R. toruloides
• Identified and deleted genes encoding 3HP degradation
• Identified and expressed genes promoting 3HP export
• Optimized process conditions for production of 3HP in lignocellulosic hydrolysate
Results
Strain engineering and process optimization led to a 3HP titer of 45.4 g/L in a fed-batch bioreactor
Significance/Impacts
The study establishes R. toruloides as a host for 3HP production from lignocellulosic
hydrolysate at high titers, and paves the way for further optimization towards enabling industrial
production of 3HP in the future. The work was supported by the BETO Agile Biofoundry and
JBEI team members advised and assisted on optimizing fermentation conditions.
Liu, D., et al. Metabolic Engineering. DOI 10.1016/j.ymben.2023.05.001

6. Office of Biological and Environmental Research
Discovery of the azaserine biosynthetic pathway uncovers
a biological route for α-diazoester production
Background/Objective
• Diazo-containing compounds have wide applications
• Biosynthetic mechanism for α-diazoester is still unknown
Results
• Hydrazonoacetic acid involves in diazo group formation
• BGC for azaserine were identified in several organisms
Significance/Impacts
Illuminating the biosynthetic route for diazo compounds will enable
people to generate more diversified diazo-containing molecules for
further applications
Van Cura, D., et al. Angew. Chem. Int. Ed. DOI: 10.1002/anie.202304646
(a) Some selected natural diazo compounds and α-diazoester. (b) Azaserine-producing
organisms encode a conserved gene cluster containing homologues of the HYAA-
cassette. (c) Proposed biosynthetic pathway for azaserine
a
b
c
Approach
By bioinformatic and biochemical methods, enzymes in the pathway
were identified and characterized for uncovering the new mechanism

7. Office of Biological and Environmental Research
JBEI Enabled Publications

8. Office of Biological and Environmental Research
A High-Quality Genome-Scale Model for
Rhodococcus opacus
Background
Rhodococcus opacus PD630 (hereafter, R. opacus) is a Gram-positive
aerobic bacterium known for its pronounced ability to produce
triacylglycerol, a biofuel precursor, from aromatic monomers.
Approach
• Here, we present iGR1773, the first genome-scale model (GSM) of R.
opacus PD630 metabolism based on its genomic sequence and
associated data.
• We predict growth rates and fluxes from transcriptomics data by
combining the model with two COBRA methods: E-Flux2 and SPOT
Significance/Impacts
iGR1773 can help the metabolic engineering community predict aromatic
substrate utilization patterns and perform computational strain design.
Roell, Garrett W., et al. ACS Synthetic Biology. https://doi.org/10.1021/acssynbio.2c00618
Figure 1: A draft version of the model was created through CarveMe, which
was then augmented with relevant uptake and biomass reactions and then
manually curated.
Results/Recommendations
• Growth rates are best predicted by E-Flux2.
• Flux profiles are more accurately predicted by E-Flux2 than flux balance
analysis (FBA) and parsimonious FBA (pFBA), when compared to 44
central carbon fluxes measured by 13C-Metabolic Flux Analysis
Figure 2: Accurate predictions of
fluxes from transcriptomics data by
E-flux2. The first R2 value does not
include ATP maintenance reaction
and the R2 value in parentheses
includes the ATP maintenance
reaction.

9. Office of Biological and Environmental Research
Background/Objective
• How organic carbon is formed and preserved in the soil has been
debated for over a century and remains controversial.
• Examine the relationship between carbon use efficiency (CUE) and
preservation of soil organic carbon (SOC), and interactions with
environmental factors.
Approach
We applied microbial explicit model, data assimilation, deep learning
and meta-analysis on 57,267 global soil profiles to investigate the
role of CUE on SOC preservation.
Results
CUE is four times as important as carbon input, decomposition or vertical
transport in determining the global SOC storage and it’s spatial variation.
Significance/Impacts
Understanding the microbial processes underlying CUE and their
environmental dependence may reduce the uncertainties in prediction of SOC
feedback to a changing climate.
Tao, F., Y. et al., Nature, https://doi.org/10.1038/s41586-023-06042-3
Figure 1: Microbial CUE favours accumulation of SOC storage
Figure 2: Global SOC stock is most sensitive to changes of microbial carbon
use efficiency
Microbial carbon use efficiency promotes global soil
carbon storage

10. Office of Biological and Environmental Research
Impacts of cover crops on soil organic carbon
in US Midwestern agroecosystems
Background/Objective
• Impacts of cover crops on soil organic carbon (SOC) are soil and
site-specific.
• Assess the impacts of winter cover crops on SOC sequestration
under different environment and management conditions.
Approach
We combined observations from field experiments with ecosys
process-based agroecosystem model to project the cover crop yields
and SOC sequestration rates across six sites in Illinois.
Results
• Cover crops can sequester SOC by 0.33 ± 0.06 MgC ha−1 year−1, and SOC
benefits are specific to legume and non-legume cover crops.
• SOC benefits from cover crops can be maximized by selecting cover crop
types and controlling growth period in US Midwest.
Significance/Impacts
This study provides practical tools and insights for practitioners and policy-
makers to design effective cover crop policies and programs.
Qin, Z., et al. Global Change Biology, doi: 10.1111/gcb.16632.
Figure 1: Ecosys model simulated and field measured average SOC
change in 0 - 0.75 m depth interval
Figure 2: Processes involved in SOC change in the cover crop
agroecosystems