Research summaries for papers published by the Joint BioEnergy Institute in October 2021

1. Progress and challenges in sorghum bio-
technology, a multi-purpose feedstock for the bioeconomy
Background
• Sorghum is the fifth most important cereal crop
globally by harvested area and production. Its drought
and heat tolerance allow high yields with minimal
input.
• It is a promising biomass crop for the production of
biofuels and bioproducts, particularly in the US.
• As an annual diploid, it’s also an excellent C4 model
system.
Approach
• Here we provide an overview of sorghum, particularly
aimed at plant researchers who may be considering
working with it.
• We review what makes sorghum such a successful
crop and identify some key traits for future
improvement.
• We assess recent progress in sorghum transformation
and highlight how transformation limitations still
restrict its widespread adoption.
• Finally, we summarize available sorghum genetic,
genomic, and bioinformatics resources.
Outcomes and Impacts
• Assist researchers who wish to work with one of
DOE’s flagship biomass species
• Identify current bottlenecks, and suggest topics for
future focused research
Silva et al. (2021) Journal of Experimental Botany, doi: 0.1093/jxb/erab450
Collaboration between Feedstocks, LEAD
Collaboration with BER project “Sorghum
Metabolic Atlas”
Figure 1. Representation of transformation methods
adopted for sorghum.

2. Background
• Enzymes are excellent catalysts, but the reactions catalyzed by
enzymes are limited, especially when compared to that by catalysts
in chemistry.
• Many artificial metalloenzymes (ArMs) have been created and
engineered for catalyzing many abiological reactions in past
decades.
• Introduction of ArMs and abiological reactions into microbes will
significantly expand the biosynthesis ability to produce new
compounds.
Approach
• We engineered the Escherichia coli (E. coli) to efficiently uptake the
artificial cofactor Ir(Me)MPIX and assemble it with a protein scaffold
to create ArMs in vivo. The ArMs were further interfaced with a
natural biosynthesis pathway to produce unnatural products.
Outcomes and Impacts
• This study demonstrates the feasibility of integration of ArMs into
natural biosynthesis pathways for producing new products, which
opens a new door for combination of synthetic chemistry and
synthetic biology to generate new valuable compounds.
• Creation of the ArMs in vivo and screening mutants with E. coli
whole cells benefits the directed evolution of ArMs by circumvention
of laborious protein purification or cell lysis.
• The ArM was evolved with the new method to be with high
diastereoselectivity by directed evolution.
Huang and Liu et al. (2021) Nature Chemistry, doi: 10.1038/s41557-021-00801-3
O
E. coli cells
M9-N, pH 7.4
N2 CO2Et
O
H
CO2Et
(A) Schematic diagram for the design. (B) Creation of ArMs in
vivo indicated by the selectivity. (C) Integration of ArMs into
a natural biosynthesis pathway to generate new products.
A
C
B
Outer membrane
Inner membrane
Periplasm
apo-CYP119 Ir-CYP119
Cytoplasm
Glucose R
Natural pathway Terpene Unnatural terpenoid
CO2R’
R
Ir-CYP119
Ir(Me)MPIX
N2 CO2R’
N2 CO2Et CO2Et
*
*
Ir-CYP119
Glucose
Introducing an Artificial Metalloenzyme into
Escherichia coli for Biosynthesis of Unnatural Products

3. Alkanolamines as Dual Functional Solvents for
Biomass Deconstruction and Bioenergy Production
Achinivu et.al (2021) Green Chemistry, doi: 10.1039/D1GC02667D
Complete conversion of waste biomass (such as sorghum) to
bioproducts (lignin and bisabolene) using an alkanolamines
Background
• This work demonstrates the feasibility of applying dual
functional solvents called alkanolamines towards the
conversion of biomass into biofuels.
• Alkanolamines have the potential to be highly effective at
biomass pretreatment while demonstrating biocompatibility;
this promotes low energy intensity, process intensification,
process consolidation and downstream conversion.
Approach
• Several key factors were considered to effectively integrate
the pretreatment technology into a biorefinery, including
solvent screening, feedstock screening, fractionation of
lignocellulose components (lignin removal), process scale up,
bioconversion efficacy, and sustainability impact assessment.
Outcomes and Impacts
• After optimization, the following conditions emerged for
optimal process economics: pretreatment of sorghum
biomass via ethanolamine (25% solvent in water) with
temperature 100 °C, time 1h and solid loading 40%.These
conditions generated yields of 90% glucose, 76% xylose,
59% lignin removal, and 73% solid recovery.
• Subsequently the process was consolidated and scaled up
(40X) to generate >99% sugar yields followed by a test of
bioconversion using the omnivorous host R. toruloides, which
converted >97% C5 and C6 sugars and phenolic into the
biofuel precursor bisabolene at a titer of 1155 mg/L.
• Economic assessment revealed that the cost of biomass
deconstruction was severely reduced (up to 50%) compared
to similar pretreatment methods, including ethanolamine
acetate and cholinium lysinate.
(A) Lignin removal, solid recovery after biomass pretreatment with
alkanolamines and (B) glucose and xylose yields recovered after
enzymatic hydrolysis of the sorghum recovered after pretreatment
with alkanolamines.

4. Non-destructive quantification of anaerobic gut
fungi and methanogens in co-culture reveals increased fungal
growth rate and changes in metabolic flux relative to mono-culture
Background
• Quantification of individual species in microbial co-cultures
and consortia is critical to understanding and designing
communities with prescribed functions. However, it is difficult
to physically separate species or measure species-specific
attributes in most multi-species systems.
• Decades of literature suggest that anaerobic gut fungal
(AGF) polysaccharide degradation and growth are
accelerated in co-culture with methanogens; however,
methods have not been available to measure concentrations
of species in co-culture.
Approach
• We present a simple, microplate-based method to measure
AGF and methanogen concentrations in co-culture based on
fluorescence and absorbance spectroscopies, enabling
calculation of growth rates and metabolic fluxes.
Outcomes and Impacts
• AGF growth rate and xylan and glucose degradation rates
were significantly greater in co-culture with methanogens
relative to mono-culture.
• AGF metabolic fluxes in co-culture differed from mono-
culture and showed increased flux through the energy-
generating hydrogenosome organelle; however
hydrogenosomal fluxes differed from widely-accepted
models.
• This method enables quantitative testing of hypotheses
related to biotechnologically promising AGF + prokaryote co-
cultures. Simple modifications to the method can extend it to
other co-cultures with different organisms and/or more than
two species.
The non-rhizoidal morphology of the AGF, Caecomyces
churrovis, enables uniform sampling of < 2% of the culture
volume for quantification of AGF and methanogen
concentrations and fluxes.
Leggieri et al. (2021) Microbial Cell Factories. doi: 10.1186/s12934-021-01684-2

5. Lepidopteran mevalonate pathway optimization
in Escherichia coli efficiently produces isoprenol
analogs for next-generation biofuels
Background
• The lepidopteran mevalonate (LMVA) pathway produces six-carbon
analogs of terpene building blocks
• These terpene building block analogs are potentially incorporated
into complex terpenes to introduce structural modifications
• The hydrolyzed products of these terpene building block analogs
are potential next-generation biofuels
• This study optimizes the lepidopteran mevalonate (LMVA) pathway
in E. coli towards high productivity
Approach
• The LMVA pathway was linked to NudB, a promiscuous
phosphatase, to produce a six-carbon analog of isoprenol (C6-
isoprenol) for GC analysis
• The LMVA pathway is redirected to start from β-oxidation (BOX) in
E. coli to transform valeric acid into C6-isoprenol
• Substrate promiscuity of the BOX-LMVA was studied
Outcomes and Impacts
• With NudB, the BOX-LMVA pathway transforms valeric acid into
390 mg/L C6-isoprenol
• The BOX-LMVA-NudB pathway also converts butanoic acid and
hexanoic acid into isoprenol and C7-isoprenol, suggesting the
BOX-LMVA pathway produces IPP and C7-IPP from the
corresponding fatty acids
• The longer chain isoprenol analogs have lower water solubilities,
similar or higher energy densities, and comparable research
octane number (RON) boosting effects to isopentenols
Pang et al. (2021) Metabolic Engineering, doi:10.1016/j.ymben.2021.10.007
The natural lepidopteran mevalonate (LMVA) pathway
depends on a thiolase to produce the key intermediate, 3-
ketovaleryl-CoA, while the pathway here employs beta-
oxidation enzymes to afford this intermediate and the analogs
thereof, to increase the productivity of the whole pathway.

6. Background
• Greenhouse gas emissions reduction, carbon sequestration, and
environmental remediation are beneficial sustainability goals for
agroecosystems and have been emphasized as research priorities
within DOE-funded bioenergy research centers (BRCs).
• BRCs are tasked with generating data to support feedstock
selection, identifying and understanding impacts of land choice for
feedstock cultivation, and developing management strategies for
bioenergy systems.
Approach
• Researchers from across the four BRCs engaged in a microbiome
workshop that focused on identifying challenges and collaboration
opportunities to better understand bioenergy-relevant plant–
microbe interactions.
Outcomes and Impacts
• Key findings of the workshop were the need to prioritize scaling
data sharing across BRCs and the broader research community
and securing collaborative infrastructure in the areas of
microbiome-ecosystem modeling and molecular plant-microbe
interactions.
• Thinking systematically, collaboratively, and interactively will best
leverage BRC research expertise and capabilities to tackle
bioenergy and sustainability challenges.
• Investments in the proposed key priorities should also provide
data, tools, and models that can be leveraged by other researchers
in the field.
Howe et al. (2021) Phytobiomes Journal, doi: 10.1094/PBIOMES-05-21-0033-MR
Summary of objectives in the production of sustainable
bioenergy feedstocks
Frontiers and opportunities in bioenergy crop
microbiome research networks

7. JBEI-Enabled Publications

8. The power of poop: Defecation behaviors and social
hygiene in insects (A review)
Background
• Although the main purpose of defecation is waste
elimination, it has also had a profound influence on the
evolution of animal behavior and ecological interactions.
• Due to the capacity of fecal material to support the growth
of microbes, insects have evolved a range of unique
behavioral and physiological adaptations, often involving
microbial symbionts and the production of secondary
metabolites to combat potential pathogens.
• Many examples are derived from wood-feeding insects
that rely on their complex microbiome to degrade
lignocellulose that may be also serve as building block for
relevant secondary metabolites including antibiotics.
Outcomes and Impacts
• Studying the composition and functional potential of fecal
microbiomes, especially from wood-feeding insects, holds
considerable promise to better our understanding of the
biology of insects, but also to the isolation of microbial
strains with novel bioactive capabilities.
Cole et al. (2021) PLOS Pathogens, doi: 10.1371/journal.ppat.1009964
The presence of antimicrobial compounds produced by
microbes in insect feces appears to have enabled the
evolution of behaviors in insects that require an intimate
contact with feces, such as its use in nest building, the use
of fecal shields to avert predators.

9. Background
• Nutrient recovery will be an important part of the future bioeconomy
to avoid harmful levels of nutrient releases to water bodies and
continued use of fossil fuels for production of nitrogenous fertilizers
• Technological options exist for recovery of nitrogen and
phosphorous from liquid waste streams, including struvite
precipitation, ammonia stripping, and thermal distillation
Approach
• Using organic waste availability and waste-to-energy conversion
scenarios for California, this study evaluates the fraction of in-state
fertilizer use that could be met by recovering nitrogen and
phosphorous from wet organic waste processing facilities
• Approach developed here can serve as the foundation of national-
level analyses for bioenergy production that account for nutrient
flows and potential recovery (as an additional important component
of any GHG mitigation strategy)
Outcomes and Impacts
• Findings suggest that nutrient recovery at wet organic waste
processing facilities could offset 11% of nitrogen and 29%
of phosphorous in synthetic fertilizer currently used in California.
• The recovered nutrients in this analysis are present as three
different end products: liquid fertilizer (38% of TN), struvite (50% of
TN, 66% of TP), and compost (12% of TN, 34% of TP)
• The recovery potential as a fraction of in-state use in other states
across the U.S. is likely to higher than the reported values here,
particularly if those states are not major agricultural producers
Orner et al. (2021) Water Resources, doi: 10.1016/j.watres.2021.117717
Mass (Mg/d) of Total Nitrogen in 2020 (A), Total Phosphorus
in 2020 (B), Total Nitrogen in 2050 (C), and Total Phosphorus
in 2050 (D) from five organic waste streams through anaerobic
digestion, separation, and nutrient recovery in the baseline
high-market scenario. Abbreviations include MSS (Municipal
Sewage Sludge), AM (Animal Manure), AC (Agricultural Crops),
AF (Agri-Food Process Residues), OMSW (Organic Municipal
Solid Waste), and AD (Anaerobic Digestion).
Fertilizer demand and potential supply through
nutrient recovery from organic waste digestate in California