Lay highlights of JBEI research papers published in August 2021

1. Enzyme for Accumulating a High-Value
Bioproduct in Plant Feedstocks
Background
With functional genomics approach and CRISPR/Cas9-mediated gene
editing, an enzyme was identified and characterized from bioenergy
crop poplar, which catalyzes the conjugation of p-hydroxybenzoate
(pHBA) to the lignin monomer, leading to the accumulation of pHBA in
woody biomass and the alteration of lignin physiochemical properties.
Approach
• A set BAHD acyltransferases expressed in woody tissues of poplar
were identified.
• In vitro functional screening with different thio-ester donors identified
a specific monolignol modification acyltransferase of interest
• CRISPR/Cas9 gene editing and overexpression provided conclusive
genetic evidence of the in-planta function of the identified enzyme
• Analytical chemical analysis and NMR studies of the cell wall verified
the enzyme-mediated p-hydroxybenzoylation of lignin
Outcomes and Impacts
While it has long been known that lignin is naturally decorated with
pHBA, a versatile chemical feedstock, the molecular basis for its
conjugation to lignin was missing. This study fills that knowledge gap and
furthers our understanding of lignin structural modification and properties.
Genetically manipulating the enzyme controlling lignin p-
hydroxybenzoylation could form the basis for accumulating this pHBA in
lignocellulosic biomass, thereby improving biofuel production, timber
durability.
Zhao Y, Yu X, Lam P-Y, Zhang K, Tobimatsu Y, and Liu C-J (2021) Monolignol acyltransferase for lignin
p-hydroxybenzoylation in Populus. Nature Plants (in press)
DOI: 10.1038/s41477-021-00975-1
The BAHD family acyltransferase member,
PtrPHBMT1, was identified to transfer p-
hydoxybenzoate from its thio-ester donor to the
sinapyl alcohol monolignol. The resulting
conjugate acts as normal lignin precursor and is
incorporated into the nascent lignin polymer,
leading to the ester-linked p-hydoxybenzoate
pendant group
O
OCH3
OH
H3CO
O
OH
OH
O SCoA
OH
OCH3
OH
H3CO
PHBMT1
O
O
O
H

2. Ionic liquid-water mixtures enhance pretreatment and
anaerobic digestion of agave bagasse
Background
• Agave bagasse (AB) is an agricultural waste generated in large
quantities and an emerging alternative for production of biofuels
bioproducts
• Although there are several pretreatment types, using certain ionic
liquids (IL) has become attractive due to its ability to dissolve the
lignocellulosic biomass under mild conditions of time and temperature
Approach
• This work aimed to evaluate the effect of pretreatment with three
diluted ILs on the A. tequilana bagasse structure and sugar yields, as
well as the potential of the enzymatic hydrolysate to produce methane
in batch mode
• Evaluated the ILs 1-ethyl-3-methylimidazolium acetate [Emim][OAc],
choline lysinate [Ch][Lys] and ethanolamine acetate [EOA][OAc]
• Monitor sugar yields and develop mass-energy balances for each
biomass-IL combination
Outcomes and Impacts
• The results showed that the IL with the best performance was
[Ch][Lys], which not only offered the highest yields of sugar production
(0.57 ± 0.03 g total sugars / g bagasse) but that it is possible to use it
at 30% aqueous solution
• Enzymatic hydrolysis of IL pretreated AB achieved 50.7 kg sugars and
49.3 kg of sugars for 90%-[Ch][Lys] and 30%-[Ch][Lys], respectively,
per 100 kg of untreated biomass
• The enzymatic hydrolysate from the 30 %-[Ch][Lys] pretreated AB
was able to achieve 0.28 L CH4/g CODfed
Perez-Pimienta et al. (2021) Industrial Crops & Products, doi: 10.1016/j.indcrop.2021.113924
Effect of agave bagasse pretreatment with IL-water mixtures
on solids recovery (top), sugar production (middle) and sugar
yield (bottom). Error bars represent the standard deviation.

3. A genomic catalog of stress response genes in
anaerobic fungi for applications in bioproduction
Background
• Anaerobic fungi are non-model fungi capable of producing a wide
array of carbohydrate active enzymes (CAZymes)
• In order to develop these organisms as a biotechnology platform to
produce CAZymes and other bioproducts, it is important to
characterize native stress pathways such as the unfolded protein
response (UPR) and heat shock response (HSR) that may be
triggered by bioproduction.
Approach
• High-quality genomes of four strains of anaerobic fungi were mined
for the components of the unfolded protein response and heat
shock repsonse.
• The transcriptomes of thermally stressed anaerobic fungi were
sequenced and used to identify transcripts of heat shock proteins.
Outcomes and Impacts
• Key genes in the unfolded protein response and heat shock
response were identified in the genomes of anaerobic fungi.
• Anaerobic fungi mostly transcribe low molecular weight heat shock
proteins (HSPs) that may help stabilize an asparagine-rich
proteome.
• Future engineering efforts can focus on alleviating these stress
responses during bioproduction using strategies similar to those
that have proven successful in model fungi such as yeasts.
Swift et al. (2021) Frontiers in Fungal Biology, doi: 10.3389/ffunb.2021.708358
The genomes of anaerobic fungi encode a greater percentage
of heat shock proteins compared to most other fungi,
including those commonly used in bioprocessing.

4. Suppression of miR168 improves yield,
flowering time and immunity
Background
• MicroRNA168 (miR168) is a key miRNA that targets Argonaute1
(AGO1), a major component of the RNA-induced silencing
complex.
• miR168 expression is responsive to fungal infection.
• How miR168 regulates immunity to infection and whether it
affects plant development was previously unknown.
Approach
• We found that the suppression of miR168 by a target mimic
(MIM168) improves grain yield, shortens flowering time and
enhances immunity to fungal infection.
• These results were validated through repeated tests in rice fields
in the absence and presence of the fungus.
• The miR168–AGO1 module regulates miR535 to improve yield
by increasing panicle number, miR164 to reduce flowering time,
and miR1320 and miR164 to enhance immunity.
Outcomes and Impacts
• Our discovery demonstrates that changes in a single miRNA
enhance the expression of multiple agronomically important traits.
• Because miR168 is widely conserved in diverse plant species,
including bioenergy crops, these results provide a strategy for
engineering bioenergy crops for sustainable agriculture.
Wang, H., Li, Y., Chern, M., Zhu Y., Zhang L.L., Lu J.H., Li X.P., Dang W.Q., Ma X.C., Yang Z.R., Yao S.Z., Zhao Z.X., Fan J.,
Huang Y.Y., Zhang J.W., Pu M., Wang J., He M., Li W.T., Chen X.W., Wu X.J., Li S.G., Li P., Li Y., Ronald P.C., Wang W.M.
(2021) “Suppression of rice miR168 improves yield, flowering time and immunity.” Nat. Plants.
A. Gross morphology and husked grains of the NPB
control plant, miR168 target mimic (MIM168)
transgenic lines and overexpression (OX168) lines
B. Model for miR168–AGO1 regulating rice immunity
and growth through three miRNAs
A
B

5. Generation of Pseudomonas putida KT2440
Strains with Efficient Utilization of Xylose and Galactose via Adaptive
Laboratory Evolution
Background
• While P. putida KT2440 has great potential for bioprocesses,
its inability to utilize the biomass abundant sugars xylose and
galactose has limited its applications.
Approach
• Heterologous enzymes (xylD and galETKM) were introduced
to construct the catabolic pathways for xylose and galactose.
• Multifaceted ALE strategies were applied depending on initial
growth of the engineered strains on these sugars.
Outcomes and Impacts
• (i) We successfully optimized poor starting strain growth
(<0.1 h−1 or none) via ALE to rates of up to 0.25 h−1 on xylose
and 0.52 h−1
on galactose.
• (ii) Whole-genome sequencing, transcriptomic analysis, and
growth screens revealed significant roles of kguT encoding a
2-ketogluconate operon repressor and 2-ketogluconate
transporter, and gtsABCD encoding an ATP-binding cassette
(ABC) sugar transporting system in xylose and galactose
growth conditions, respectively.
• (iii) We demonstrated 3.2 g/L and 2.2 g/L of indigoidine
production from 10 g/L of either xylose or galactose by using
the evolved strains as microbial platforms.
• (iv) The generated KT2440 strains have the potential for
broad application as optimized platform chassis to develop
efficient microorganism-based biomass-utilizing bioprocesses.
Lim et al. (2021) ACS Sustainable Chemistry & Engineering https://doi.org/10.1021/acssuschemeng.1c03765
Overall approach to enable xylose and
galactose utilization
DNA/RNA-seq analysis to understand mechanisms

6. Background
• Filamentous fungi are excellent lignocellulose degraders, which
they achieve through producing carbohydrate active enzymes
(CAZymes). CAZyme production is highly orchestrated and gene
expression analysis has greatly expanded understanding of this
important biotechnological process.
• Thermophilic fungus Thermoascus aurantiacus secretes highly
active thermostable enzymes that enable saccharifications at
higher temperatures; however, the genome-wide measurements of
gene expression in response to CAZyme induction are not
understood.
Approach
• In this study, cultivation conditions were established to perform
gene expression experiments on the T. aurantiacus response to C5
and C6 sugars derived from biomass.
Outcomes and Impacts
• A fed-batch system with plant biomass-derived sugars D-xylose, L-
arabinose and cellobiose established that these sugars induce
CAZyme expression in T. aurantiacus. The C5 sugars induced both
cellulases and hemicellulases, while cellobiose specifically induced
cellulases (Figure 1).
• It was found that d-xylose and L-arabinose strongly induced a wide
variety of CAZymes, auxiliary activity (AA) enzymes and
carbohydrate esterases (CEs), while cellobiose facilitated lower
expression of mostly cellulase genes.
• This work provides paths to produce broad spectrum
thermotolerant enzymatic mixtures.
Gabriel et al. (2021) Biotechnology for Biofuels, 14, 169 doi: 10.1186/s13068-021-02018-5
Figure 1. Response of T. aurantiacus to
C5 and C6 sugars
CAZymes from the thermophilic fungus Thermoascus
aurantiacus are induced by C5 and C6 sugars

7. Faster, Better, and Cheaper: Harnessing Microfluidics and
Mass Spectrometry for Biotechnology
Background
• High-throughput screening technologies are widely used for
elucidating biological activities. These typically require trade-offs
in assay specificity and sensitivity to achieve higher throughput.
• Microfluidic approaches enable rapid manipulation of small
volumes and have found a wide range of applications in
biotechnology providing improved control of reaction conditions,
faster assays, and reduced reagent consumption. The integration
of mass spectrometry with microfluidics has the potential to create
high-throughput, sensitivity, and specificity assays.
Approach
• This review highlights recent microfluidic approaches that have
been successfully integrated with mass spectrometry analysis for
high-throughput bioassays to improve throughput, sensitivity, and
specificity. This review introduces the major ionization techniques
of mass spectrometry that have been integrated with various
microfluidic systems.
• Additionally, this work discusses recent applications and shares
future outlooks on multiple aspects of these technologies.
Outcomes and Impacts
• The major ionization techniques of mass spectrometry have been
successfully integrated with various microfluidic systems for high-
throughput biological assays.
• Recently, high-impact applications in single-cell analysis,
compound screening, and the study of microorganisms have been
demonstrated.
• Broad adoption of these techniques will require robust
implementation of strategies for the stable sample storage,
containment, and sample tracking.
Ha et al. (2021) RSC Chemical Biology, DOI: 10.1039/d1cb00112d
Integration of mass spectrometry with microfluidics for
high-throughput bioassays
(Article & image are featured on the journal back cover)

8. Production Cost and Carbon Footprint of
Biomass-Derived Dimethylcyclooctane as a
High-Performance Jet Fuel Blendstock
Background
• Near-term decarbonization of aviation requires energy-dense,
renewable liquid fuels.
• Biomass-derived 1,4-dimethylcyclooctane (DMCO), a cyclic
alkane with a volumetric net heat of combustion up to 9.2%
higher than Jet A, has the potential to serve as a low-carbon,
high-performance jet fuel blendstock that may enable paraffinic
bio-jet fuels to operate without aromatic compounds.
Approach
• We developed detailed process configurations for DMCO
production to estimate the minimum selling price and life-cycle
greenhouse gas (GHG) footprint considering three different
hydrogenation catalysts and two bioconversion pathways. All
modeling was done in SuperPro Designer and life-cycle GHG
inventory used our previously-developed BioC2G model
Outcomes and Impacts
• The platinum-based catalyst offers the lowest production cost
and GHG footprint of $9.0/L-Jet-Aeq and 61.4 gCO2e/MJ, given
the current state of technology.
• When the supply chain and process are optimized,
hydrogenation with a Raney nickel catalyst is preferable,
resulting in a $1.5/L-Jet-Aeq cost and 18.3 gCO2e/MJ GHG
footprint if biomass sorghum is the feedstock.
• Because increased gravimetric energy density of jet fuels
translates to reduced aircraft weight, DMCO also has the
potential to improve aircraft efficiency, particularly on long-haul
flights.
Baral et al. (2021) ACS Sustain. Chem. Eng., doi: 10.1021/acssuschemeng.1c03772
Figure 2. Minimum selling price of DMCO under different scenarios:
(a) current state of technology (SOT) with the MVA pathway; (b)
improved MVA pathway with 90% of the theoretical isoprenol yield;
(c) improved MEP pathways with 90% of the theoretical isoprenol
yield; and (d) optimal future case with the MVA pathway.
Figure 1. Overview of 1,4-dimethylcyclooctane (DMCO) synthesis
processes from the biomass-derived glucose and xylose.

9. A Biological Parts Search Portal and Updates
to the ICE Parts Registry Software Platform
Background
• Modern biological part registries need additional features to address
the needs of advances in synthetic biology.
• The synthetic biology community will benefit from a tool providing easy
accessibility to the large amounts of biological part data available in the
public domain.
Approach
• We updated the ICE parts registry platform to include several
significant new features.
• We also developed a new search portal (bioparts.org) that indexes
publicly available biological parts and enables searching using keywords
or sequence fragments (Figure 1).
Outcomes and Impacts
• We highlighted the significant features that were developed for ICE
since its introduction over a decade ago. This includes the
implementation of the web of registries concept which creates a single
distributed parts database out of multiple independent ICE instances.
• We established a search portal that sources data from registries and
repositories such as IGEM, NCBI GenBank, SynbioHub, ICE, AddGene
and Genscript (Figure 2). The availability of an API enables third-party
applications to utilize features such as automatic sequence annotation.
Plahar, et al., “BioParts - A Biological Parts Search Portal and Updates to the ICE Parts Registry Software Platform”,
ACS Synthetic Biology. doi: 10.1021/acssynbio.1c00263
Figure 1
Figure
2

10. JBEI Enabled Papers

11. Plant single-cell solutions for energy and the
environment
Background
• Single-cell transcriptomics has led to fundamental new insights
into animal biology, such as the discovery of new cell types
and cell type-specific disease processes
• The application of single-cell approaches to plants, fungi,
algae, or bacteria (environmental organisms) has been far
more limited, largely due to the challenges posed by
polysaccharide walls surrounding these species’ cells
Approach
• In this perspective, we discuss opportunities afforded by
single-cell technologies for energy and environmental science
and grand challenges that must be tackled to apply these
approaches to plants, fungi and algae
• Highlight the need to develop better and more comprehensive
single-cell technologies, analysis and visualization tools, and
tissue preparation methods
Outcomes and Impacts
• advocate for the creation of a centralized, open-access
database to house plant single-cell data.
• These efforts should balance the need for deep
characterization of select model species while still capturing
the diversity in the plant kingdom. Investments into the
development of methods, their application to relevant species,
and the creation of resources to support data dissemination
will enable groundbreaking insights to propel energy and
environmental science forward
Cole et al. (2021) Commun Biol. doi: 10.1038/s42003-021-02477-4
Using single-cell methods in bioproducts
and biomaterials applications.

12. Co-cultivation of anaerobic fungi with rumen bacteria
establishes an antagonistic relationship
Background
• The rumen microbiome efficiently degrades lignocellulosic biomass
through its wide array of bacterial and fungal carbohydrate active
enzymes (CAZymes).
• Little is known about how these microorganisms cooperate or
compete in order to degrade biomass.
• Fungal interactions with rumen bacteria are not well characterized.
Approach
• Synthetic co-cultures of anaerobic fungi and rumen bacteria were
cultivated on switchgrass (a bioenergy crop) and crystalline
cellulose (Avicel®
) for comparison.
• This study uses ‘omics’ technologies (genomics, transcriptomics,
and metabolomics) to illustrate the antagonistic relationship
between anaerobic fungi and bacteria.
Outcomes and Impacts
• Biosynthetic genes of fungal secondary metabolites were
upregulated during bacterial challenge, and rumen bacteria
responded by upregulating drug efflux pumps and toxin-antitoxin
systems.
• Transcriptional studies and western blotting affirmed that the
secondary metabolism of anaerobic fungi is likely regulated by
chromatin remodeling.
• The fundamental understanding provided by this study is an
important step toward engineering synthetic microbial consortia for
biomass degradation.
Swift et al. (2021) mBio, doi: 10.1128/mBio.01442-21
Helium ion microscopy of the anaerobic fungus Anaeromyces
robustus and the bacteria Fibrobacter UWB7 illustrates close
contact of these rumen microorganisms in a synthetic co-
culture.

13. Plant genome engineering from lab to field—
a Keystone Symposia report
Background
• Enhancing crop resiliency and yield via genome engineering will be a key
part of tackling food source challenges posed by a growing global
population and a warming climate.
• New tools such as CRISPR/Cas have ushered in significant advances in
plant genome engineering, but several challenges remain, including
efficient transformation and plant regeneration for most crop species,
low frequency of some editing applications, and high attrition rates.
Approach
• Experts in plant genome engineering and breeding from academia and
industry met virtually for the Keystone eSymposium “Plant Genome
Engineering: From Lab to Field” to discuss advances in genome editing
tools, plant transformation, plant breeding, and crop trait development,
all vital for transferring the benefits of novel technologies to the field.
Outcomes and Impacts
• Presenters described novel tools for gene and base editing, gene
targeting by homology-directed repair, transcriptional regulation,
epigenetic editing, and induced chromosomal rearrangements.
• Presenters also discussed applications of genome editing in agriculture,
including improving potato traits with CRISPR/Cas, improving cereal crop
transformation, and increasing fruit and vegetable consumption with
genome editing.
Cable et al. (2021) Annals of the New York Academy of Sciences, doi: 10.1111/nyas.14675
A comprehensive analysis of plant core promoters.