Lay highlights of the papers published by the Joint BioEnergy Institute in July 2021

1. Integration of acetic acid catalysis with one-pot protic
ionic liquid configuration to achieve high-efficient
biorefinery of poplar biomass
Background
• Recyclable biocatalysts and high-efficiency lignocellulose deconstruction
are the crucial factors for cost-effective conversion of biomass into
biofuels and bioproducts.
• Acetic acid-based catalytic hydrolysis is not effective on woody biomass,
such as poplar.
• One way to improve conversion performance of this process is to
integrate it with other effective processes, such as pretreating biomass
with protic ionic liquids (PILs).
Approach
• An integrated acetic acid based one-pot ethanolamine acetate
pretreatment (HAc-[EOA][OAc]) was developed for the efficient
depolymerization of poplar polysaccharides.
Outcomes and Impacts
• Compared to previous studies on poplar biorefining, the new process has
three distinct differences:
• (1) simultaneously removal of ~88% of the hemicellulose and selective
extraction of up to ~46% of the lignin from lignocellulosic biomass;
• (2) yields of over 80% enzyme-hydrolyzed glucose that was attributed to
an increase in the accessible surface area of cellulose to the hydrolytic
enzymes;
• (3) HSP and COSMO-RS analysis indicate that [EOA][OAc] is a good
lignin solvent, which leads to the higher delignification of biomass.
• Overall, this study demonstrates that the integration of IL with acid
pretreatment is a promising strategy for conducting effective pretreatment
on woody lignocellulose.
Huang et al. (2021) Green Chemistry, https://doi:10.1039/d1gc01727f
Schematic of process design differences
between HAc and HAc-[EOA][OAc]
pretreatment
Poplar
Saccharification
Hydrolysis
<60%
Glucose Yield
HAc
XOS
Liquid
Slurry
A
B
Poplar
Prehydrolysis
HAc
Recycling >80%
Glucose Yield
HAc
XOS
Liquid
One-pot PIL pretreatment
and saccharification
PIL Recycling
Water
wash
Slurry
Consolidated process
Individual process
0 10 20 30 40 50 60 70 80 90 100
pH adjustment*
Water wash
Control- IL
Control- HAc
Sugar yield (%)
Xylose
Glucose

2. Towards Understanding of Delignification of Grassy
and Woody Biomass in Cholinium-based Ionic Liquids
Background
• Lignin is one of the major constituents of the lignocellulosic biomass along with
cellulose and hemicellulose, and is the most-abundant bio-renewable source of
aromatics.
• Fractionation of lignin remains a major challenge for biorefineries due its
recalcitrance, heterogeneity, strong interactions, and hydrophobicity.
• Recent studies have shown that it is more difficult to extract lignin from
softwood biomass using conventional pretreatment technologies.
Approach
• To understand the mechanism of delignification during biomass pretreatment
with cholinium-based ionic liquids, we employed COSMO-RS and molecular
dynamics simulations to evaluate the molecular interactions between lignin-like
model compounds and the IL components.
Outcomes and Impacts
• The dissolution of lignin was carried out in six different cholinium-based ILs
• The van der Waals (vdW) interaction energies between lignin and anion were
found to be more important for lignin dissolution than electrostatic interactions
• From the experimental and computational approaches, [Ch][Lys] was found to
be the best solvent for lignin extraction due to the stronger interactions,
formation of multiple hydrogen bonds, higher dissociation constant (pKa), and
lower viscosity of [Ch][Lys]
• The simulation data suggested that [Ch][Lys] has higher affinity for ether
linkages of lignin (e.g., β‒O‒4) than for C‒C linkages, which explains the
higher delignification of hardwood and grassy biomasses (60-80% C‒O‒C
linkages) than softwood in [Ch][Lys]
• The results presented in this study provide fundamental insights into the
dissolution of lignin in cholinium-based ILs and opens a path for the design of
new IL to improve the biomass delignification efficacy.
Mohan et al. (2021) Green Chemistry, https://doi.org/10.1039/D1GC01622A
β‒O‒4 surrounded by
the lysinate anion
5‒5 surrounded by
the lysinate anion
H-bond Network
Spatial distribution functions

3. Review of advances in the development of
laccases for the valorization of lignin
Background
• Lignin valorization remains one of the biggest challenges
(and opportunities) in terms of lignocellulosic biofuels and
bioproducts.
• Effective and affordable lignin depolymerizing enzymes,
such as laccases and peroxidases, are an essential step in
transforming lignin into bioavailable substrates that can be
converted into biofuels and bioproducts by microorganisms.
Approach
• In this review, we highlight the different methods employed
for lignocellulose depolymerization and the importance of
lignin valorization for biofuels and bioproducts production in
each.
• We also highlight the potential of laccase enzymes for the
depolymerization of lignin in a biorefinery setting.
Outcomes and Impacts
• Currently, laccases are still too expensive to be used at the
biorefinery scale.
• Despite all the bottlenecks and challenges that exist, recent
research on laccases has resulted in significant innovations
and impacts that improve the outlook of this approach.
• We have presented on new approaches that can provide a
roadmap for the scientific community to work towards
achieving the true potential of lignin.
Curran et al. (2021) Biotechnology Advances, doi:10.1016/j.biotechadv.2021.107809
Word cloud generated from the titles of the latest 100 review
papers on laccases in PubMed. The size of the words is
proportional to the number of times the word appears in the
paper titles. Blue pie chart: Proportion of journals publishing on
laccases in Pubmed. Pink graph: The number of reviews
published about laccases in PubMed since 1967.

4. Metabolic engineering strategies for sesquiterpene
production in microorganism
Background
• Sesquiterpenes are a large variety of terpene natural products,
widely existing in plants, fungi, marine organisms, insects, and
microbes.
• Value-added sesquiterpenes are extensively used in industries such
as: food, drugs, fragrances, and fuels.
• Advances in synthetic biology provide new strategies on the creation
of desired hosts for sesquiterpene production.
Approach
• This review presents a summary of metabolic engineering strategies
on the hosts and pathway engineering for sesquiterpene production.
• Challenges and future perspectives of the bioprocess for translating
sesquiterpene production into practical industrial work are discussed.
Outcomes and Impacts
• Compared to previous review papers on sesquiterpene production,
three differences are here involved:
• (1) describing the metabolic engineering strategies on sesquiterpene
production from three levels of hosts, pathways, and compounds
more detailed;
• (2) analyzing the strategies for dramatically improving sesquiterpene
production resulting from regulation of global metabolic networks of:
cell growth, nutrient uptake, energy utilization, and gene expression;
• (3) outlooking the challenges and the possible addressed approaches
in the bioprocess of translating bio-sesquiterpenes into practical
industrial work more detailed and comprehensively.
• Overall, the biosynthesis of sesquiterpenes with engineered
microorganisms is a worthy-studied theme for their widely promising
applications and values
Liu et al. (2021) Critical Reviews in Biotechnology,
https://doi.org/10.1080/07388551.2021.1924112
Schematic of strategies for sesquiterpene
production in E. coli and S. cerevisiae

5. Paladin, a tyrosine phosphatase-like protein, is
required for XA21-mediated immunity in rice
Background
• Plants and animals use immune receptors to recognize and respond to
microbes. Understanding the underlying signaling pathways can guide
efforts to engineer enhanced resistance in agricultural crops.
• Rice plants carrying the XA21 immune receptor exhibit robust resistance
to strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae
(Xoo). However, the detailed molecular and physiological mechanisms
underlying XA21 resistance have yet to be fully determined.
Approach
• To identify components regulating XA21-mediated immunity, we
generated and screened a mutant population of fast-neutron-
mutagenized rice expressing Ubi:Myc-XA21 for those susceptible to Xoo.
We report the characterization of one of these rice mutants, sxi2.
Outcomes and Impacts
• Whole-genome sequencing revealed that sxi2 carries a deletion of the
PALADIN (PALD) gene encoding a protein with three putative protein
tyrosine phosphatase-like domains (PTP-A, -B,and -C).
• A deletion of the PALD gene is responsible for the susceptible phenotype
observed in the sxi2 mutant. Introduction of PALD rescues the
susceptible phenotype of sxi2.
• PALD, a previously uncharacterized class of phosphatase, functions in rice
innate immunity, and the conserved cysteine in the PTP-A domain of
PALD is required for its immune function.
Chen et al. (2021) Plant Communications, doi: 10.1016/j.xplc.2021.100215
A chromosome 3 deletion co-segregates with the sxi2
susceptible phenotype in an F2 population.

6. JBEI Enabled Publications

7. Accurate prediction of protein structures and
interactions
Background
• The ab initio prediction of protein structures from sequence alone has
been a longstanding goal in structural biology.
• Metabolic pathways for the production of biofuels and bioproducts
can be improved using enzyme engineering. These efforts usually
depend on structural information being available.
Approach
• Machine learning approaches were applied to information at the 1D
sequence level, the 2D distance map level, and the 3D coordinate
level to predict the structures of proteins, and protein complexes.
• JBEI data was used to demonstrate that these predictions were
accurate enough to solve recently collected crystallographic data for
a lignin processing enzyme.
Outcomes and Impacts
• The engineering of biofuels and bioproducts related enzymes should
be enhanced by tools that make it possible to predict structures,
leading to hypotheses about enzyme function and specificity.
• The detailed structural studies still required to completely reveal the
atomic resolution details of enzyme mechanism and specificity will be
greatly enabled by accurate starting models.
• The ability to predict protein/protein complex structures will likely be
transformative for large modular enzymes such as polyketide
synthases.
• Overall: Accurate protein structure modeling enables rapid solution of
structure determination problems and provides insights into biological
function.
Baek et al. (2021) Science,
https://doi.org/10.1126/science.abj8754
Comparison of predicted structure (blue/red) and
crystallographic structure (grey) for JBEI enzyme
Comparison of predicted protein complexes (right)
and crystallographic structures (left)