Research summaries for papers published by the Joint BioEnergy Institute in January 2022

1. The impact of biomass pretreatment methods on the
structure and arrangement of plant cell wall polymers
Background
• Efficient separation of polymers during biomass
processing is challenging.
• Many deconstruction methods can introduce
additional recalcitrance due to cell wall polymer
rearrangement.
• This is challenging to study, as many analytical
methods also rely on a physical pretreatment
step.
Approach
• Here, we discuss how solid-state Nuclear
Magnetic Resonance (NMR) approaches show
that even minimal physical processing can
disrupt the 3D organization of the cell wall.
• We use 13C-labelled sorghum stem to illustrate
this.
Outcomes and Impacts
• The organization of these polymers in the
secondary plant cell walls varies across biomass
sources and sample preparation methods.
• These structural differences will contribute to
variable efficiencies for biofuels and bioproducts
downstream processing.
• We propose that these methods can be applied
to follow biomass deconstruction, and
understand polymer rearrangements that can
lead to poor yields.
Munson et al. (2021) Frontiers in Plant Science, doi: 10.3389/fpls.2021.766506
Figure 1: 13C-enriched plant stems are harvested, frozen in liquid nitrogen, sectioned on a
dry ice cooled surface, and subjected to vibrational ball milling using a zirconium chamber
and balls.
Figure 2: Changes in the rigid structure of sorghum stem tissue after 2 min of ball milling.
(A) Percent difference in integrated signal intensities from the 2 min ball-milled sample,
relative to the unprocessed control sample. Due to the nature of the CP-INADEQUATE
experiment, two signals are observed for most sites. (B) The CP-INADEQUATE spectrum
of the unprocessed control sample.

2. Background
• Generating value from lignin through depolymerization and
biological conversion is limited by lack of cost-effective
depolymerization methods, toxicity within the breakdown products,
and low bioconversion of the breakdown products.
• High yield depolymerization of natural lignins requires cleaving
carbon-carbon bonds in addition to ether bonds.
Approach
• To address that need, we report that a chelator-mediated Fenton
reaction can efficiently cleave C-C bonds in sulfonated polymers at
or near room temperature
Outcomes and Impacts
• This method was used to depolymerize lignosulfonate from Mw =
28,000 g/mol to Mw = 800 g/mol. The breakdown products were
characterized by SEC, FTIR and NMR and evaluated for
bioavailability.
• A panel of nine organisms were tested for the ability to grow on the
breakdown products. Growth at a low level was observed for
several monocultures on the depolymerized lignosulfonate (LS) in
absence of glucose.
• Much stronger growth was observed in the presence of 0.2%
glucose and for one organism we demonstrate doubling of melanin
production in the presence of depolymerized LS.
• The results suggest that this chelator-mediated Fenton method is a
promising new approach for biological conversion of lignin into
higher value chemicals or intermediates.
Martinez et al. (2022) Green Chemistry, DOI: 10.1039/d1gc03854k
Figure 1. Gel permeation chromatograms before and
after treatment of lignosulfonate with chelated Fe(II) and
H2O2.
Depolymerization of lignin for biological conversion through
sulfonaton and a chelator-mediated Fenton reaction
Figure 2. Growth of microbes on depolymerized
Lignosulfonate in the presence and absence of glucose
Figure 5. Molecular weight distribution for [LS] = 5 mg/ml before and after
reaction with [FeCl3] = [DHB] = 0.5 mM and [H2O2] = 0.5% at RT measured at a)
210 nm and b) 270 nm.
a) b)
Figure 10. Growth of monocultures in the LS breakdown stream or base medium in the presence or
absence of glucose. Bars represent the average of three replicates and the error bars indicate the
standard deviation.

3. Assembly of Artificial Metalloenzymes in Escherichia
coli Nissle 1917 for Selectively Catalyzing Reactions
Background
• Artificial metalloenzymes (ArMs) are increasingly important in
catalyzing abiological reactions with many examples in recent
years,
• Methods for assembling and screening ArMs are still laborious
and time-consuming.
• Assembly of ArMs in cells and screening them with the whole
cell will significantly simplify and facilitate the creation and
engineering processes.
Approach
• We used Escherichia coli Nissle 1917 (EcN) to create ArMs in
vivo by expressing apo-P450 and importing the target artificial
cofactor into cytoplasm, via a native transporter.
• This strain was able to assemble the ArMs in vivo and
selectively catalyze the reactions and provided the system to
screen the directed evolution mutants of the ArMs.
Outcomes and Impacts
• This study developed an approach using a non-pathogenic E.
coli strain to assemble ArMs in vivo, and significantly simply the
process for creation of new ArMs and their improvement.
• Catalyzing reactions and conducting directed evolution with this
new microbial platform was tested and proved to be an efficient
method.
• A transporter able to import the target artificial cofactor was
identified and can be used for further applications.
Liu et al, J. Am. Chem. Soc., 2022, 144, 883-890
(A) Schematic diagram for the assembly of ArM in the
EcN cells. (B) EcN cells harboring ArMs catalyzing site-
selective C-H activation. (C) Identification of the
cofactor transporter.
A
B
3 para-4 meta-4
+
EcN harboring
Ir-CYP119
N2
CO2Et
O
30 oC, 4 h
O
CO2Et
R
O
R
R
CO2Et
+
3a R = Br
CYP119-PL1
para:meta = 3.1:1
TON 2302±27
CYP119-ML1
para:meta = 1:3.2
TON 3241±54
3b R = Cl
CYP119-PL1
para:meta = 2.8:1
TON 3407±234
CYP119-ML1
para:meta = 1:3.4
TON 4643±103
3c R = OMe
3d R = tBu
3a R = Br
3b R = Cl
CYP119-PL1
para:meta = 4.9:1
TON 1634±36
CYP119-PL2
para:meta = 4.8:1
TON 938±140
C
1 2
+
BL21(DE3) harboring
Ir-CYP119
N2
CO2Et
O
30 oC, 4 h
O
CO2Et

4. JBEI Enabled Publications

5. Heinz resistant tomato cultivars exhibit a lignin-
based resistance to field dodder (Cuscuta
campestris) parasitism
Background
• Cuscuta species (dodders) are agriculturally destructive, parasitic
angiosperms. These parasitic plants use haustoria as physiological
bridges to extract nutrients and water from hosts.
• While some wild tomato relatives are resistant, cultivated tomatoes
are generally susceptible to C. campestris infestations. However,
some specific Heinz tomato (Solanum lycopersicum) hybrid
cultivars exhibit resistance to dodders in the field, but their defense
mechanism was previously unknown.
Approach
• We discovered that the stem cortex in these resistant lines
responds with local lignification upon C. campestris attachment,
preventing parasite entry into the host. LIF1 (Lignin Induction
Factor 1, an AP2-like transcription factor), SlMYB55, and CuRLR1
(Cuscuta R-gene for Lignin-based Resistance 1, a CC-NBS-LRR)
are identified as factors that confer host resistance by regulating
lignification.
Outcomes and Impacts
• This work demonstrates how plants can utilize lignification to
prevent parasitism by field dodder.
• This discovery provides a foundation for investigating multilayer
resistance against Cuscuta species and has potential for
application in other essential crops attacked by parasitic plants.
Jhu et al. (2022) Plant Physiology, doi: 10.1093/plphys/kiac024