JBEI Highlights January 2016

1. Arabidopsis Plasma Membrane Proteome
Outcomes
• A collection of over 700 high-confidence plasma membrane proteins
were identified which included many soluble peripheral proteins
• This included members of the recently-characterized TPLATE
complex, which is required for clathrin-mediated endocytosis in plants
• A host of cell wall biosynthetic proteins were identified, including
cellulose synthases members, glycoside hydrolases and glucan
synthase-like proteins.
1) Breakdown of the FFE plasma membrane proteome by spectral
count.
The approach was able to enrich highly purified PM vesicles with minimal
contaminants (20%) demonstrating the power of the free-flow electrophoresis
approach. We have previously used a similar purification approach to enrich Golgi
membranes from plants.
de Michele et al. (2016) “Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning
Enhances the Quality of the Proteome from Arabidopsis Seedlings”, J Prot Res., doi, 10.1021/acs.jproteome.5b00876
Background
• The plant plasma membrane is the cells
interface to the environment regulating
transport into the cell and external signals.
• The plasma membrane is also the site of
cellulose biosynthesis and a barrier to the
deposition of cell wall hemicellose synthesized
in the Golgi which needs to be crossed.
Significance
• Defining novel constituents and major players involved in cell wall
biosynthesis processes provides potential targets for the future
manipulation of plant biomass.
2) Fluorescent tags (YFP) were used to validate newly
assigned plasma membrane proteins.
The YFP fluorophore was attached to a number of proteins identified
in the proteome and transiently expressed in tobacco to confirm their
subcellular localizations. In this example, the protein AT1G12080
was confirmed to localize to the plasma membrane when the signal
merged with the control pBullet-PM (CFP – blue). The presence of
Hechtian strands after plasmolysis confirms the PM localization for
the protein
Approach
• To further characterize components of this
structure, we employed an advanced organelle
separation technology (free-flow electrophoresis)
to enrich a plasma membrane vesicles with
peripheral components still attached.

2. Overexpression of Rice Wall-Associated
Kinase 25 (OsWAK25) Alters Resistance
to Bacterial and Fungal Pathogens
Outcomes
• Overexpression of OsWAK25 confers resistance to Xanthomonas
oryzae pv. oryzae and Magnaporthe oryzae
• OsWAK25 overexpression plants are more susceptible to
Cochliobolus miyabeanus and Rhizoctonia solani
• Overexpression of XB15 compromises resistance to Xoo conferred by
OsWAK25 overexpression
Harkenrider et al. (2016). "Overexpression of Rice Wall-Associated Kinase 25 (OsWAK25) Alters
Resistance to Bacterial and Fungal Pathogens”, PLoS ONE, doi: 10.1371/journal.pone.0147310
Background
• Wall-associated kinases (WAKs) comprise a subfamily of proteins
within the receptor-like kinase (RLK) superfamily, and have been
extensively studied in Arabidopsis
• WAKs have critical roles in pathogen responses in other species
including tomato, wheat, and the model grass, rice
• OsWAK25 is one of the 67 WAK-RLKs identified in rice and encodes
a 76 kDa protein with one EGF-like repeat in the extracellular
domain
Significance
• Provides new insights into the roles and functions of WAKs in rice and
provides the foundation for engineering bioenergy crops with
enhanced pathogen resistance
Approach
• Generated Kitaake rice plants overexpressing OsWAK25
• Assessed resistance to hemibiotrophic and necrotrophic pathogens
and assayed expression of defense-related genes
Overexpression of XB15 compromises OsWAK25-
mediated resistance to Xoo. Transgenic lines NTAP-XB15
Ox 17A-3 and OsWAK25 Ox 3–17,
both in the Kitaake background, were crossed and
segregating F2 progeny of the double overexpression
line were inoculated with Xoo strain PXO99 along
with Kitaake and parental lines as controls

3. Enabling Xylose Utilization in
Saccharomyces cerevisiae
Outcomes
• A mutation in the glucose
transporter Hxt7 allowed for
significantly improved growth on
xylose medium
• This mutant transporter displays
improved xylose uptake rates that
allow for partial co-utilization in a
mixed carbon cultivation.
Background
• Xylose uptake remains the major
limitation to complete lignocellulosic
carbon utilization in S.cerevisiae
Approach
• We allowed S. cerevisiae to evolve
for growth and uptake of xylose,
and sequenced the evolved strain.
Significance
• Laboratory evolution is a powerful method to obtaining desired phenotypes
• We obtained the first xylose transporter that allows for growth and uptake of
xylose from a low-copy plasmid in a minimally engineered background
• Enables more efficient production of advanced biofuels from all sugar types
present in lignocellulose
Strains expressing
Hxt7(F79S)
consume more
xylose than their
wild-type
counterparts.
Reider Apel et al. (2016) “Evolved hexose transporter enhances xylose uptake and glucose/xylose co-
utilization in Saccharomyces cerevisiae”, Sci. Rep., doi: 10.1038/srep19512
Hxt7(F79S) shows a two‐fold
increase in xylose transport
velocity as assayed by
radioactive xylose uptake.
The evolved
S.cerevisiae
strain was
resequenced at
the DOE Joint
Genome Institute.
Strains expressing Hxt7(F79S) from a single
copy plasmid grow on xylose medium.

4. Multifunctional Cellulase Catalysis Targeted
by Fusion to Different Carbohydrate-binding
Modules
Outcomes
• Several CelEcc-CBM fusions showed enhanced hydrolytic activity with
different substrates relative to the fusion to CBM3a.
• Binding studies and quantitative catalysis studies using nanostructure-
initiator mass spectrometry show that enhanced enzyme reactivity was
correlated with moderate binding affinity of the CBM.
• Numerical analysis of reaction time courses showed that CelEcc-CBM44
gave the fastest rates for the hydrolysis of both the hexose and pentose
fraction of ionic-liquid pretreated switchgrass.
1) Affinity gel electrophoresis of
GFP_CBM fusions. Thirty-nine GFP_CBM
fusions were tested for biding specificities in
native polyacrylamide gels containing CaCl2
and either lichenan, galactomannan,
beechwood xylan or arabinoxylan. A “No
substrate” gel is shown for comparison with the
substrate gels. Red stars indicate where
binding was detected. Soybean trypsin
inhibitor (STI) was used as control for no
binding.
Walker et al. (2016) “Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-
binding modules”, Biotechnol Biofuels, doi: 10.1186/s13068-015-0402-0
Background
• Carbohydrate binding modules (CBMs) bind
polysaccharides and help target glycoside
hydrolases catalytic domains to their
appropriate carbohydrate substrates.
Significance
• This fusion approach provides a new avenue to improve reactivity of
simple combinations of enzymes and lower costs of GH mixtures needed
for biofuel production
2) Domain structures and relative sizes of CelE_CBM hybrids
aligned with the yield of hexose and pentose products after 24 h
reaction in 50 mM phosphate, pH 6.0, at 60 ˚C. All reactions
contained 0.32 µmol of enzyme active sites and 1 mg of biomass.
Approach
• To better understand how CBMs can improve
cellulolytic enzyme reactivity, representatives
from each of the 18 families of CBM found in
Ruminoclostridium were fused to the
multifunctional GH5 catalytic domain of CelE
(Cthe_0797, CelEcc), which can hydrolyze
numerous types of polysaccharides including
cellulose, mannan, and xylan.

5. Suppression of Bacterial Infection in Rice
by Treatment with a Sulfated Peptide
Outcomes
• Post-inoculation treatment of XA21 plants with the sulfated RaxX peptide
suppresses the development of Xoo infection.
• The treated plants display restricted lesion development and reduced
bacterial growth.
Wei et al. (2016) “Suppression of bacterial infection in rice by treatment with a
sulfated peptide”, Molecular Plant Pathology, doi: 10.1111/mpp.12368.
Background
• The rice XA21 receptor kinase confers robust
resistance to bacterial blight disease caused by
Xanthomonas oryzae pv. oryzae (Xoo).
• A tyrosine-sulfated peptide from Xoo, called
RaxX, triggers XA21-mediated immune
responses, including production of ethylene,
reactive oxygen species and induction of defense
gene expression.
Significance
• Our findings demonstrate that application of sulfated peptide activates a
cell-wall localized receptor in planta, and provide a potential strategy for
controlling bacterial disease in bioenergy feedstocks and other crops.
Post-inoculation treatment with sulfated RaxX
enhances resistance to PXO99ΔraxX strain in XA21
plants. (a) Rice leaves from Ubi::XA21 (XA21) and
Kitaake (Kit) plants 13 days after inoculation (dai). (b)
Lesion lengths of Ubi::XA21 (open bars) and Kitaake
(closed bars) at 13 dai. Five-week-old hydroponic
Ubi::XA21 and Kitaake plants were inoculated with
PXO99ΔraxX, and treated with water (Mock), 1 µM 21-
amino-acid RaxX-Y or RaxX-sY for 6 hours at 2 dai. (c)
Bacterial population was quantified as the number of
colony-forming units (cfu) per inoculated leaf at 13 dai.
Different letters in (b, c) indicate significant differences
between the samples (Tukey’s HSD test, α < 0.05).
Approach
• Determine if these immune responses confer
effective resistance to Xoo in planta using a
newly established post-inoculation treatment
assay.

6. Metabolic Engineering of Escherichia coli
for the Biosynthesis of 2-Pyrrolidone
Outcomes
• Protein modification, such as MBP fusion, increased the activity of
expressed ORF27
• Metabolic engineering and process optimization collectively improved 2-
pyrrolidone titer from glutamate
• 1.1g/L of 2-pyrrolidone was produced from 7.7g/L of glutamate,
representing a 25% yield of theoretical maximum
Zhang et al. (2016) “Metabolic engineering of Escherichia coli for the biosynthesis of 2-
pyrrolidone”, Metabolic Engineering Communications, doi: 10.1016/j.meteno.2015.11.001
Background
• 2-Pyrrolidone was identified by the US Department of Energy as an
important C4 “Top Value-Added Chemical from Biomass” that can
potentially be derived from glutamate
• There is no route available today for the efficient biosynthesis of 2-
pyrrolidone
• Missing step in the biosynthetic pathway for the enzymatic ring
closing of γ-aminobutyrate (GABA) into 2-pyrrolidone
Significance
• Discovery of ORF27, coupled with metabolic engineering of E. coli,
enabled the production of 2-pyrrolidone, a potentially important co-product
for biorefineries
Effect of pH on production of 2-pyrrolidon in
engineered E. coli fed 10 mM glutamic acid
Approach
• Discovered ORF27 in S. cerevisiae, an auxiliary enzyme in the
linearmycin A biosynthetic cluster that performs the GABA activation
step to form 2-pyrrolidone under mild fermentation conditions
• Expressed ORF27 in E. coli to complete biosynthetic pathway for the
production of 2-pyrrolidone
E. coli production of 2-pyrrolidone from glutamic acid
(closed triangle), OD600 of E. coli growth (closed circle),
glutamic acid consumption (open circle), and GABA
intermediate accumulation (closed diamond).