JBEI Research Highlights - April 2019

1. Conversion of depolymerized sugars and aromatics
from engineered feedstocks by two oleaginous red
yeasts
Background
• The use of several species of fungi with the natural
ability to assimilate lignocellulose-derived components
has recently gained interest because they can attain
high cell densities under controlled conditions and are
tolerant to inhibitory compounds.
• In particular, some oleaginous red yeasts from the
phylum Basidiomycota are known to consume
hexoses, pentoses, organic acids and aromatic
monomers, while efficiently transforming them into cell
biomass that is rich in lipids and carotenoids
Approach
• The red yeasts Rhodosporidium toruloides and
Rhodotorula mucilaginosa were evaluated for their
ability to assimilate sugars and aromatic compounds
extracted from two engineered lines of Arabidopsis
thaliana with modified lignin or the wild-type using ionic
liquid, acid or alkaline pretreatments.
Outcomes and Impacts
• Biomass from engineered Arabidopsis lines yielded
aromatics that can be assimilated in both red yeasts.
• Genetically-engineered strains of the two red yeasts
successfully converted the depolymerized products
into the biofuel precursor bisabolene when cultivated
on hydrolysates or synthetic media containing specific
sugars, acids and aromatics found in the hydrolysates.
Rodriguez et al. (2019) Biores. Technol., doi: 10.1016/j.biortech.2019.121365
Workflow employed to generate and characterize
lignocellulosic hydrolysates for the production of bisabolene
with recombinant red yeasts.

2. Guanidine-riboswitch-regulated efflux transporters
protect bacteria against ionic liquid toxicity
Background
• Ionic liquids are a promising pretreatment for the conversion
of plant biomass to biofuels and biochemicals.
• Previous work had identified MFS efflux pumps that confer
ionic liquid tolerance to E. coli.
Approach
• An isolate collection from compost samples was interrogated
for tolerance to imadiazolium-based ionic liquids. Two Bacillus
isolates were found to be highly tolerant to ionic liquids and
the molecular mechanism of IL tolerance investigated.
Outcomes and Impacts
• Genomic libraries constructed from B. cereus and B.
licheniformis isolates conferred IL tolerance to E. coli
• Analysis of the genomic libraries indicated that Small Drug
Resistant (SMR) pumps from the Bacilli conferred tolerance to
E. coli
• Deletion of an SMR pump in B. thuringiensis generated a
strain that was sensitive to ILs, establishing a genetic basis for
IL tolerance.
• The gene for the SMR pimp form B. licheniformis was shown
to contain a guanadinium-activated riboswitch.
• SugE, an SMR pump in E. coli, was also shown to contain a
guanadinium-activated riboswitch, and IL tolerance was
conferred to E. coli by co-incubation of the IL with
guanadinium.
Higgins et al. (2019) Journal of Bacteriology, doi: 10.1128/JB.00069-19.
0.00
0.25
0.50
0.75
1.00
0
5
10
15
Time (Hours)
Celldensity(OD600
)
Strain
B. cereus JP5
B. cereus Type
B. licheniformis Type
B. licheniformis Z98
E. coli DH10b
P. lignolyticus SCF1
A
Max. Growth Rate (μ max)
Max. Cell Density (K)
B.cereusJP5
B.cereusType
B.licheniformisType
B.licheniformisZ98
E.coliDH10b
P.lignolyticusSCF1
0.0
0.3
0.6
0.9
0.00
0.05
0.10
0.15
Strain
mean
B

3. Methyl ketone production by Pseudomonas
putida is enhanced by plant-derived amino acids
Background
• Conversion strategies often use a fraction of the total biomass,
focusing on sugars from cellulose and hemicellulose. Strategies
that use plant components such as plant-derived aromatics and
amino acids have the potential to improve the efficiency of overall
biomass conversion.
• Methyl ketones derived from fatty acids are a promising diesel
blendstock and have been produced by multiple microbial
platforms.
Approach
• We engineered Pseudomonas putida to produce methyl ketones by
introducing a truncated beta-oxidation pathway. We tested the
strain on pure substrates and plant hydrolysates containing
significant amounts of plant-derived aromatics.
Outcomes and Impacts
• Engineered P. putida produced methyl ketones from glucose and
lignin-related aromatics.
• Methyl ketone production by P. putida increased with plant
hydrolysates as substrates in comparison to sugar controls, in
contrast to E. coli, from which production was lowered.
• Metabolomics and proteomics demonstrated that the increased
methyl ketone production was due to plant derived-amino acids
• Inculsion of a defined amino acid mixture increased methyl ketone
production 9-fold relative to a sugar-only control.
Dong et al. (2019) Biotechnology and Bioengineering, doi: 10.1002/bit.26995

4. Biosynthesis of a sulfated microbial peptide
that promotes growth and cell expansion
Outcomes
 Proteolytic cleavage of the N-terminal leader is necessary
for export of RaxX so it can interact with host receptors.
 A raxX-associated peptidase-containing secretion system
carries out processing and secretion of RaxX.
 A second peptidase-containing secretion system can
partially process and secrete RaxX.
Luu et al. (2019) Proc Natl Acad Sci USA, 116(17): 8525-8534, doi: 10.1073/pnas.1818275116
Approach
• Targeted proteomics was used to detect RaxX in Xoo
supernatants.
• RaxX secretion in planta was assessed based on bacterial
colonization and activation of XA21-mediated immunity.
Background
• The rice pathogen Xanthomonas oryzae pv. oryzae (Xoo)
produces a sulfated peptide named RaxX, which activates
XA21-mediated immunity in rice and is predicted to activate
cell-wall remodeling enzymes that loosen the cellulose
network.
• RaxX is genetically clustered with a tyrosine sulfotransferase
and components of a peptidase-containing secretion system.
Significance
 RaxX is proteolytically processed and secreted as a mature
sulfated peptide.
 The mature peptide sequence and biosynthetic strategy
resembles that of plant peptides involved in cell-wall
remodeling.
RaxX is secreted as an immunogenic proteolytically processed
peptide. (A) RaxX is ribosomally synthesized as a precursor peptide
(proRaxX) containing an N-terminal 40-residue leader sequence. proRaxX is
detected in cell lysates (B) but only the proteolytically processed peptide is
detected in Xoo supernatants (C). Mutation of residues preceding the leader
cleavage site on proRaxX (D) or catalytic residues of the
peptidase/transporter RaxB (E) compromises secretion of RaxX and the
ability of the bacterium to be recognized by the plant XA21 immune receptor.

5. Approaches for more efficient biological conversion of
lignocellulosic feedstocks to biofuels and bioproducts
Background
• Optimizing biomass to biofuels/bioproducts conversion pathways
requires cross-coordination among biologists, chemists,
agronomists, and engineers.
• This study surveys recent advancements in bioenergy crop
engineering, lignocellulosic biomass deconstruction and
fractionation, catabolism of biomass-derived sugars and aromatics,
and biological conversion to fuels and products
Approach
• We organize major research approaches into broad categories and
comment on which strategies offer synergies or trade-offs in the
context of different biorefinery process strategies to improving the
economics and carbon-efficiency of advanced biofuels and
bioproducts.
Outcomes and Impacts
• A successful bioeconomy must make use of every carbon source
available and generate renewable fuels and products for markets in
which few or no viable alternatives exist.
• Research is required into the cultivation, harvest, and long-term
storage of low-input, and high yield and quality biomass.
• Simpler hydrolysates with lower concentrations of inert materials
will benefit biorefinery configurations across the spectrum, from
consolidated one-pot processes to extensively fractionated,
targeted conversion of each plant-derived component.
• Utilization of a more diverse range of carbon sources beyond
glucose, and conversion of those substrates to fuels and products
will enable biorefineries of the future to derive the greatest value
from biomass.
Baral et al. (2019) ACS Sus. Chem. Eng., doi: 10.1021/acssuschemeng.9b01229
Selected Research Approaches
Biorefinery Process Strategies

6. Gupta et al. (2019) J. Biol. Chem., doi: 10.1074/jbc.RA119.007592
X-ray radiolytic labeling reveals the molecular basis of
orange carotenoid protein photoprotection
XFMS determined high resolution model of FRP-CTD
complex
XFMS provided model for OCP-photoprotection
Background
• In cyanobacterial photoprotection, the orange carotenoid protein (OCP) is
photoactivated under excess light conditions and binds to the light-
harvesting antenna, triggering the dissipation of captured light energy.
• In low light, the OCP relaxes to the native state, a process that is
accelerated in the presence of fluorescence recovery protein (FRP).
• Despite the importance of the OCP in photoprotection, the precise
mechanism of photoactivation by this protein is not well understood.
Approach
• X-ray radiolytic labeling (X-ray Footprinting) and Mass Spectrometry
(XFMS) methods can directly quantify solvent accessibility (SA) of amino
acids in proteins under native solution environments.
• We combined steady-state and time-resolved XFMS to study OCP in
various states and the native condition. The solution structural data at
single residue level is used for restraint base docking simulations.
Outcomes and Impacts
• XFMS probed real-time solvent accessibility changes at key OCP
residues during photoactivation and relaxation. We observed a biphasic
photoactivation process in which carotenoid migration preceded domain
dissociation. Using steady-state XFMS, we identified sites of interaction
between the FRP and OCP.
• This study revealed new mechanistic details of the key events in OCP
photoprotection, providing both a spatial and temporal view of site-
specific conformational changes in the OCP.