The document discusses standard flow liquid chromatography for shotgun proteomics in bioenergy research. The approach assesses the suitability of this proteomics platform using samples from Escherichia coli and Arabidopsis thaliana, which are commonly used model systems. Nearly 800 proteins from E. coli samples and over 1,000 proteins from A. thaliana samples were reliably identified. The results indicate this is a robust approach for analyzing complex bioenergy samples, providing reproducibility between replicates.

1. Background
• Functional gene networks have been demonstrated to serve as
powerful approaches for generating holistic models of pathways in
many organisms including plants.
• RiceNet, was demonstrated to be useful in identifying genes that are
involved in biotic stress responses. (Over 70 citations)
Approach
• The gold standard co-functional gene pair used for network training
was generated by pairing Oryza. sativa ssp. japonica genes that share
the same pathway annotations by at least one of the four databases.
Outcomes
• Improved algorithms to infer co-functional links from gene
neighborhood.
• New associalogs from the latest networks for other species.
• Substantially larger amount of expression data derived from Gene
Expression Omnibus (GEO) database.
Significance
• RiceNet v2 is substantially improved in terms of both genome
coverage and network accuracy, leading to enhancement in prediction
power.
RiceNet v2: an improved network
prioritization server for rice genes
Lee et al. (2015). “RiceNet v2: an improved network prioritization server for rice genes”.
Nucleic Acids Res., doi, 10.1093/nar/gkv253
www.inetbio.org/ricenet/

2. Background
• Xylan constitute about 30% of biomass and is composed
largely of xylose
• The key substrate for xylan biosynthesis is UDP-xylose, which
is produced both inside the Gogli apparatus and in the
cytoplasm
• UDP-xylose made in the cytoplasms must be transported into
the Golgi by specific transporters
Approach
• Nucleotide sugar transporters were screened by (1)
heterologous expression in yeast and transport assays and
(2) by examining the cell wall phenotype of mutants and
overexpressors in Arabidopsis
Outcomes
• Identification of three UDP-xylose transporters
• UXT1 is rate limiting for xylan biosynthesis in stems, but not for
xyloglucan biosynthesis
• The cytoplasmic UDP-xylose synthesis is essential for xylan
biosynthesis
Identification and Characterization of a Golgi-
Localized UDP-Xylose Transporter Family from
Arabidopsis
Ebert et al, (2015). "Identification and Characterization of a Golgi‐ Localized UDP‐Xylose
Transporter Family from Arabidopsis". Plant Cell. doi, 10.1105/tpc.114.133827
Significance
• Nucleotide sugar transporters are powerful tools for cell wall
engineering
• The specificity of the transporters suggest substrate channeling
Many proteins are needed
to make xylan, and we still
do not know all of them.
UDP-Xyl transporter(s)
(arrow) are essential for
delivering substrate to xylan
synthase.
The UXT transporters are
related to the URGT1 (UDP-
Gal/UDP-Rha) transporters
previously identified at JBEI The three UDP-Xyl
transporters are located
in the Golgi membranes
Loss-of-function mutants in UXT1 are deficient in cell wall xylose.
Xylan is specifically reduced (LM10, LM11, UX1 and AX1
antibodies) while xyloglucan and cellulose are not affected.

3. Blending Municipal Solid Waste with Corn Stover
for Sugar Production Using Ionic Liquid Process
1Linshiz, et al., “PaR-PaR: Laboratory Automation System.” ACS Synth. Biol. 2:216-222 (2013).
2Linshiz, et al., “PR-PR: Cross-Platform Laboratory System.” ACS Synth. Biol. Article ASAP (2014).
Background
• Municipal solid waste (MSW) has the advantage of year-
round availability, an established collection infrastructure
and potential availability at negative cost. An efficient
use of MSW would not only benefit biofuel industry but
also reduce landfill disposal
• Feedstock costs could be reduced by blending MSW with
other high quality feedstocks
• Among the various options of biomass pretreatment
strategies, ionic liquid (IL) pretreatment with imidazolium-
based ILs has been proven to be one of the most
effective ways for biomass processing
Approach and Outcomes
• MSW can be blended into corn stover (CS) providing
lower cost biorefinery feedstock inputs that are easily
pretreated using the IL pretreatment technology
• After acetate based IL pretreatment, up to 84% glucose
and 75% xylose are released. Pretreatment in Cl based
IL followed by acidolysis is also efficient with maximums
of 80% glucose yield and 90% xylose yield
Significance
• MSW can be blended into corn stover providing lower cost
biorefinery feedstock inputs that are easily pretreated using the IL
pretreatment technology
Sun et al. (2015). "Blending municipal solid waste with corn stover for sugar production using ionic liquid process".
Bioresour Technol, 186(0), 200‐206. doi, 10.1016/j.biortech.2015.02.087 .
DOE target
70$/ ton 80$/ ton >100$/ ton
Acidolysis sugar yields
Delivered feedstock costs

4. Divergent Mechanistic Routes for the
Formation of gem-Dimethyl Groups in the
Biosynthesis of Complex Polyketides
1Linshiz, et al., “PaR-PaR: Laboratory Automation System.” ACS Synth. Biol. 2:216-222 (2013).
2Linshiz, et al., “PR-PR: Cross-Platform Laboratory System.” ACS Synth. Biol. Article ASAP (2
Background
• The gem-dimethyl groups in polyketide-derived
natural products add steric bulk and, accordingly, lend
increased stability to medicinal and commodity
compounds, however, our ability to rationally
incorporate this functional group in modified natural
products is limited.
Approach and Outcomes
• To characterize the mechanism of gem-dimethyl
group formation, with a goal toward engineering of
novel compounds containing this moiety, the gem-
dimethyl group producing polyketide synthase
modules of yersiniabactin and epothilone were
characterized using mass spectrometry.
• The work demonstrated, contrary to the canonical
understanding of reaction order in PKSs, that
methylation can precede condensation in gem-
dimethyl group producing PKS modules.
Significance
• PKS engineering strategies to incorporate gem-dimethyl groups using
combinatorial biosynthesis should swap whole modules, instead of
swapping individual MT domains into modules unaccustomed to a
particular order of reactions
Poust et al. (2015). "Divergent Mechanistic Routes for the Formation of gem‐Dimethyl
Groups in the Biosynthesis of Complex Polyketides". Angewandte Chemie 127, 2400‐2403.
OH
OO
SACP
Acyl-KS
R
OO
SACP
yPKS
OH
OO
SCoA
ACP
Sfp
O
S
O
R
KS

5. Wu et al. (2015) “Genomic Analysis of Xylose Metabolism in Members of the Deinoccocus‐
Thermus Phylum from Thermophilic Biomass‐Deconstructing Bacterial Consortia”. Bioenergy
Research, 1‐8. doi 10.1007/s12155‐015‐9600‐7
Genomic Analysis of Xylose Metabolism in Members
of the Deinoccocus-Thermus Phylum from
Thermophilic Biomass-Deconstructing Bacterial
Consortia
Background
• Members of the phylum Deinoccocus-Thermus are capable of growing under
extremes of temperature and radiation and may have broad applications in
biotechnology. However, the specific roles of members of Deinoccocus-
Thermus in plant biomass deconstruction remains largely unknown.
• Adaptations of thermophilic communities to grow on plant biomass
substrates as the sole carbon source have consistently produced consortia
with abundant populations affiliated with the Deinoccocus-Thermus, which
enables us to take a closer look at these bacterial populations.
Outcomes
• Two bacterial populations recovered from an adapted culture grown on
xylan-rich substrates, NIC-1 (distantly related to Truepera radiovictrix)
and Thermus thermophilus, were relatively abundant in this microbial
community.
• A putative operon for xylose utilization was identified on the NIC-1
genome but was absent from the Thermus thermophilus genome.
• Inspection of metagenomic datasets for adapted communities indicates
that the xylose-utilization genes were present on the plasmid of the T.
thermophilus populations but may be lost upon isolation.
Phylogenetic trees of the Deinococcus‐Thermus
population, NIC‐1, isolated from the adapted
microbial community grown on xylan‐rich substrate.
Significance
• The Deinococcus-Thermus genomes recovered from the adapted community allow researchers to understand the roles of
this phylum in plant biomass deconstruction.
• The operon for xylose utilization found in the NIC-1 genome lends support to the hypothesis that the Deinococcus-Thermus
populations may be a secondary consumer of xylose in adapted communities.
‐ The putative operon for xylose utilization (top)
‐ the xylose‐utilization genes found on the plasmid of
the Thermus thermophilus population (bottom)

6. An Investigation on the Economic Feasibility of
Macroalgae as a Potential Feedstock for Biorefineries
Background
• Macroalgal biomass has been considered as a prospective
feedstock for biofuel production as, among other benefits, it is an
abundant source of renewable sugars and its growth does not
require arable land, fresh water, or intense care.
• Successful commercial deployment of macroalgae-based
biorefineries, however, depends on their economic viability at
industrial scales.
Approach
• A detailed technoeoconomic analysis (TEA) of a macroalgae
biorefinery (Fig. 1) was carried out to understand the economic
potential and cost drivers of macroalgae as a feedstock for the
production of biofuels and biochemicals.
Outcomes
• Based on the TEA of the base case macroalgae-to-ethanol
biorefinery, the MESP was $8.5/gal, with costs notably influenced
by the macroalgae price, the overall yield, the solids loading in
the process and the enzyme loading in hydrolysis (Fig. 2, left).
• While the co-production of alginate was observed to improve the
overall economics of the biorefinery, the expected production of
alginate from a single biorefinery (i.e., 130,000-220,000 MT/yr)
was far greater than its global demand (i.e., 26,500 MT/yr).
Significance
• TEA has successfully identified the key cost bottlenecks associated with macroalgae biorefineries.
• To bring the MESP to $2.5/gal or less, multiple advances (i.e., ≥80% yield, ≥20% solids loading,
≤10 mg/g enzyme loading, and a feedstock price of $26/MT) need to be realized (Fig. 2, right).
Konda et. al. (2015). "An Investigation on the Economic Feasibility of Macroalgae as a Potential
Feedstock for Biorefineries". BioEnergy Research. doi, 10.1007/s12155‐015‐9594‐1
Fig. 1 Simplified representation of the macroalgae biorefinery
Fig. 2 Cost breakdown (left) and sensitivity analysis (right)

7. Transgenic Expression of the Dicotyledonous Pattern
Recognition Receptor EFR in Rice Leads to
Ligand-Dependent Activation of Defense Responses.
Background
• Plant pattern recognition receptors (PRRs) confer
quantitative resistance to pathogens after recognizing
conserved molecules. The dicot EFR-TU receptor (EFR)
activates an immune response after recognizing the elf18
epitope. XA21 is a monocot PRR from rice that confers
qualitative resistance to the rice pathogen Xanthomonas
oryzae pv oryzae (Xoo).
• Functionality of dicot immune receptors expressed in
monocots has not been well established.
Approach and Outcomes
• We expressed the EFR protein in rice as well as a
chimeric EFR::XA21 protein to determine if EFR and
EFR::XA21 are functional after elf18 activation and if they
confer resistance to Xoo.
• We confirmed elf18 specific activation of immune- related
molecular events such as reactive oxygen species (ROS)
production, MAPK cascade activation, and stress gene
induction in EFR and EFR::XA21 rice.
• EFR and EFR::XA21 confer quantitative resistance to
weakly virulent strains of Xoo. EFR interacts with and
requires XB24 and OsSERK2, which are also required for
XA21-mediated immunity to Xoo in rice.
Significance
• We show that the dicot derived EFR protein functions when expressed in rice. We provide
evidence that overlapping genes are required for dicot- and monocot- derived PRR function in rice.
Schwessinger et al., 2015. Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice
Leads to Ligand‐Dependent Activation of Defense Responses. PLoS Pathogens, 11(3), p.e1004809.
elf18 treatment in EFR and EFF::XA21 rice, but not
WT Kitaake, leads to ROS production
EFR rice exhibit
induction of stress
related gene, Pr10b,
after elf18 treatment.
However, not in rice
expressing EFR and
OsSERK2 RNAi
knockdown
transgenes.
Pr10b

8. E. coli
A. thaliana
Standard flow liquid chromatography for
shotgun proteomics in bioenergy research
Background
• Advances in biofuels research focusing on feedstock
characterization and the genetic manipulation of
microbes have progressed significantly in the last few
years, yet analytical capabilities required to efficiently
monitor and assess these changes are lagging
behind.
• Most biotechnology research challenges are not
constrained by the sensitivity or resolution of an
assay, rather they depend on accurate identification
and quantitation of target molecules for a large
number of samples.
Approach and Outcomes
• We assessed the suitability of the standard flow
proteomics platform using samples from Escherichia
coli and Arabidopsis thaliana, organisms commonly
used as model systems for biofuels research.
• Nearly 800 proteins from E. coli samples were found
and over 1,000 proteins for A. thaliana reliably
identified (Table 1).
• Venn diagrams of identified proteins and peptides for
individual replicates indicate high reproducibility.
Significance
• Standard flow liquid chromatography for shotgun proteomics provides a robust approach for the
analysis of complex samples.
González et al. (2015). "Standard flow liquid chromatography for shotgun proteomics in
bioenergy research". Front. Bioeng. Biotechnol., 3:44, doi: 10.3389/fbioe.2015.00044.
Table 1