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/

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 xylansynthase.

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.

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 infrastructureand potential availability at negative cost. An efficientuse of MSW would not only benefit biofuel industry butalso reduce landfill disposal

• Feedstock costs could be reduced by blending MSW withother high quality feedstocks

• Among the various options of biomass pretreatmentstrategies, ionic liquid (IL) pretreatment with imidazolium-based ILs has been proven to be one of the mosteffective 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

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, lendincreased stability to medicinal and commoditycompounds, however, our ability to rationallyincorporate this functional group in modified naturalproducts is limited.

Approach and Outcomes• To characterize the mechanism of gem-dimethyl

group formation, with a goal toward engineering ofnovel compounds containing this moiety, the gem-dimethyl group producing polyketide synthasemodules of yersiniabactin and epothilone werecharacterized using mass spectrometry.

• The work demonstrated, contrary to the canonicalunderstanding of reaction order in PKSs, thatmethylation 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‐DimethylGroups 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

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 ConsortiaBackground• 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‐Thermuspopulation, 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)

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)

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 recognizingconserved molecules. The dicot EFR-TU receptor (EFR)activates an immune response after recognizing the elf18epitope. XA21 is a monocot PRR from rice that confersqualitative resistance to the rice pathogen Xanthomonasoryzae pv oryzae (Xoo).

• Functionality of dicot immune receptors expressed inmonocots 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 andEFR::XA21 are functional after elf18 activation and if theyconfer resistance to Xoo.

• We confirmed elf18 specific activation of immune- relatedmolecular events such as reactive oxygen species (ROS)production, MAPK cascade activation, and stress geneinduction in EFR and EFR::XA21 rice.

• EFR and EFR::XA21 confer quantitative resistance toweakly virulent strains of Xoo. EFR interacts with andrequires XB24 and OsSERK2, which are also required forXA21-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 RNAiknockdown transgenes.

Pr10b

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 ofmicrobes have progressed significantly in the last fewyears, yet analytical capabilities required to efficientlymonitor and assess these changes are laggingbehind.

• Most biotechnology research challenges are notconstrained by the sensitivity or resolution of anassay, rather they depend on accurate identificationand quantitation of target molecules for a largenumber of samples.

Approach and Outcomes• We assessed the suitability of the standard flow

proteomics platform using samples from Escherichiacoli and Arabidopsis thaliana, organisms commonlyused as model systems for biofuels research.

• Nearly 800 proteins from E. coli samples were foundand over 1,000 proteins for A. thaliana reliablyidentified (Table 1).

• Venn diagrams of identified proteins and peptides forindividual 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