Biochemical characterization of

Arabidopsis APYRASE family reveals their

roles in regulating endomembrane

NDP/NMP homeostasis

Background • Plant apyrases are nucleoside triphosphate diphosphohydolases

implicated in an array of functions, including the regulation of

extracellular ATP.

• Arabidopsis encodes a family of seven membrane bound

apyrases, with only 2/7 functions known.

Approach • Systematic study of all seven apyrases to determine their

subcellular localizations and substrate specificities.

• Combined phylogenetic and biochemical screening to elucidate

roles and functions, and compared activities to known yeast and

human apyrases and endo-apyrases, respectively.

Outcomes • All Arabidopsis members localize internally within the cell and have

an NDPase preferences. Their roles are thus likely to be involved

in ensuring lumenal UDP / GDP from GT reactions is efficiently

converted to UMP / GMP.

Chiu et al., (2015). "Biochemical characterization of Arabidopsis APYRASE family

reveals their roles in regulating endomembrane NDP/NMP homeostasis”, Biochem J.,

doi:10.1042/bj20150235

Significance • No real evidence in Arabidopsis of an apoplastic apyrase

(NTPase) either subcellular localization or biochemically. This is

contrary to research for the past 20 years implicating 2 members

as having an involvement in external ATP signaling.

cytosol

extracellular matrix

Golgi

NH2 COOH NH2

COOH

Ca2+ , Mg 2+

ER

Ca2+ ,Mg 2+

AtAPY6 NH2 COOH

endosome

AtAPY3

NH2

COOH

AtAPY7 NH2

COOH

NH2

COOH

NH2

COOH

AtAPY1 AtAPY2 AtAPY4

plasma membrane

NDP→NMP UDP→UMP NTP→NDP

?

AtAPY5 NTP → NDP

NDP/NTP → NMP/NDP

Schematic diagram summarizing the subcellular localization, putative topology and major specific

activity of the Arabidopsis apyrase family.

Development of a high throughput

platform for screening glycoside

hydrolases based on oxime-NIMS

Background • Biomass composition varies significantly both in terms of

chemistry and bond moieties.

• Enzyme costs remain significant and there is a need for

simpler, high-performing enzyme mixtures for bioamass

deconstruction.

• desirable to have a reliable high throughput enzyme assay

methods and standardized panels of substrates and

conditions.

Approach • Leverage oxime-NIMS capability and screen three cellulases

against 12 substrates of interest to GLBRC and JBEI.

Outcomes • CelEcc_CBM3a is a multifunctional enzyme that has

cellulase, mannanase and hemicellulase activities.

• CelEcc_CBM3a produced 8x more of hexose products with

IL-SG than AFEX-SG.

• CelRcc-CBM3a has only cellulase activity.

Deng, K. et al. (2015). "Development of a High Throughput Platform for Screening Glycoside Hydrolases

based on Oxime-NIMS”, Frontiers in Bioengineering and Biotechnology, doi:10.3389/fbioe.2015.00153

Significance • This platform automates the handling of both solid biomass and

soluble substrates, the introduction of enzymes as individuals or

a combinations, and the recovery of products for high sensitivity

and high resolution mass spectral analysis

Workflow of oxime-NIMS automation developed to study GHs. 1) Solid biomass was dispersed by Labman. 2) Liquid handling was performed by Biomek Automation Workstation, including

setup of enzyme and oxime bioconjugation reactions. 3) Sample array on the NIMS chip was generated by an acoustic printer

(ATS Acoustic Liquid Dispenser) 4) Mass spectrometry imaging (MSI) provided the readout of experimental assay results.

Labman biomass dispensing robot

Biomek fluid handling robot

Acoustic printer + NIMS

Plate A

Plate B Surface acoustic printer

Characterization of protein N-glycosylation

by tandem mass spectrometry using

complementary fragmentation techniques

Background • The identification, characterization and quantification of

post-translational modifications (PTMs) in proteins are a

major challenge in the field of proteomics.

• The development of different fragmentation techniques

available in the current generation of MS instruments may

provide an alternative approach to study PTMs.

Approach • Used three common fragmentation techniques, namely

CID, HCD,and ETD, to analyze a glycopeptide.

• Develop an integrated fragmentation approach to identify

the modified residue and characterize N-glycan on a

peptide.

Outcomes • This complementary approach identified the peptide

HLTYKENFTDAKAEADHQR with a complex N-linked

glycan.

Ford, K. L., Zeng, W., Heazlewood, J. L., & Bacic, A. (2015). "Characterization of protein N-glycosylation by

tandem mass spectrometry using complementary fragmentation techniques". Front Plant Sci, 6, 674.

doi:10.3389/fpls.2015.00674

Significance • Helps build a vision for a future instrument that simultaneously

perform a variety of fragmentation processes during a standard

proteomics workflow.

• Would enable a global analysis of PTMs in conjunction with

protein identification and label free quantification.

Fragmentation spectra of a plant glycopeptide using (a) CID, (B) HCD, and (C) ETD.

A method to constrain genome-scale

models with 13C labeling data

Background • While metabolic fluxes constitute the most direct

window into a cell’s metabolism, their accurate

measurement is non trivial.

• JBEI has developed a rigorous, self-consistent

method that uses the full amount of information

contained in 13C labeling data to constrain fluxes for a

genome scale model.

Approach • The simultaneous use of 13C labeling data and

genome-scale models combines the advantages of

both FBA and 13C MFA: two-scale 13C Metabolic Flux

Analysis (MFA).

Outcomes • Fluxes calculated through 2S-13C MFA are robust with

respect to their experimental accuracy of the 13C

labeling data.

• Confidence intervals obtained through 2S-13C MFA

immediately identify two types of fluxes, constrained

and loosely constrained, and show that the use of

13C labeling experimental data produce much

narrower confidence intervals than those produced by

FBA.

Garcia Martin, H., Kumar, V. S., Weaver, D., Ghosh, A., Chubukov, V., Mukhopadhyay, A., Arkin, A., &

Keasling, J. D. (2015). "A Method to Constrain Genome-Scale Models with 13C Labeling Data". PLoS Comput

Biol, 11(9), e1004363, doi:10.1371/journal.pcbi.1004363

Significance • This approach will be a tool of extreme utility in bioengineering,

at a time when a variety of different frameworks for flux

prediction for genome-scale models are becoming available

Algorithm flow diagram for 2S-13C MFA showing a recursive procedure to achieve self-consistent results. The full model

consists of a genome-scale model (iJR904 in this case) to which information on carbon transitions for the core sets of

reactions is added.

Calorimetric evaluation indicates that

lignin conversion to advanced biofuels is

vital to improving energy yields

Background • Biomass conversion studies have typically focused on

mass yields rather than calculating energy yields

• Typically do not capture the overall efficiencies and areas

for improving biomass conversion.

Approach • Energy density measurements using bomb calorimetry

were applied along with mass yields to calculate energy

yields from combinations of individual processes and

lignocellulosic feedstocks using ionic liquid pretreatment.

Outcomes • Mass yield from switchgrass (68.0% theoretical) after IL

pretreatment was lower than energy yield (61.6%

theoretical).

• In contrast, energy yield (68.0% theoretical) after IL

pretreatment of eucalyptus, was lower than MY (74.3%

theoretical)

• Due to differences in initial lignin present in the feedstock.

Gardner, J. L., He, W., Li, C. L., Wong, J., Sale, K. L., Simmons, B. A., Singh, S., & Tanjore, D.

(2015). "Calorimetric evaluation indicates that lignin conversion to advanced biofuels is vital to

improving energy yields". RSC Advances, 5(63), 51092-51101, doi:10.1039/c5ra01503k

Significance • Strong correlation between lignin concentrations in pretreated

solids and energy densities.

• New analytical method to establish energy yield as a function of

mass yield of fermentable sugars from biomass conversion

Mass balance and energy yields from mixed feedstocks after [C2C1Im][OAc] pretreatment and subsequent

enzymatic hydrolysis; *calculated values. Note: all energy values are reported after adjusting energy density for ash

and moisture content in the sample.

Local and global structural drivers for the

photoactivation of the orange carotenoid

protein

Background • Photosynthetic organisms have evolved a protective

mechanism known as nonphotochemical quenching (NPQ)

to dissipate excess energy,

• Cyanobacteria, in contrast, use a relatively simple NPQ

mechanism governed by the water soluble orange

carotenoid protein (OCP), and this protein is not well

understood.

Approach • Used multiple analytical techniques to generate new

insights into the mechanism of the OCP.

Outcomes • Light activation causes a global conformation change that

results in the complete separation of the two domains of the

OCP.

• Identified local structural changes in residue solvent

accessibility and roles for structural water molecules in

activation of the OCP.

Gupta, S., Guttman, M., Leverenz, R. L., Zhumadilova, K., Pawlowski, E. G., Petzold, C. J.,

Lee, K. K., Ralston, C. Y., & Kerfeld, C. A. (2015). "Local and global structural drivers for the

photoactivation of the orange carotenoid protein”, Proc Natl Acad Sci,,

doi:10.1073/pnas.1512240112

Significance • By combining small-angle scattering, hydrogen-deuterium

exchange, and X-ray hydroxyl radical footprinting studies, we

were able to construct a model of the structural changes during

the activation of the OCP with an unprecedented level of detail.

Local structural changes in the OCP upon activation monitored by XF-MS. (A) Solvent accessibility changes in response to photoactivation, (B) Modified residues are represented by sticks on the X-ray crystal

structure of OCPO (PDB ID code 3MG1), (C) Solvent accessibility changes at the major and minor interface, (D) Solvent accessibility

changes in the NTD. The color codes are the same as in A.

Refining the phylum Chlorobi by

resolving the phylogeny and metabolic

potential of the representative of a deeply

branching, uncultivated lineage

Background • Chlorobi have been found in microbial communities

associated with high rates of biomass deconstruction

and ionic liquid tolerance, but role and function remains

elusive.

Approach • Phylogenetic trees of the Chlorobi strain NICIL-2 were

built using 16S ribosomal RNA genes and a comparison

was carried out using bioinformatics resources at JBEI.

Outcomes • Despite its abundance as part of a biomass-

deconstructing consortium, NICIL-2 has limited

metabolic potential for the deconstruction of complex

biomass.

• NICIL-2 is likely a secondary consumer of simple sugars

or amino acids produced during biomass deconstruction

by community members.

Hiras, J., Wu, Y.-W., Eichorst, S. A., Simmons, B. A., & Singer, S. W. (2015). "Refining the phylum

Chlorobi by resolving the phylogeny and metabolic potential of the representative of a deeply

branching, uncultivated lineage”, ISME J., doi:10.1038/ismej.2015.158

Significance • The targeted recovery of genomes from uncultivated organisms

related to the NICIL-2 provides a promising route to build a more

detailed evolutionary model linking the Bacteroidetes and the

Chlorobi.

Reconstructed metabolism of NICIL-2 inferred from the reassembled genome. Red text represents enzymes or

biosynthetic pathways that are missing from the genome. Blue, green and purple arrows indicate ATP,

NADH and NADPH flow, respectively.

Assay for lignin breakdown based on

lignin films: insights into the Fenton

reaction with insoluble lignin

Background • The valorization of lignin is currently one of the greatest

challenges in developing economically viable industries based

on lignocellulosic biomass.

• One challenge is a rapid and robust diagnostic assay for

insoluble lignin breakdown by enzymes, catalysts, or microbes.

Approach • Developed a robust technique based on the amount of mass

released from the film as soluble fragments that is quantified

through the decrease in film thickness measured by

ellipsometry.

Outcomes • The method is quantitative and highly sensitive, as film

thickness can be determined to within ±20 Å.

• While the film format precludes interrogation by NMR, chemical

bonding within the film can be studied by surface analytical

techniques such as FTIR-ATR or XPS, and the chemical nature

of the fragments released can be identified by mass

spectrometry, FTIR, and UV-vis

Kent, M. S., Avina, I. C., Rader, N., Busse, M. L., George, A., Sathitsuksanoh, N., Baidoo, E., Timlin, J., Giron,

N. H., Celina, M. C., Martin, L. E., Polsky, R., Chavez, V. H., Huber, D. L., Keasling, J. D., Singh, S., Simmons,

B. A., & Sale, K. L. (2015). "Assay for lignin breakdown based on lignin films: insights into the Fenton reaction

with insoluble lignin”, Green Chem., doi:10.1039/c5gc01083g

Significance • With a high sensitivity and multiplexed format, this new assay

should be useful for rapid assessment of catalytic, enzymatic,

and microbial degradation of insoluble lignin

(a) Photo of a lignin film-coated silicon wafer after use in multiplexed format. The reaction mixtures were incubated against the lignin film for 18 h at RT. (b) Decrease in lignin film thickness as a function of FeCl2 concentration corresponding to the image in (a).

Theoretical insights into the role of water

in the dissolution of cellulose using

IL/water mixed solvent systems Background • Ionic liquid (IL) pretreatment is promising, but there

remain unanswered questions into the exact

mechanisms between molecules that give rise to desired

performance during pretreatment.

Approach • Use computational modeling (all atom MD) to

understand the fundamental interactions between water-

IL-cellulose to determine dominant forces that govern

cellulose dissolution. The IL studied is 1-ethyl-3-

methylimidazolium acetate ([C2C1Im][OAc]).

Outcomes • Interaction of the IL [C2C1Im][OAc] and water at certain

concentrations plays as physicochemical driving forces

assisting the cellulose dissolution process.

• Preferred solvation of [OAc]- energetically favors their

accumulation around the cellulose hydroxyl groups of

the glucan chains and disrupts inter-chain H-bonds

• Increases in water concentration leads to spontaneous

solvation of both [C2C1Im][OAc] and cellulose, which in

turn disrupts the overall pretreatment process efficacy.

Parthasarathi, R., Balamurugan, K., Shi, J., Subramanian, V., Simmons, B. A., & Singh, S.

(2015). "Theoretical Insights into the Role of Water in the Dissolution of Cellulose using

IL/Water Mixed Solvent Systems". J Phys Chem B., doi:10.1021/acs.jpcb.5b02680.

Significance • Knowledge gained from this study provides a better

understanding of the dual role played by the water (as a co-

solvent/antisolvent) in dissolving cellulose.

The final structural snapshots of the simulated system for 100 ns at 433K.

Analytics for metabolic engineering

Background • There have been significant advances in the fields of

systems biology and metabolic engineering over the past

decade.

• Multiple applications in energy, environment, and health

if true potential is realized

• Current approaches, although sometimes very

successful, are energy, resource, and bandwidth

intensive, with very little translation of knowledge

between institutions and projects.

Outcomes • This review article highlights the need for an advanced

Design-Build-Test-Learn (DBTL) cycle, with the following

analytical attributes:

Advanced gene and pathway design

Pathway selection and analysis

Multiple robust hosts

Metabolomics

Proteomics

High-throughput screening

Automation

Machine learning

Petzold, C. J., Chan, L. J., Nhan, M., & Adams, P. D. (2015). "Analytics for Metabolic

Engineering”, Front Bioeng Biotechnol, doi:10.3389/fbioe.2015.00135

Significance • Informs the broader scientific community on the gaps,

challenges, and opportunities for advances in metabolic

engineering and advanced biomanufacturing.

The design–build–test–learn cycle of metabolic engineering highlighting important parts of each of the components. The Design component identifies the problem, selects the desired pathway and host; the Build component selects, synthesizes, and assembles parts

for incorporation into the host; the Test component validates the engineered strains for target molecule production, transcripts,

proteins, and metabolites; the Learn component analyzes the Test data and informs subsequent iterations of the cycle.

Structural features affecting the

enzymatic digestibility of pine wood

pretreated with ionic liquids

Background • Pinus radiata (pine) is a promising feedstock worldwode

for the production of advanced biofuels and renewable

chemicals, but is notoriously difficult to process due to

terpene content and lignin.

• Certain ionic liquids (ILs) are promising but relatively

unproven for processing pine, and the important factors

in determining sugar yields are not well known.

Approach • Evaluate two different ionic liquids over a range of

temperatures to determine impact on structure,

chemistry, and enzyme accessibility.

Outcomes • Detailed analysis of the substrates revealed that the

most important change brought about by the IL

pretreatments was an increase in accessible surface

area.

• Loss of cellulose crystallinity only occurred for the more

severe [C2C1Im][OAc] pretreatments.

• Maximum glucose yields of 84% were obtained.

Torr, K. M., Love, K. T., Simmons, B. A., & Hill, S. J. (2015). "Structural features affecting the enzymatic

digestibility of pine wood pretreated with ionic liquids”, Biotechnol Bioeng., doi:10.1002/bit.25831

Significance • Provides new insights into the mechanism of IL

pretreatment and the most significant factors that

generate high yields of fermentable sugars.

Field emission scanning electron microscopy images of Pinus radiata wood untreated (A), pretreated with [C2C1Im]Cl at 80

oC (B) and 120 oC (C), and pretreated with [C2C1Im][OAc] at 80 oC (D), 100 oC (E), and 120 oC (F). Scale bar =10 um.