JBEI Highlights - August 2014

1. A high-throughput targeted proteomics
toolkit for metabolic engineering
Outcomes
• We developed a toolkit for >400 E. coli proteins
with a high-throughput proteomics method.
• We constructed 18 synthetic genes to make
over 800 peptide standards for absolute
quantification of E. coli proteins.
1) Target
proteins
1Batth, et al., “A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli
proteins.” Metab. Eng. (2014) DOI: 10.1016/j.ymben.2014.08.004.
Background
• Recent developments in
metabolic engineering have
accelerated pathway construction
producing novel engineered
organisms more rapidly than ever
before.
• Yet, the throughput of proteomic
technologies have lagged far
behind
Approach
• Develop targeted proteomics
toolkit to rapidly quantify protein
amounts of engineered microbes1
Significance
• This toolkit provides an invaluable resource for metabolic engineering by simplifying proteomic
analysis via increased sample throughput and reduced development time.
2) Make peptide
standards
3) Quantify protein
amounts
4) Absolute quantification proteins from
E. coli grown on glucose and xylose

2. Environmental Lifecycle Assessment
(LCA) of Aviation Biofuels
Outcomes
• We determined the environmental impacts for the three routes, as shown in the result tables above (from ref. 1)
• The impacts depend on the assessment method, which highlights the need to consider multidimensional metrics
Results of the LCA via the system expansion and economic allocation methods (ref. 1)
References:
1. Cox, K., Renouf, M., Dargan, A., Turner, C. and Klein-Marcuschamer, D. (2014), Environmental life cycle assessment (LCA) of aviation biofuel from microalgae,
Pongamia pinnata, and sugarcane molasses. Biofuels, Bioprod. Bioref., 8: 579–593. doi: 10.1002/bbb.1488.
Background
• Air transport consumes ~10% of
global transportation energy and
the aviation industry is looking to
reduce their GHG emission
contribution
• Lifecycle Assessment (LCA) is
used to measure the environmental
impact of different production
routes and was applied to biojet
fuel production from sugarcane,
Pongamia, and microalgae
• This project was spearheaded and
supported by Boeing R&T Australia
Approach
• We modeled the environmental
impacts of the three routes using
attributional LCAs, applying both
economic allocation and system
expansion
Significance
• The study provides a thorough LCA in the oftentimes ignored area of air transport. Aviation
fuels make up a significant fraction of transportation energy needs and biofuels provide the
only viable route for fueling air transport using renewable resources

3. Comparison of enzymatic reactivity of
corn stover solids prepared by dilute acid,
AFEX, and ionic liquid pretreatments
Gao, X., Kumar, R., Singh, S., Simmons, B. A., Balan, V., Dale, B. E., & Wyman, C. E. (2014). "Comparison of enzymatic reactivity of corn stover solids prepared by dilute acid, AFEX, and ionic liquid
pretreatments". Biotechnol Biofuels, 7(71), 71. doi, 10.1186/1754-6834-7-71.
Background
BESC-JBEI-GLBRC is
collaborating to understand how
three different pretreatment
technologies (Dilute acid, ionic
liquid and AFEX) being
developed at BRCs influence
substrate features and sugar
yields.
Approach
In this study, corn stover was
pretreated by DA, AFEXTM, IL,
and enzymatic digestion was
performed on the pretreated
solids at low to high protein
loadings with ratios of cellulase,
xylanase, and pectinase
enzymes optimized for each
pretreatment.
Results & Significance
• Substrate reactivity and digestibility were affected by substrate features and interactions between substrate and enzymes
for all three pretreatment technologies.
• Ionic liquid pretreated corn stover displayed the highest initial reactivity at all enzyme loadings and the highest final
digestibility for a low enzyme loading of 3 mg protein/g glucan in the raw material.
• Increasing the enzyme loading to 12 mg/g glucan resulted in dilute acid and AFEXTM pretreated
corn stover attaining higher cellulose digestions.
• No single factor accounts for enzymatic digestion performance for the three pretreated materials.

4. A peptide-based method for 13C metabolic
flux analysis in microbial communities
Outcomes
• A new method to infer intracellular metabolic fluxes from the labeling of peptides.
• This approach has the advantage that peptides can be assigned to each species in a community in a high-throughput
fashion through modern proteomic methods.
• We computationally tested this method with a well-characterized simple microbial community consisting of two species.
1Amit Ghosh, et al., “A peptide-based method for 13C metabolic flux analysis in microbial communities.”
PLOS Computational Biol. (2014) Volume 10, Issue 9, e1003827.
Background
•Microbial communities underlie a
variety of important biochemical
processes ranging from
underground cave formation to gold
mining or the onset of obesity. The
most authoritative method to
measure fluxes for pure cultures
consists of feeding the cells a
labeled carbon source and deriving
the fluxes from the ensuing
metabolite labeling pattern (typically
amino acids). Since we cannot
easily separate cells of metabolite
for each species in a community,
this approach is not generally
applicable to microbial communities.
Significance
• We have shown, by using this method, it is theoretically possible to recover the same
amount of information as through the standard approach, if enough peptides were used.
Mixed Culture
C13
C12
Microbial Community 13C isotope labeled Peptide Flux profile
AAGSE
AAGSE
HLLEG
HLLEG
13C labeled
Glucose
http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1003827

5. Developing a foundation for assessing
and mitigating the environmental risks of
microbial metabolic engineering
Outcomes
• Capacity to culture a wastewater treatment plant microbial community and maintain adequate diversity and repeatability to
test engineered bacteria survival and gene transfer rates in a laboratory-scale model system.1
• Chloroacetate, D-threonine, and the ionic liquid [C2mim]Cl could serve as effective selective media in future experiments.
Microbial composition of wastewater treatment plant
and laboratory-cultured samples.
1Lee et al., “Characterization of wastewater treatment plant microbial communities and the effects of
carbon sources on diversity in laboratory models.” PLoS One 9(8), e105689 (2014).
Background
• We are developing a
laboratory model to improve
our capacity to assess the
biological risks of engineered
bacteria in the environment.
• An industrial bioreactor
failure could introduce
engineered bacteria to a
downstream wastewater
treatment plant.
Approach
• Investigate how the waste-
water microbial community
changes when propagated in
the laboratory under a variety
of growth media conditions.
Significance
• Establishes a foundation for assessing and mitigating the risks of future large-scale
microbial metabolic engineering projects, including those beyond the bioreactor.
Overview of experiments.

6. Site-Directed Mutagenesis of IRX9, IRX9L and IRX14
Proteins Involved in Xylan Biosynthesis:
Glycosyltransferase Activity Is Not Required for IRX9
Function in Arabidopsis
Outcomes
• Mutated IRX9 and IRX14 can support xylan
biosynthesis
• The role of the proteins is not catalytic
Ren, Y., Hansen, S. F., Ebert, B., Lau, J., & Scheller, H. V. (2014). "Site-Directed Mutagenesis of IRX9, IRX9L and IRX14
Proteins Involved in Xylan Biosynthesis: Glycosyltransferase Activity Is Not Required for IRX9 Function in Arabidopsis".
PLoS One, 9(8), e105014. doi, 10.1371/journal.pone.0105014
Background
• Three different putative glycosyltransfereases
(IRX9, IRX10 and IRX14) are required to
make xylan
• This raises questions about the role of the
individual proteins and the mechanism of
xylan biosynthesis
Approach
• The IRX9 and IRX14 proteins were
mutagenized to destroy putative active sites
• The ability of the mutant proteins to function
in xylan biosynthesis was investigated.
Significance
• Provides a better understanding of xylan
biosynthesis
• Suggest the presence of a xylan synthase
protein complex and new strategies to
engineer bioenergy crops
IRX10
IRX9 IRX14
0
100
200
300
400
500
Inflorescencestemheight(mm)
d debde bd beab
ab a
a
c
Xylan biosynthesis takes
place in the Golgi apparatus
and requires a large number
of proteins
All mutated versions of IRX9 complemented
the irx9-2 mutant
IRX9 and IRX14 most likely
function by anchoring the
xylan synthase protein IRX10
in the membrane

7. Identification of a Sphingolipid
α-Glucuronosyltransferase that is Essential
for Pollen Function in Arabidopsis
Outcomes
• A gene named IPUT1 was shown to encode an enzyme that glycosylates GIPC lipids.
• Plants lacking a functional copy of IPUT1 are unable to make functional pollen, indicating that GIPCs are essential.
Rennie, et al., “Identification of a Sphingolipid α-Glucuronosyltransferase that is Essential for Pollen Function in
Arabidopsis. Plant Cell 26:3314-3325 (2014). http://www.plantcell.org/content/26/8/3314.short
Background
• Plants contain an important
lipid component called
glycosyl inositol
phosphorylceramides
(GIPCs).
• GIPCs make up about 25%
of plant membranes, but
almost nothing is known
about their synthesis and
functions.
Approach
• Demonstrate the function of
GIPC biosynthetic genes by
engineering yeast to make
plant-type GIPC lipids.
Significance
• Provides genetic tools with which to study an important, abundant type of plant lipid.
Parent genotype Selfing TEOvule TEPollen
iput1‐1 / + 0.98 : 1 95% 1%
iput1‐2 / + 0.93:1 92% 2%
Genetic analysis indicates that only 1-2% of Arabidopsis pollen is functional without GIPCs
Cytosol Golgi
UDP-Glc
UDP-GlcA
UGD2
hUGTrel7
GDP-Man
IPC
SUR1
MIPC
UDP-GlcA GlcA-
IPCIPUT1
Saccharomyces cerevisiae
Plant GIPC pathway engineered into yeast includes:
- Removal of endogenous glycosyltransferase (SUR1)
- Dehydrogenase (UGD2)
- Sugar transporter (hUGTrel7)
- Glycosyltransferase (IPUT1) MIPC
GlcA-IPC
m/z
m/z
WT yeast
Engineered
yeast
Mass spectrometry shows engineered
yeast make GlcA-IPC (plant lipid)
instead of MIPC (yeast lipid)

8. Structure of the OsSERK2 leucine rich
repeat extracellular domain
Background
• OsSERK2 is an integral membrane receptors involved in the
regulation of development and immune responses in plants.
• OsSERK2 is essential for XA21-, BRI1-, FLS2-, and EFR-mediated
responses.
Approach
• We determined the crystal structure of the extracellular domain of
OsSERK2 and a point mutant (D128N) that is predicted to have
altered binding to coreceptors.1
• The extracellular domain was fused to the variable lymphocyte
receptor from hagfish to facilitate expression and crystallization.1
Outcomes
• The structures of OsSERK2 and the D128N mutant reveal local
structural changes which suggest a mechanism for the altered
interaction with coreceptors in the D128N mutant.1
Significance
• Understanding SERK structure and function will help optimize the
growth-defense balance to maximize crop yields.
1McAndrew, et al., 2014, Structure of the OsSERK2 leucine rich repeat extracellular
domain, Accepted for publication in Acta Crystallographica Section D

9. Ionic Liquids (ILs) derived from biomass –
provide an effective biomass pretreatment
Background
Ionic Liquids (ILs) have been shown to be an excellent pretreatment solvent
for biomass; however, the availability and cost of the ILs remains an
issue.
Approach
First investigation of synthesis and evaluation of a series of new ILs from
monomers obtained directly from lignin and hemicellulose.
Tertiary amine‐based ILs were synthesized from aromatic aldehydes
derived from lignin and hemicellulose. Molecular modeling was used to
compare IL solvent parameters with experimentally obtained
compositional analysis data.
Effective pretreatment using these new ILs of switchgrass was investigated
by
• powder X-ray diffraction showing structural changes in cellulose and
• glycome profiling showing changes in the extractability of hemicellulose
epitopes.
Socha, et al., “Efficient biomass pretreatment using ionic liquids derived from lignin and hemicellulose,” PNAS, published online
August 18, 2014, http://www.pnas.org/content/early/2014/08/15/1405685111.full.pdf+html
Outcomes
• Reductive animation of aromatic aldehydes followed by treatment with phosphoric acid provided three
biomass‐derived ILs in excellent yields without the need for chromatographic purification.
• Renewable ILs generate comparable high sugar yields after pretreatment + saccharification relative to current
imidazolium‐based ILs.
• Cost projections of renewable ILs are $4/kg, much lower than top performing conventional ILs.
Significance
• Deriving ILs from lignocellulosic biomass shows significant potential for the realization of a “closed‐loop”
process for future lignocellulosic biorefineries and has far‐reaching economic impacts for other IL‐based
conversion technology currently using ILs synthesized from petroleum sources.
Process scheme for a closed‐loop biorefinery using ILs derived
from lignocellulosic biomass