The document describes a study that screened 168 yeast strains for tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2Ciim][OAc]). 13 strains were found to be tolerant to 5% [C2Ciim][OAc], with Galactomyces geotrichum being the most tolerant. G. geotrichum exhibited enhanced growth in the ionic liquid medium. Several yeasts were also identified that were capable of rapid growth and high cell density in 5% of the ionic liquid.

1. Yeast tolerance to the ionic liquid 1-ethyl-
3-methylimidazolium acetate
Outcomes
• 13 yeast strains tolerant to 5% [C2Ciim][OAc] were identified.
• Galactomyces geotrichum was the most tolerant yeast. This yeast actually exhibited enhanced growth in IL medium.1
Several yeasts capable of rapid growth and high cell density in 5% IL were identified.
1Sitepu, et al., “Yeast tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate.” FEMS Yeast Research
(in press)
Background
• Pretreatment of biomass with
ionic liquids (ILs) can
improve hydrolysis. However,
residual IL on pretreated
biomass can inhibit many
fermentative
microorganisms.
• There is a need for yeasts
that can tolerate up to 4% of
the IL 1-ethyl-3-
methylimidazolium acetate
([C2Ciim][OAc]), a promsing
IL for pretreatment.
Significance
• These [C2Ciim][OAc]-tolerant yeasts could be used for fermentation of hydrolysates
from IL-pretreated biomass or serve as a source of yeast IL-tolerance pathways.
Finalcultureabsorbanceat600nm
Approach
• 168 strains of yeasts spanning multiple phyla from the Phaff Yeast Culture Collection at UC Davis were screened by
culturing in media containing varying levels of [C2Ciim][OAc].

2. Expression of a bacterial 3-dehydroshikimate dehydratase
(QsuB) reduces lignin content and improves biomass
saccharification efficiency.
Outcomes
• Arabidopsis plants expressing QsuB show drastic lignin reductions (~50%), enrichment of H-units, and reduced lignin DP.
• The biomass from the engineered Arabidopsis lines display a two-fold increase of saccharification efficiency.
1 Eudes, A., Noppadon, S., Baidoo, E., George, A., Liang, Y., Yang, F., Singh, S., Keasling, J., Simmons, B., Loque, D. (2014).
Expression of a bacterial 3‐dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification
efficiency. Plant Biotechnology Journal.
Background
• Lignin is a polymer that
confers recalcitrance to plant
biomass.
• Novel approaches to reduce
lignin in bioenergy crops
without affecting biomass
yield are desired for
economical production of
second-generation biofuels
Approach
• Identify enzymes whose
expression in Arabidopsis
leads to lignin reduction1.
Such enzymes will be
expressed in a tissue-specific
manner in bioenergy crops to
avoid the adverse effects of
lignin reduction.
Significance
• Expression of 3-dehydroshikimate dehydratase is a novel strategy to reduce lignin.
• Crops expressing QsuB under the control of tissue-specific promoters are being developed.
3-dehydroshikimate
shikimate
phenylalanine
p-coumaroyl-CoA
p-coumaroyl-shikimate
feruloyl-CoA
p-coumaryl alcohol
coniferyl alcohol
sinapyl alcohol
H-unit
G-unit
HCT shikimate
PCA
QsuB
phenylalanine
coniferaldehyde
sinapaldehydeS-unit
phosphoenol pyruvate
+
p-coumaraldehyde
erythrose-4-phosphate
1) The lignin pathway and expression of QsuB
PCA, protocatechuate
0
2
4
6
8
10
12
14
16
18
WT
qsuB‐1
qsuB‐3
qsuB‐6
qsuB‐7
3) Lignin content, composition (2D‐NMR), and structure (SEC) in qsuB transgenic lines
qsuB-1 WT qsuB-3 qsuB-6 qsuB-7
2) QsuB plants do not show drastic biomass reduction
Lignin (% Cell wall)
H: 3.8%
S: 20.0%
G: 76.2%
H: 27.2%
S: 30.2%
G: 42.6%
0
50
100
150
200
250
300
350
400
450 WT qsuB‐1
qsuB‐3 qsuB‐6
qsuB‐7
4) Biomass saccharification
Sugars (µg mg‐1biomass)
0.00
0.05
0.10
0.15
0.20
0.25
5 10 15 20 25
Elution time (min)
Normalized intensity
m > 22 kDa 22 kDa > m > 0.74 kDa
WT
qsuB-1
m < 0.74 kDa

3. Background
• Protein-protein interactions are important for
enzyme function, including cell wall
biosynthesis
• Current methods to assess interactions have
limitations
Approach
• Develop and validate split luciferase as
method for determining interactions between
plant membrane proteins
• Compare with other methods (BiFC, Y2H)
Outcomes
• Split luciferase was demonstrated to be a
robust method for assessing protein
interactions
A reversible Renilla luciferase protein
complementation assay for rapid identification of
protein-protein interactions reveals the existence of
an interaction network involved in xyloglucan
biosynthesis in the plant Golgi apparatus
Lund, C. H., Bromley, J. R., Stenbaek, A., Rasmussen, R. E., Scheller, H. V., & Sakuragi, Y. (2014). "A
reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein
interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant
Golgi apparatus". J Exp Bot, 18. doi, 10.1093/jxb/eru401
Significance
• The method is an important tool for understanding
cell wall biosynthesis and engineer improved
biofuel crops
FUT1*
XXT2*
XXT5XXT1
CSLC4
*
MUR3
*
Golgi apparatus
When protein fusions
with the N- and C-
termini of luciferase
interact, a functional
luciferase is generated
The method can be used with proteins
transiently expressed in tobacco
The method was used to investigate
interactions between
glycosyltransferases involved in
xyloglucan biosynthesis

4. OH
O
P
O
O
OH
OH
D-glyceraldehyde 3-phosphate
O
O
O-
pyruvate
HO
OH
OHO
P
O
O
OH
OH
D-ribulose 5-phosphate
OH
OHO
P
O
O OH
OH
1-deoxy-D-xylulose 5-phosphate (DXP)
CO2
2C-methyl-D-erythritol 4-phosphate (MEP)
4-diphosphocytidyl-2C-methyl D-erythritol (CDP-ME)
Dxr (ispC)
Dxs
nDXP
(ribB mutants
or yajO)
IspD
IspE
4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate (CDP-MEP)
IspF
2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcPP)
IspG
IspH
(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP)
IPP DMAPP
Idi
Enhancing terpene yield from sugars via
novel routes to DXP (1-deoxy-D-xylulose 5-phosphate)
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
• Terpene production via the DXP pathway entails condensation
of pyruvate and G3P by DXP synthase (Dxs). Carbon is lost
as CO2 in the process.
• To avoid this carbon-loss and circumvent allosteric regulation
of DXS we searched for alternative routes to DXP from a
pentose phosphate.
Approach
• The dxs gene was knocked out in E. coli and we used both
directed evolution and rational gene selection approaches to
discover alternative routes to DXP.
Significance
• The theoretical pathway yield to terpene biofuels has been increased significantly, almost reaching
the maximum biochemical yield. nDXP provides a direct route from C5 sugars to terpenes.
Metabolic route γP Bisabolene (g/g)
Maximum Biochemical Yield ‐ 0.324
Glu, mevalonate 77.8 0.252
Glu, DXP via Dxs 86.2 0.279
Glu, DXP via nDXP 95.4 0.309
Xyl, DXP via Dxs 73.3 0.237
Xyl, DXP via nDXP 79.6 0.258
Kirby et al., “Enhancing Terpene Yield from Sugars via Novel Routes to 1‐Deoxy‐D‐Xylulose 5‐Phosphate”
Appl. Environ. Microbiol. 81: AEM.02920‐14 (2014).
Genome sequencing at the
JGI identified ribB mutants
that complemented a dxs
knockout in E. coli
Outcomes
• Two novel routes to DXP
(nDXP) were discovered
and used to increase
terpene titers in E. coli
grown on either glucose or
xylose.

5. Understanding the Role of Histidine in the GHSxG
Acyltransferase Active Site Motif: Evidence for Histidine
Stabilization of the Malonyl-Enzyme Intermediate
Outcomes
• A histidine to alanine mutation (H640A) in the GHSxG motif yersiniabactin acyltransferase results in an approximately
seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity.
Poust et al., “Understanding the Role of Histidine in the GHSxG Acyltransferase Active Site Motif: Evidence for
Histidine Stabilization of the Malonyl-Enzyme Intermediate.” PloS One. 9: e109421 (2014).
Background
• The role of the conserved
histidine in the active site
motif of acyltransferase
active sites is unclear,
contradictory findings exist in
the literature
Approach
• Utilize high resolution mass
spectrometry and
colorimetric kinetic assays to
determine the acyltransfer
activity and hydrolysis rates
of active site knockouts of
the yersiniabactin
acyltransferase
Significance
• Suggests that acyltransferases have evolved to protect acyl intermediates, functionally diverging from their α/β
hydrolase relatives

6. A droplet-to-digital (D2D) microfluidics
platform for single cell assays1
Outcomes
• A microfluidic device was successfully used to screen the effects of ionic liquids (16 conditions) on cell growth and biofuel
production (comparable to well-plate methods).
• This is a first step towards a higher-throughput microfluidic method which will screen over 400 ionic liquid conditions on
enzymes and biofuel producing microorganisms.
1Shih et al., “A droplet-to-digital (D2D) microfluidic device for single cell assays.” Lab on a Chip. DOI:
10.1039/c4lc00794h
Background
• Ionic liquid (IL) pretreatment
has been shown to efficiently
dissolve biomass and
produce higher lignin-free
cellulose.
• Some IL are toxic to most
enzymes and organisms
used for sacchrification and
fermentation.
Approach
• Develop a microfluidic device
to isolate and to analyze
single cells for toxicity
towards four types of ILs in
various concentrations.
Significance
• This is automated microfluidic method which allows fast determination of toxic IL
with 600-fold reduction in volumes (compared to well-plate methods) and with only
6 pipetting steps.
Microfluidic device (D2D)
Microfluidic automation system
Single yeast cell grown on a
microfluidic device
Growth curves / biofuel production