The document describes research that engineered the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in the yeast Saccharomyces cerevisiae. Key findings include: - An engineered strain was able to grow in the absence of mevalonate, reaching 80% of growth compared to strains with mevalonate added. - The DXP pathway became functional enough to support moderate growth without the mevalonate pathway, but growth on agar without mevalonate was not achieved. - This work established a foundation for further optimizing yeast to produce bioenergy-relevant isoprenoids through the DXP pathway under industrial conditions.

1. Engineering a functional 1-deoxy-D-xylulose
5-phosphate (DXP) pathway in
Saccharomyces cerevisiae
Outcomes
• One engineered strain grew in the absence of
mevalonate, reaching an OD around 80% of that of
the same strain with 15 mM mevalonate
• The DXP pathway became functional to the point
where it could sustain moderate growth of S.
cerevisiae in the absence of the MEV pathway but
we were unable to achieve growth on agar in the
absence of mevalonate
Kirby et al. (2016) “Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in
Saccharomyces cerevisiae”. Metab Eng. pii: S1096-7176(16)30191-4
Background
• Isoprenoids are a valuable class of biofuels and
renewable chemicals
• There are two microbial pathways to produce
these compounds – mevalonate (MEV) and DXP
• S. cerevisiae uses MEV pathway, but it may be
advantageous to use DXP instead
Approach
• Addressed barriers to DXP pathway in S.
cerevisiae using synthetic biology, biochemistry
and metabolomics
• IsG and IsP (both Fe-S) proteins in DXP pathway
are critical “pinch points” that must be addressed
Growth of S. cerevisiae
supported by the DXP
pathway. Strain Y6084-a
is Y6084 containing
pAM2398 (BtispG), and
strain Y6084-b is Y6084
containing pAM2398-
ispD (adding a second
copy of ispD).
Significance
• First expression of DXP pathway in S. cerevisiae
• Establishes the foundation for further optimization and engineering of yeast for the
production of bioenergy relevant isoprenoids at industrially relevant conditions
Improvement of flux via the DXP
pathway. (A) Improvement of
flux via the DXP pathway (as
shown by percentage of
ergosterol that is 13C-labeled)
by reduction of aeration and
addition of an extra copy of
ispD. Y6084 is the parent strain,
containing all of the DXP
pathway genes except ispG.
Y6084-a is Y6084 containing
pAM2398 (BtispG), and Y6084-
b is Y6084 containing
pAM2398-ispD (adding a
second copy of ispD). Culture
shaking speeds are indicated.
(B) Abundance of DXP pathway
intermediates (relative to strain
Y6084) in response to
increasing DXP pathway flux.

2. Engineering bacteria to catabolize sarin:
teaching E. coli to eat isopropanol
Outcomes
• The engineered E. coli consumed 65% of isopropanol
compared to no-cell controls and was able to grow on
isopropanol as a sole carbon source
• Reconstitution of the large (370 kDa) ACX complex in E.
coli allowed us to study this otherwise cryptic enzyme in
more detail than would have been possible in the less
genetically tractable native Xanthobacter system
Significance
• First example of heterologous expression of a functional
ACX
• Highlights the power of working with model organisms for
the study and deployment of biological solutions to CBW
agent decontamination and recovery
Brown et al. (2017). "Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli
to Eat Isopropanol". ACS Synth Biol, 5(12), 1485-1496. doi, 10.1021/acssynbio.6b00115
Background
• Sarin is a highly potent chemical and biological
weapon (CBW) agent
• Isopropanol is produced by the decomposition of
sarin and can be used as a mock CBW agent
• Isopropanol can be transformed into acetyl-CoA by
enzymatic conversion with a key reaction performed
by the acetone carboxylase complex (ACX)
Approach
• Engineered the heterologous expression of the ACX
complex from Xanthobacter autotrophicus PY2 into E.
coli
(Left) Isopropanol‐only growth comparison. Expression of
ACX at 10 μM IPTG using pBbS5a‐CB‐CA‐RN‐EC (JBEI‐
14130) vs the control pBbS5a‐CB‐CA (JBEI‐14129). (Right)
HPLC analysis of isopropanol from four day old cultures
grown in the presence of only isopropanol. CB, CBadh; CA,
carbonic anhydrase; RFP, red fluorescent protein; IPA,
0.75% isopropanol; glc, 0.2% glucose

3. Streamlining the Design-to-Build transition with
Build-Optimization Software Tools (BOOST)
Outcomes
• BOOST web application (available at https://boost.jgi.doe.gov) provides easy and interactive access to all functionalities
• BOOST supports community standard data-exchange formats including FASTA, GenBank32, and SBOL
Significance
• For DNA foundry operations that closely monitor cost and cycle time metrics,
using the BOOST tools can result in very significant efficiency gains
Oberortner et al. (2016) “Streamlining the Design-to-Build transition with Build-Optimization
Software Tools (BOOST)”. ACS Synth. Biol., DOI: 10.1021/acssynbio.6b00200
Background
• Even state-of-the-art methods
cannot synthesize all DNA
sequences
• Current biological computer-aided
design and manufacture
(bioCAD/CAM) tools do not
consider DNA synthesis limitations
Approach
• Develop Build OptimizatiOn
Software Tools (BOOST), a suite
of tools to automate the design of
DNA constructs ready for
commercial DNA synthesis
• Include tools for reverse-
translation, codon juggling,
detection and resolution of DNA
synthesis constraint violations,
and partitioning of DNA
sequences into shorter fragments

4. Genome-resolved metagenomics defines a
community response to ionic liquid challenge
Outcomes
• Increasing levels of ionic liquid amendment in the cultures selects for a Firmicutes-dominated communities that retain the
ability to produce glycoside hydrolases up to 2% [C2C1im][OAc]. At 2% [C2C1im][OAc], the community switches to acetate
metabolism
• The combination of genome-resolved metagenomics (>100 genomes were recovered from the metagenomic dataset) and
metatranscriptomics allowed the identification of individual community members that were tolerant of [C2C1im][OAc] and
were able to produce cellulases and xylanases in the presence of [C2C1im][OAc]
Significance
• This study provides insights into a community-level reponse to ionic liquid challenge and demonstrates the use of
genome-resolved metagenomics to identify active microbes that can tolerate industrially relevant amounts of ionic liquids
used in the deconstruction of lignocellulose.
Wu et al. (2017) "Ionic Liquids Impact the Bioenergy Feedstock-Degrading Microbiome and Transcription of
Enzymes Relevant to Polysaccharide Hydrolysis". mSystems, 1(6). doi, 10.1128/mSystems.00120-16
Background
• Previous work at JBEI has defined the response
of bacterial isolates to ionic liquids
• Cultivation of biomass-deconstructing microbial
consortia in response to ionic liquid challenge
would illuminate community responses to these
promising pretreatment chemicals
Approach
• A consortia of microbes was adapted to grow on
switchgrass under solid state cultivation and this
adapted community inoculated into switchgrass
cultures amended with increasing levels of
[C2C1im][OAc]. The response of the community to
this challenge was measured by respiration,
enzymatic assays and metatranscriptomics.

5. On-chip integration of droplet microfluidics
with nanostructure initiator mass spectrometry
for enzyme assay
Outcomes
• Fabrication of enzyme arrays with 640 assays available
per 5 cm chip
• Built a device that can manipulate150 nl droplets with
subsequent deposition onto the NIMS surface, achieving
a significant reagent reduction vs. previous work
requiring a 20 μl dead volume
Significance
• Disruptive technology based on a grid of 50 μm
resolution that enables the possibility of >100 000 pads
per 5 cm2 NIMS array when scaling the current design
• Potential significant impact on enzyme engineering,
DNA manipulation, microbial screening for bioenergy
applications
Heinemann et al. (2017) "On-chip integration of droplet microfluidics and nanostructure-initiator mass spectrometry for
enzyme screening". Lab Chip. doi, 10.1039/c6lc01182a http://pubs.rsc.org/en/content/articlepdf/2017/lc/c6lc01182a.
Background
• Current enzyme engineering strategies typically require
many assays to be effective
• Assay technology is expensive and time consuming to
implement
Approach
• Designed and fabricated microfluidics chip for assaying
enzyme activity with 1000x fold reduction in reagent
consumption.
• Integrated microfluidics with NIMS to establish a new
screening platform
μNIMS assembly and operation. A. Electrode and fluidics design, B. Digital
microfluidics chip, compression sealed to the NIMS array, C. the stack for holding
the layers together. D. operation workflow E. Inject: chip is filled with droplets,
load: flow is stopped and droplets are loaded onto the NIMS array for incubation
and probe deposition, eject: the droplets are incubated for 10, 20, 30, and 40
minutes over six successive pads and then actuated into the central chamber
where they are then evacuated, F. workflow of NIMS array removal and analysis.

6. Development of an integrated approach for
α-pinene recovery and sugar production
from loblolly pine using ionic liquids
Outcomes
• [C2C1Im][OAc] is very efficient at extracting
terpenes (i.e., α-pinene) from loblolly pine while
generating a carbohydrate-enriched stream suitable
for bioconversion into renewable biofuels and
chemicals
• Techno-economic analysis (TEA) revealed that the
α-pinene recovery after IL pretreatment could
reduce the minimum ethanol selling price (MESP)
by $0.6-1.0/gal
Papa et al. (2016) “Development of an integrated approach for α-pinene recovery and sugar
production from loblolly pine using ionic liquids", Green Chemistry, DOI: 10.1039/C6GC02637K”.
Background
• Terpenes are produced in high concentrations from
loblolly pine (Pinus taeda L.) that could represent a
valuable supplement to bioenergy production chains
Approach
• We investigated imidazolium-based ionic liquid (IL)
[C2C1Im][OAc] pretreatment in conjunction with
different analytical protocols using GC–MS, to extract
α-pinene and simultaneously pretreat the pine to
generate high yields of fermentable sugars after
saccharification
Terpene recovery (left) and glucose release (right) of different tissues of loblolly pine
Significance
• This integrated terpene extraction/lignocellulose pretreatment approach may provide a
compelling model for a biorefinery, reducing costs and increasing commercial viability
• ILs can be used to selectively extract volatile compounds from plants during pretreatment

7. Implications of U.S. Biofuels Policy for
Sustainable Transportation Energy in Maine and
Northeast
Outcomes
• The EPA's current definition of “renewable biomass” is unclear, especially in the case of naturally regenerated forest
biomass. EPA’s definition of biomass favors biomass from plantations regardless of actual ecological impacts on
biodiversity, soil and water quality. In Maine and northeast, over 90% of Maine's forests are naturally regenerated.
• According to the current EPA definition, only a very small percentage of Maine forestland (~27%) would qualify as
renewable biomass This significantly reduces the availability of RFS2 compliant biomass in Maine and Northeast.
• Though drop-in biofuels are a promising next generation fuel, efficient conversion technologies with higher fuel yield and
subsequent lower production cost are key to commercial production of these fuels.
Significance
• In contradiction to EPA's goal to promote renewable fuel, it is limiting the domestic supply of forest-based biofuel in Maine
through its current definition under RFS2.
• Considering the unique nature and forest management practices in Maine, the EPA and Congress should consider revising
this RFS2 definition to allow more naturally regenerating forests to qualify as renewable biomass.
Neupane, B., & Rubin, J. (2016) “Implications of US biofuels policy for sustainable transportation energy in Maine and
the Northeast”. Renewable and Sustainable Energy Reviews. DOI: http://dx.doi.org/10.1016/j.rser.2016.11.253
Background
• Sustainable production of biofuels requires analyzing policies that directly or indirectly affect biofuels production.
• Biofuels produced from forest biomass face conflicting definitions of renewable biomass that adversely impact the viability
of biofuel production in Maine's and other northeastern forests despite a long history of using those same forests for pulp
and paper production.
Approach
• Reviewed current status of RFS2 policy and potential challenges in implementing this policy.
• Examined the potential of drop-in biofuel production from forest biomass in Maine and Northeast.
• Reviewed broader environmental, economic and social implications of drop-in biofuel production, in particular looking at
RFS2 policy and forest biomass availability in Northeast U.S.

8. Strategy for extending the stability of bio-oil
derived phenolic oligomer via mild hydrotreatment
with ionic liquid stabilized nanoparticles
Kim et al. (2016) “Strategy for extending the stability of bio-oil derived phenolic oligomer via mild
hydrotreatment with ionic liquid stabilized nanoparticles”. Chemsuschem, DOI: 10.1002/cssc.201601515
Background
• Development of catalytic transformations and
processes is essential to utilize bio-oil and lignin
derivatives
• Metal nanoparticles (NPs) stabilized in ionic liquid (IL)
are promising for catalytic hydrotreating of bio-oil and
phenolics
Approach
• Ruthenium NPs were synthesized with copolymers in
1-ethyl-3-methylimidazolium acetate
• Mild hydrotreating of phenolic oligomer was performed
in the presence of synthesized NP catalyst at 100 °C
for 6 hrs with the goal of producing a stable phenolics
Outcomes
• Hydrotreating of phenolic oligomer over NPs in IL
significantly increased aliphatic carbons, resulting in
alkylphenol units with improved thermal stability
• The catalyst system employed in this work was highly
effective in stabilizing reactive phenolic oligomer
Significance
• The findings of this work provide insight into
hydrotreating mechanisms of phenolic oligomer and
whole bio-oil, which will be useful for development of
improved bioenergy processes in the future
OH
H3CO
OH
O
OCH3
O
OH
OCH3
O
N N
N
O
x y
OH
H3CO
OH
O
OCH3
OH
OH
OCH3
O
Mild hydrotrea ng