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 pointwhere it could sustain moderate growth of S. cerevisiae in the absence of the MEV pathway butwe 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. cerevisiaesupported by the DXPpathway. Strain Y6084-ais Y6084 containingpAM2398 (BtispG), andstrain Y6084-b is Y6084containing pAM2398-ispD (adding a secondcopy 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 DXPpathway. (A) Improvement offlux via the DXP pathway (asshown by percentage ofergosterol that is 13C-labeled)by reduction of aeration andaddition of an extra copy ofispD. Y6084 is the parent strain,containing all of the DXPpathway genes except ispG.Y6084-a is Y6084 containingpAM2398 (BtispG), and Y6084-b is Y6084 containingpAM2398-ispD (adding asecond copy of ispD). Cultureshaking speeds are indicated.(B) Abundance of DXP pathwayintermediates (relative to strainY6084) in response toincreasing DXP pathway flux.

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

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 gainsOberortner 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

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.

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. Digitalmicrofluidics chip, compression sealed to the NIMS array, C. the stack for holdingthe 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 incubationand probe deposition, eject: the droplets are incubated for 10, 20, 30, and 40minutes over six successive pads and then actuated into the central chamberwhere they are then evacuated, F. workflow of NIMS array removal and analysis.

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 suitablefor 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

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.

Strategy for extending the stability of bio-oil derived phenolic oligomer via mild hydrotreatmentwith 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

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