YEAR 8 ANNUAL REPORT - JBEI · JBEI recorded 28 records of invention (ROI) disclosures, filed 26 patent applications, had 3 patents issued, executed 11 licensing agreements, and initiated

J O I N T B I O E N E R G Y I N S T I T U T E

YEAR 8 ANNUAL REPORT

SEPTEMBER 15, 2015

CONTAINS PROPRIETARY INFORMATION

J B E I 2 Y e a r 8 A n n u a l R e p o r t

TABLEOFCONTENTSOverview................................................................................................................................................3

1. FeedstocksDivision.........................................................................................................................41.1 Introduction...........................................................................................................................................................................................41.2 ScientificProgress...............................................................................................................................................................................41.3 MajorResearchHighlights...............................................................................................................................................................81.4 MajorResearch&PersonnelChanges........................................................................................................................................91.5 CollaborativeResearch&IndustrialInteractions.................................................................................................................91.6 ImpactsofResearch............................................................................................................................................................................91.7 LinkagestoFuturePlans................................................................................................................................................................10

2. DeconstructionDivision.................................................................................................................102.1 Introduction.........................................................................................................................................................................................102.2 ScientificProgress.............................................................................................................................................................................102.3 MajorResearchHighlights.............................................................................................................................................................152.4 MajorResearch&PersonnelChanges......................................................................................................................................152.5 CollaborativeResearch&IndustrialInteractions...............................................................................................................152.6 ImpactsofResearch..........................................................................................................................................................................162.7 LinkagestoFuturePlans................................................................................................................................................................16

3. FuelsSynthesisDivision.................................................................................................................163.1 Introduction.........................................................................................................................................................................................163.2 ScientificProgress.............................................................................................................................................................................173.3 MajorResearchHighlights.............................................................................................................................................................213.4 MajorResearch&PersonnelChanges......................................................................................................................................223.5 CollaborativeResearch&IndustrialInteractions...............................................................................................................223.6 ImpactsofResearch..........................................................................................................................................................................223.7 LinkagestoFuturePlans................................................................................................................................................................22

4. TechnologiesDivision....................................................................................................................234.1 Introduction.........................................................................................................................................................................................234.2 ScientificProgress.............................................................................................................................................................................234.3 MajorResearchHighlights.............................................................................................................................................................264.4 MajorResearch&PersonnelChanges......................................................................................................................................264.5 CollaborativeResearch&IndustrialInteractions...............................................................................................................264.6 ImpactsofResearch..........................................................................................................................................................................274.7 LinkagestoFuturePlans................................................................................................................................................................27

5. Bibliography..................................................................................................................................28


OVERVIEWIn Year 8, JBEI had 75 research and review publications that are referenced throughout this report out of a total of 502 publications since the program’s inception. In the past year, JBEI researchers received 19 awards, including a Fel-lowship of the National Academy of Inventors, The Economist 2014 Innovation Award in the Bioscience category, LBL Director’s Award for Exceptional Achievement and a Chalmers University Honorary Doctorate. This fiscal year JBEI recorded 28 records of invention (ROI) disclosures, filed 26 patent applications, had 3 patents issued, executed 11 licensing agreements, and initiated 6 funds-in projects (CRADAs and WFOs) with industry partners. In Year 8, JBEI has been highlighted over 111 times in the press and has hosted more than 104 tours of its facility. JBEI’s inte-grated research programs are cross-divisional in nature and bring multiple disciplines together to address key prob-lems in JBEI’s feedstocks to fuels efforts, as indicated by the fact that ~45% of the Year 8 publications involved multi-ple teams at JBEI. JBEI’s research program has played a leading role in developing the scientific underpinnings for the conversion of biomass to feedstocks to advanced biofuels and renewable chemicals. Highlights from the four di-visions include:

Feedstocks Division: • Discovered UDP-xylose, GDP-fucose, GDP-mannose transporters and determined their role in cell wall bio-

synthesis • Discovered a wide range of HCT inhibitors while exploring its catalytic promiscuity • Established Cas9 based bioediting tools to generate hybrid-plants and reduce lignin in fiber cells • Engineered plants with novel pathways to create dominant low lignin and high-saccharification traits • Engineered plants with decreased lignin, increased hexose to pentose ratio, and increased wall density • Identified new glycosyltransferases involved in glycan biosynthesis • Identified and characterized 10 novel rice lines altered in growth, stress tolerance and development • Optimized and generated new tools for whole genome network analysis and functional genomics of grasses

Deconstruction Division:

• Established new low cost, biomass derived bionic liquids that generated high fermentable sugar yields • Discovered a novel process that consolidates pretreatment, saccharification and fermentation into one unit

operation through the use of a biocompatible ionic liquid • Discovered a library of microbial pumps that impart ionic liquid tolerance in E. coli and S. cerevisiae • Discovered a bacterial non-cellulosomal cellulase complex with significant hydrolytic activity • Established multiple routes for enzymatic and microbial lignin depolymerization and conversion into ad-

vanced biofuels and renewable chemicals • Identified promising mutants for improved recombinant protein expression in Aspergillus niger

Fuels Synthesis Division:

• Engineered organisms with moderately high yields for several advanced biofuels, including isopentenol (56% of maximum theoretical yield), methyl ketones (40%), and bisabolene (34%)

• Established multi-omics capabilities that facilitated metabolic engineering • Discovered tolerance mechanisms to enable improved production of isopentenol and tolerance to limonene. • DIVA adopted by the NationalUniversityofSingapore; 1,642 users of j5 at 435 institutions worldwide • Quantitatively accurate predictions on biological systems obtained through 2S-13C MFA and 13C MOMA • Discovered a native E. coli transporter that confers tolerance to isopentenol and also increases production

Technologies Division:

• Established high-throughput technologies for large scale analysis of plant and microbial metabolites and en-zyme activities

• Designed and established chips for rapid screening lignocellulose degrading enzyme functions • Established proteomics pipelines for biofuels systems biology • Established high- and medium-resolution imaging of plant biomass and biofuel pathway enzymes • Established the Experiment Data Depot developed to support enzyme engineering and genetic regulatory

component characterization data


1. FEEDSTOCKS DIVISION 1.1 Introduction The primary goal of the Feedstocks Division is to generate basic knowledge about plant cell wall biosynthesis and modification to facilitate the engineering of a new generation of feedstocks that produce high yields of fermentable sugars and are less recalcitrant to deconstruction. This is accomplished by:

• Identifying new genes, alleles and metabolic pathways controlling cell wall composition, growth, develop-ment, stress tolerance and recalcitrance to saccharification

• Establishing and validating approaches for predictive models and systems biology analysis in plants • Developing tools for functional genomics analysis of monocots and dicots and engineering desirable biomass

traits to bioenergy crops Cost efficient conversion of lignocellulosic biomass into biofuels requires the development of genetically improved bioenergy crops, such as switchgrass, sorghum, poplar and eucalyptus, which are optimized for biomass production and agronomically useful properties. To accomplish this, the genes involved in cell wall synthesis, modification, deg-radation and stress tolerance need to be identified in tractable model plant species, such as Arabidopsis and rice. This will provide the basic knowledge necessary to develop a new generation of bioenergy crops. JBEI’s Feedstocks Division uses model species to develop new strategies to facilitate deconstruction for sugar production, and to pro-vide a source of lignin for conversion to high-value products. These approaches will facilitate inter-species transfer of high value traits through conventional breeding and genetic engineering.

1.2 Scientific Progress 1.2.1 Plant Systems Biology In FY2015, the plant systems biology group focused on developing the tools and discovering the knowledge to enrich the C6 content of biomass by targeting the secondary cell wall polysaccharide, glucomannan. This is an extractable polymer which is the dominant hemicellulose in the secondary wall of gymnosperms, such as pine, but is a minor component in the cell wall of most angiosperms. The GT family responsible for its synthesis has been identified (a sub-clade of GT2, the CSLAs) but attempts to increase the polysaccharide content by simply increasing the enzyme quantity has failed. A systems biology approach is being used to investigate how to increase the flux of carbon into this polymer. Topics include altering the synthase activity, increasing pools of substrate available to the synthases and identifying regulatory proteins that may boost synthase activity. Metabolomics of cell wall polysaccharide precursors. To boost substrate availability for polysaccharide biosynthe-sis it is critical to understand the regulation and synthesis of the nucleotide diphosphate linked monosaccharides (NDP-sugars) substrates. Our work, using the NDP-sugar analytical platform developed at JBEI [1], in collaboration with the Universities of Saitama and Cambridge, has identified two enzymes that strongly enhance the production of GDP-Man by directly interacting with the GDP-Man pyrophosphorylase. GDP-Man is an excellent target since its synthesis is also a limiting factor in vitamin C biosynthesis and protein glycosylation, and advances will have wider impacts beyond cell wall biosynthesis. These techniques are also valuable for future work looking to build metabolic models describing the interconversion of NDP-sugars. Nucleotide sugar transporters. Nucleotide sugar transporters have been proposed to be a control point for the flow of the substrates for cell wall biosynthesis into the Golgi. Studies have shown that the CSLA proteins have an active site localized in the Golgi lumen, which would require a GDP-sugar transporter. Only four transporters in Arabidop-sis are predicted to be able to transport GDP-sugars, but our previous work had shown that one candidate, GONST1, was a GDP-Man transporter specifically providing substrates for glycosylated sphingolipids (GIPCs). We have now characterized two homologs; GONST2, which appears to be a GIPC-specific transporter and in collaboration with the University of Melbourne, GONST4 which transports GDP-fucose in vitro and the engineered mutants have a reduced fucose phenotype. This leaves either GONST3 or another as yet unidentified GDP-Man transporter candidate as a potential target for cell wall dependent GDP-Man transport. Glycosyltransferases (GTs) and GT-regulating proteins. Despite having a relatively simple structure, xylan has been revealed to require a large number of proteins for its synthesis, although it is not clear why. In collaboration with the University of Cambridge, we identified a novel type of xylan in the primary wall of Arabidopsis, which re-quires only a subset of known xylan-related GTs for its synthesis [2]. This xylan has more in common with grass xy-lan than typical dicot xylan, and could provide a useful model for studying xylan synthase complexes. We have also established a Pichia system for expressing mannan synthases, in order to screen CSLAs from different species to de-


termine whether specificity and rate is dependent on the protein. A bioinformatics pipeline was developed to identi-fy candidate proteins which may regulate GT function. A yeast 2 hybrid system and in planta luciferase system have been established to test protein-protein interactions between mannan synthase enzymes and candidate regulatory proteins, and screening is ongoing [3]. Tools for cell wall analysis. Ongoing work to improve the reproducibility, throughput and quality of cell wall ana-lytics has included the establishment of a cell wall structural analysis platform (PACE) at JBEI. We have also halved the required HPAEC-PAD runtime required for monosaccharide analysis of all plant cell wall sugars, enabling us to work with the Deconstruction Division to look at the effects of microbial communities on different Feedstocks. Final-ly, we have been developing plant compatible mass spectrometry approaches with the Technology Division to ena-ble more efficient analyses to be conducted. This has included an organelle profiling technique to assist in profiling subcellular preparations from plants [4] and a high-flow proteomics technique for shotgun proteomics of complex samples [5]. In a complementary approach to the tools described above, we have developed methods to characterize intact biomass. FT-IR polarizing spectroscopy can capture structural information from highly organized components of the cell wall such as cellulose [6]. Building on this technique, we leveraged the world-class facilities at LBNL, and with collaborators at the Advanced Light Source (ALS) we have used high-resolution synchrotron IR spectromicros-copy (SR-FT-IR) to obtain high resolution (3 µm) and tissue specific information about the cell wall composition and structure. Application of these tools to a mixed-linkage glucan (MLG) deficient rice mutant (cslf6) [7], revealed that cellulose deposition was altered in cross sections of rice three-day-old seedling mesophyll cells and the bonds associ-ated with cellulose and arabinoxylan had a lower energy state [8].

1.2.2 Cell Wall Biosynthesis Substrate biosynthesis and transport. The Cell Wall Biosynthesis team is working closely together with the Systems Biology team in the characterization of nucleotide sugar transporters. In FY2015, we published our work on three UDP-xylose transporters [9]. Mutants in the most highly expressed transporter, UXT1, showed a substantial reduc-tion in xylan, but did not have any change in xylose in xyloglucan or protein N-glycans [10]. This result demonstrates that cytosolic UDP-xylose synthesis is important for xylan biosynthesis. The UDP-xylose synthase in the Golgi lumen is insufficient for providing UDP-xylose for xylan biosynthesis and may be more involved in providing UDP-xylose for other glycans. Understanding the substrate channeling of these transporters in vivo remains an important chal-lenge. Work on a GDP-fucose transporter was also completed and submitted for publication in FY2015. The single GFT1 (GONST4) transporter is essential and simultaneously provides GDP-fucose for synthesis of proteins, xyloglu-can and pectin. In FY2016, we will continue the completion of the nucleotide sugar transporter studies. Transporters of UDP-arabinose, UDP-apiose and UDP-glucuronic acid have been identified and publications are in preparation. RWA proteins are putative transporters of acetyl-CoA and we are currently studying their function in vitro using the protocol we developed for nucleotide sugar transporters. Efficient expression of the proteins in yeast was challeng-ing but has been achieved after codon optimization. In FY2015, we investigated the role of RWA proteins in patho-gen defense that we previously observed [11]. It turns out that the resistance to Botrytis in the rwa2 mutant is related to changes in the cuticle and to a massive reprogramming of the transcriptome with induction of genes related to response to both abiotic and biotic stress [12]. Biosynthetic enzymes. A key objective of JBEI is to develop technologies and resources to rapidly advance our un-derstanding of cell wall biosynthesis. The Feedstocks and Technology Divisions cloned nearly 90% (400 out of 450) of the GT genes in Arabidopsis, as well as about 15% of those encoded by the rice genome [13]. All clones have been made available to the community via ABRC. In collaboration with the University of Melbourne, we have now cloned a further 30 GTs and 80 GT-like enzymes and now have nearly 95% of GTs encoded by Arabidopsis (http://gt.jbei.org/). Xylans are the major non-cellulosic polysaccharides in biomass, but the biosynthesis is still not well understood. We have extended our studies of the GUX family of xylan glucuronosyltransferases. In vivo the two major forms, GUX1 and GUX2, are responsible for remarkably different patterns of xylan substitution in second-ary walls [14] and we showed GUX3 to be specifically involved in synthesis of xylan in the primary wall [2]. The closest homolog of the GUX proteins turned out to also be a glucuronosyltransferase, but its acceptors are inositol phsophoryl ceramide sphingolipids, a major class of plasma membrane lipids in plants [15]. We showed this activity by expressing the Arabidopsis proteins, designated IPUT1, in yeast that was modified to produce UDP-GlcA and to transport it into the Golgi. The IPUT1 protein is essential for pollen tube guidance and fertilization. Synthesis of the backbone of xylan requires at least four proteins; IRX9, IRX10, IRX14 and IRX15 (or their paralogs IRX9L, IRX10L, IRX14L, and IRX15L) [14]. In FY2014, we concluded a site directed mutagenesis study of IRX9, IRX9L and IRX14 and concluded that the IRX9 and IRX14 proteins have a main role that is not catalytic [16]. We think it is most likely that the proteins function in a protein complex together with the IRX10/IRX10L proteins, which have been shown to have xylan synthase activity in vitro. To probe protein-protein interactions, we developed a split-luciferase assay [3]. This method suffers less from false positive of the commonly used BiFC assay. We can demonstrate interactions between xyloglucan biosynthetic proteins with the luciferase assay.


Polysaccharide engineering. Increased C6/C5 sugar ratio in the cell wall is a desirable trait and in FY2015 we com-pleted studies of plants overexpressing both β-1,4-galactan synthase and the UDP-glucose epimerase that ensures supply of UDP-galactose. In the resulting plants, cell wall galactose content in stems was increased by almost 100% [17]. The discovery in FY2014 of UDP-galactose transporters opened the possibility to additionally overexpress the transporters. Preliminary experiments confirmed that the transport would be limiting in galactan engineered plants. We therefore developed plants expressing all three proteins, and included plants that also contained a construct for decreased lignin content. The galactan engineered did not show any alteration in growth and development. In col-laboration with the Cell Wall Engineering team, we expanded this project with the expression of a callose synthase gene (PMR4) under the control of a lignin promoter (pC3H) to accumulate glucose-based polymers (callose) in sec-ondary cell walls together with the density trait and the lignin reduction approach developed last year that targets the availability of cytosolic shikimate [18]. We selected a lignin promoter to accumulate callose in the epidermis to enhance plant pathogen tolerance. Plants harboring these three traits perform better than plants harboring only the low lignin and density traits. Preliminary data show that plants harboring all traits exhibit outstanding saccharifica-tion efficiency. The best stacking approach will be transferred into switchgrass and sorghum after confirmation of these data and validation of biomass densification. Environmental interactions. The ability to grow plants that can maintain a high yield on marginal lands with mini-mal inputs is important for the success of bioenergy feedstocks. Especially important is stress tolerance and efficient nutrient acquisition. We have therefore initiated research to identify useful stress tolerance genes. A drought re-sistant Arabidopsis mutant has been identified and is currently studied to determine the mechanism of the resistance and its usefulness under a variety of growth conditions. We also initiated research to investigate the role of cell wall properties in plant interactions with arbuscular mycorrizal fungi [19] and nitrogen fixing bacteria. Working with the Cell Wall Engineering team, a Medicago-Rhizophagus-Rhizobium model system has been established and is currently used to study the importance of a number of cell wall proteins for the interactions. Moreover, a screen of free nitro-gen fixing bacteria (endophytes and rhizospheric) allowed the identification of a new endophyte Raoultella terrigena R1Gly [20] that can fix nitrogen, produce auxin and other signaling molecules such as of 2,3-butanediol and acetoin volatiles based on genome analysis and promoter plant growth in tobacco and switchgrass.

1.2.3 Grass Genetics Rice mutant collection for functional analysis of grass genes. Rice serves as a model for other grass species, includ-ing switchgrass and sorghum. To facilitate using rice in bioenergy research, we generated a mutant population con-taining over 7,000 M1 lines using FN mutagenesis in Kitaake, an early flowering rice variety, with a rapid generation time of nine weeks [21]. To date, we have sequenced and analyzed over 1,000 mutants in collaboration with JGI. The genes affected in these mutants have been identified and cataloged using a newly established sequence analysis pipeline. To make this mutant collection publicly accessible to the research community, we constructed KitBase, a database to track the mutants. Kitbase provides genomic and phenotypic data and information on the number of seeds available. This resource will allow researchers to quickly identify deleted genes in the mutant population to accelerate discovery of grass genes controlling cell wall growth, development, immunity and other biological pro-cesses. Using this new information, we identified and characterized 15 mutants altered in cell wall composition, stress tolerance, and development and isolated genes corresponding to mutations. The identified genes encode two cell wall localized cysteine rich receptor kinases, a novel regulatory microRNA, a Dicer-like protein, a kinesin-4 pro-tein controlling grass cell wall composition, and a RNA polymerase subunit or antiphosphatase protein. These re-sults demonstrate that the FN-mutagenized collection can be used to efficiently identify genes corresponding to mu-tations that control important biological pathways. Switchgrass functional genomics and proteomics. To facilitate genetic analysis of switchgrass, we built on knowledge established for rice to identify switchgrass genes predicted to control biomass and stress-response-related traits. We screened 96,000 clones from two switchgrass BAC libraries [22]. Full-length sequencing of 311 BAC clones revealed sequence for ~3.2% (52 Mb) of the switchgrass genome, coding for 3,948 genes (4,217 gene models). Gene annotation and ontology analysis revealed 695 genes belonging to gene families targeted in the screening. These in-clude 350 kinase, 203 glycosyltransferase (GT), 109 glycoside hydrolase (GH) and 33 ethylene responsive transcrip-tion factor (ERF) family genes. Rice orthologs of 65 genes have demonstrated roles in bioenergy-relevant traits. This list includes 14 GT2 family genes that regulate synthesis of cellulose and hemicelluloses. The switchgrass BAC li-brary was also screened for clones carrying genes to be involved in lignin biosynthesis resulting in the isolation of more than 80 BAC clones. The sequence analysis of these BAC clones is in progress. This switchgrass genome analy-sis provides a foundation for detailed characterization, breeding and engineering of genes that regulate bioenergy-related traits in switchgrass. In parallel with the genomic analysis, we worked with the Plant Systems Biology group and the Technology Division to characterize the endomembrane proteome of switchgrass coleoptiles [23]. We iden-tified 1,750 unique proteins from two biological replicates. These data have been deposited in the ProteomeXchange with the identifier PXD00135. The proteomic dataset will be used for advancing switchgrass genome annotation.


Structural studies of cell wall sensing proteins. Proteins associated with cell wall, including many important cell wall sensing proteins, are important for cell wall properties. Leucine-rich repeat-receptor kinases (LRR-RKs) in the plant cell wall are critical for many innate immune responses. The immune response often involves the dimerization of LRR-RKs with common somatic embryogenesis receptor kinases (SERKs). Rice has two members of the SERK fam-ily: OsSERK1 and OsSERK2 [24]. OsSERK2 interacts with the rice brassinosteroid receptor OsBRI1 and is critical for brassinosteroid-mediated development. OsSERK2 is a functional homolog of BAK1 in rice, serving as a common mediator of various LRR-RK signaling pathways. To better understand how this receptor kinase works, we used X-ray to determine the crystal structures of parts of the extracellular domains of OsSERK2 and a D128N mutant of Os-SERK2 [25].[25]. These structures reveal how the OsSERK2 receptor kinase functions in rice.

Identification of sorghum mutants in genes known to control cell wall associated functions. Sorghum (Sorghum bicolor), an important biofuel crop, has a small genome (-730Mb) and is easy to transform, making it an attractive tar-get for genetic improvement. To advance knowledge on sorghum cell wall structure, we identified sorghum TILL-ING mutants [26] carrying mutations in orthologs of three well-characterized rice genes: OsAT10, XAX1, and CslF6.

Assembly of the genome of the model rice variety Kitaake. Compared with the rice Japonica variety, Nipponbare, and the Indica rice variety 93-11, of which the genomes are available, the Kitaake Japonica rice variety has a smaller stature, is easier to transform and has a much shorter life cycle (ca. 9 weeks vs. 3 months) [21]. For these reasons, Ki-taake has become the preferred model for rice genetic analysis. To facilitate genetic analysis of grasses, we collabo-rated with JGI to generate a Kitaake genome de novo assembly. Our team generated multiple libraries with insert siz-es ranging from 500 bp to140 kb. The sequencing depth from the Illumina sequence reads alone is 270-fold. The Ki-taake genome sequence version 0.5 is now available with release of version 1 planned for year nine.

Tools for whole genome network analysis of grasses. Diverse proteomics and genomics datasets for grasses are increasingly becoming available. Our team undertook an intensive effort to extend the functional significance of this vast amount of information establishing RiceNetv1 and v2 [27, 28]. RiceNet takes advantage of the observation that genetic modifiers of the same gene often cluster in gene networks. Thus, novel genes involved in cell wall biosynthe-sis and degradation can be effectively identified and prioritized based on local connectivity in gene networks. By incorporating recently generated data, we updated RiceNet v1 to RiceNet v2, providing a network of 25,765 genes (70.1% of the coding genome) and 1,775,000 co-functional links. The updated RiceNet v2 enable users to explore genes in more plant species and effectively identifies candidate genes involved in grass biological pathways. We have also updated and generated new phylogenomic databases for glycosyltransferases, glycoside hydrolases, kinas-es, transcription factors, transporters, and cytochrome P450 monooxygenases to facilitate cell wall research in grasses [29].

1.2.4 Cell Wall Engineering Lignin engineering. Based on our previous lignin engineering results [30, 31], our focus remained on developing novel dominant technologies to reduce lignin content without adversely affecting plant development. HCT is an im-portant enzyme in the lignin pathway as it controls the phenylpropanoid flux toward the production coniferyl and sinapyl alcohols, major backbone of lignin. We continued developing approaches to target this enzymatic step and characterized HCT promiscuity to identify potential competitive inhibitors and explore the potential use of this en-zyme to redirect part of the phenylpropanoid flux toward added-value chemicals while reducing biomass recalci-trance. Out of 20 aromatic based metabolites tested, we identified eight new substrates, from which some were able to inhibit strongly HCT activity. We engineered Arabidopsis lines to produce one of the identified inhibitors (proto-catechuate) from chorismate in lignifying tissues and showed that these lines exhibit ~30% lignin reduction and sac-charification improvement >30% without growth penalty compared to control plants. As pre-translation study, we also explored how well this promiscuity was conserved and showed that it seems to be conserved across all land plants since HCT enzymes from a bryophyte (Physcomitrella patens), a lycophyte (Selaginella moellendorffii), a conifer-ous gymnosperm (Pinus radiata), a monocot angiosperm (Panicum virgatum), and two dicot angiosperms showed the same substrate promiscuity [32]. Therefore, we are highly confident that we could transfer this approach to reduce lignin content in various crops and are currently setting up a collaboration with Dr. R. Dixon (BESC) to translate it into switchgrass. More recently, we started a different approach to manipulate lignin composition and content that focuses on the availability of SAM (S-adenosylmethionine) essential substrate for COMTs and cell wall methylases. Our preliminary data revealed that lignin content in plants expressing a SAM hydrolase in secondary cell wall was reduced by ~20% and saccharification improved by ~20% without growth penalty compared to control plants. The Cell Wall Engineering group worked closely together with the Cell Wall Biosynthesis group on gene stacking (de-scribed above under Polysaccharide Engineering). Development of standardized tools. The standardization of components and analytic methods is very important for reproducibility and rapid scientific progress. Last year we started the development of a rapid method for gene as-sembly using Golden Gate cloning [33] and gene stacking using yeast homologous recombination [34]. Because Golden Gate cloning has been widely adopted in plant research laboratories, together with a group of plant synthetic


biologists from across the world, we established a common syntax for 12 fusion sites used to assemble various parts to assemble genes [33]. This syntax will also facilitate the distribution and utilization of all parts that are generated in the feedstock division. We expanded our tool portfolio with bioediting tools to be able to generate hybrid plants [35]. This approach is based on the expression of endonuclease such as CRISPR/Cas9 to generate allelic variants in target loci only in desired tissues. We are currently using the CRISPR/Cas9 system and developed a rapid method to assay it on various target DNA. Finally, we generated several switchgrass lines to characterize the strength and tissue-specific activity of six promoters from various species using GUS as the reporter gene. Such characterization is essen-tial to facilitate engineering and fine-tune gene expression in energy-grasses, and to support our translation path from model systems into energy crops (switchgrass and sorghum). Translation to biofuel crops. We have made significant progress in trait translation from model species (rice and Arabidopsis) to potential biofuel crop by engineering switchgrass, sorghum and poplar plants with our best lignin competitive pathway approach [18]. Several lines were generated for each plant species using various promoters. Transformation of poplar and sorghum is performed in collaboration with Dr. S. Mansfield and Ceres Inc., respec-

tively. Poplar trees from the first set of transformation were exhibiting severe growth inhibition, thus new constructs with different promoters and new trans-formations were performed to alleviate the growth is-sues. Switchgrass engineer-ing is done partly by JBEI and partly in collaboration with BESC and with Afingen Inc. Some of the engineered plants display highly prom-ising growth and processing properties (Figure 1).

1.3 Major Research Highlights K E Y A C C O M P L I S H M E N T S F U R T H E R C H A L L E N G E S

Identified and characterized GDP-fucose and UDP-Xyl transporters

Characterization of additional transporters involved in transport of other nucleotide sugars. Understanding the mechanism for restricted transporter specificity in vivo.

Stacking of multiple genes for increased C6 sugars biosyn-thesis (galactan and callose) and decreased lignin.

Translation of best constructs to crop plants

Established model system for investigating the role of plant cell walls in mycorrhizal interactions

Using model system to characterize candidate genes

Generated novel dominant approaches to reduce lignin content without impacting plant biomass yield

Translation to crops

Identified 695 switchgrass genes predicted to control cell wall biosynthesis, modification and stress responses

Generation of grasses altered in expression and charac-terization of biomass and growth properties of plants

Identified 12 sorghum mutants altered in genes known to control cell wall associated functions

Assess growth and saccharification phenotypes

Identified and characterized genes and mutants controlling growth, development, immunity and drought tolerance

Determine mechanisms of action

Sequenced and analyzed 1000 rice mutants Complete collection of 4000 mutants so that the grass genetics community can efficiently determine function of genes predicted to control key traits

Optimized and generated new tools for whole genome network analysis of grasses.

Use RicenetV2 to identify genes predicted to control bio-logical pathways controlling stress tolerance and cell wall traits

Figure 1. JBEI technologies for en-gineering low lignin and increased wall density have been transferred to switchgrass in collaboration with the company Afingen Inc. Several lines show much more vigorous regrowth after hedging than con-trol plants (A) and have substan-tially increased plant height and tillering (B). The plants also have substantially improved saccharifi-cation. Photo and data courtesy of Afingen Inc.


K E Y A C C O M P L I S H M E N T S F U R T H E R C H A L L E N G E S

Developed negative regulator to enhance tissue specific expression in plants Crystallized a key cell wall sensing protein

Translations to monocot plant species. Determine crystal structure of complex with other cell wall proteins

Identified and characterized drought tolerance genes in rice and Arabidopsis

Determine biological mechanism for function and assess potential in bioenergy crops

1.4 Major Research & Personnel Changes The research goals and directions of the Feedstocks Division in Year 8 have largely followed the plan with more fo-cus on sustainability and performance of plants under field conditions while improved saccharification and yield of fermentable sugars remains important focus areas. A new Director for Plant Systems Biology, Dr. Jennifer Mortimer, was hired on October 1, 2014. A new Deputy Director for Grass Genetics, Dr. Guotian Li, was also appointed on Oc-tober 1, 2014.

1.5 Collaborative Research & Industrial Interactions The Feedstocks Division has extensive external collaborations including with industry in all areas of our research program. Major collaborations are listed in the table below.

I N S T I T U T I O N C O L L A B O R A T O R S T O P I C S

BESC M. Udvardi, Y. Tang, R. Dixon, M. Hahn

Switchgrass genomics and transformation. Glycome profiling

JGI D. Rokhsar, C. Penacchio, K. Bar-ry

Switchgrass, Arabidopsis, and rice sequencing

GLBRC N. Santoro, J. Ralph High throughput rice saccharification screen Lignin modification

HudsonAlpha Institute of Biotechnology

J. Grimwood, J. Schmutz Switchgrass genomics

Univ of Copenhagen, Denmark

W. Willats, P. Ulvskov, Y. Sa-kuragi,

Analysis of cell walls, pathogen responses to cell wall alterations, and evolution of cell walls

University of Melbourne J. Heazlewood Nucleotide sugar transporters Technical Univ. of Den-mark

M.H. Clausen, R. Madsen Chemical synthesis of oligosaccharide substrates

Univ.of Cambridge P. Dupree Polysaccharide profiling

Univ. of Saitama, Japan T. Kotake, T. Ishikawa Nucleotide sugar biosynthesis, MRM-analysis of mu-tants

Univ. of British Columbia S. D. Mansfield Translation of lignin competitive pathway approach

Colorado State Univ. J. Leach Rice natural variation impact on saccharification

Ceres R. Pennell Translation of traits to sorghum

Futuragen M. Abramson Translation of traits to eucalyptus and poplar

Afingen Inc. A. Oikawa Translation of traits to switchgrass

USDA sorghum Tilling Team, Texas

Zhanguo Xin

Translation of traits to sorghum

1.6 Impacts of Research The cell wall engineering has been highly successful and additional publications and patents have been made in Year 8. Afingen Inc. is a JBEI startup located in Emeryville and working on commercialization of the artificial positive

J B E I 1 0 Y e a r 8 A n n u a l R e p o r t

feedback loop technology to develop improved switchgrass varieties and yeast strains. Afingen has 4 full time em-ployees and is funded through two SBIR grants. The switchgrass are in greenhouse trials. Futuragen is an Israeli-Brazilian company that is commercializing the APFL technology and other technologies in eucalyptus and poplar from the Feedstocks Division. Engineered eucalyptus is already in field trials. Bridgestone Americas is working with JBEI through a CRADA to develop guayule plants for rubber production using JBEI technology. In addition to these companies that have obtained exclusive licenses, another company has obtained a non-exclusive license, and one company is testing our constructs in sorghum through a collaborative agreement.

1.7 Linkages to Future Plans In addition to transferases, it is clear that substrate interconverting enzymes and nucleotide sugar transporters are key proteins, and powerful tools for rational engineering of bioenergy crops. In Year 9, we will continue to study the stacking of genes for improved biomass composition and saccharification, as well as immunity and drought toler-ance. During Year 9, we will translate the most promising combinations to bioenergy crops, and we expect to grow some bioenergy crops in field studies. Establishment of the rice mutant population, construction of Kitbase and RiceNetv2 represent enabling technologies that will advance basic knowledge on grass biology and facilitate engi-neering of biofuel crops. These resources establish a firm foundation for reverse and forward genetics analyses to identify genes controlling cell wall properties, stress tolerance and carbon allocation. In addition, we will use our efficient method for nucleotide sugar metabolomics to investigate potential bottlenecks in engineered plants and will investigate metabolic fluxes using 13C-labeled precursors. These efforts will allow more rational design of improved bioenergy crops in the future and be important especially for developing crops that can be used to generate coprod-ucts in addition to fuels. We will continue to work with the Deconstruction and Fuels Synthesis Divisions to evaluate the impact of the engineered plants on fermentable sugar production and biofuel yields. With an increased focus on sustainability and the ability to grow plants under field conditions, we will continue to develop research on the in-teractions of plants with mycorrhizal fungi and with pathogens. In particular we will investigate the role of the cell wall in such interactions.

2. DECONSTRUCTION DIVISION 2.1 Introduction The efficient deconstruction of lignocellulose into fermentable sugars is one of the key steps in the biological conver-sion of biomass to fuels. Several challenges must be overcome before this process can be fully realized at the com-mercial scale: (1) lignocellulose is a complex material that requires significant energy inputs to liberate high yields of fermentable sugars; (2) crystalline cellulose is difficult to hydrolyze, and the enzymes required to do so are expen-sive; (3) the presence of lignin occludes enzyme accessibility to polysaccharides; and (4) pretreatment can produce inhibitory compounds that are toxic to fuel-producing organisms. The primary mission of the Deconstruction Divi-sion is to develop new process technologies that address each of these challenges. The goals of the Deconstruction Division are: • Provide the scientific and technological basis for an affordable and scalable integrated biomass conversion tech-

nology based on ionic liquids (ILs) • Discover lignocelluloytic enzymes with desired operating characteristics through the targeted study of microbial

communities • Develop enzyme mixtures and engineer enzymes for optimal performance under targeted pretreat-

ment/saccharification conditions (temperature, presence of ILs, high solids loading) • Demonstrate a cost-effective route for production of heterologous enzymes in Aspergillus niger; this system will

be used for cellulase and ligninase production that enables enzyme cocktail development at JBEI.

2.2 Scientific Progress 2.2.1 Biomass Pretreatment Process integration for biomass to biofuels using IL. The integration of IL pretreatment with enzymatic saccharifi-cation and microbial fermentation is challenging due to the toxicity of the ILs currently used for pretreatment, re-quiring extensive water wash. In FY15, we developed a one-pot, integrated process for the production of fuels direct-ly from lignocellulose without removal of IL or any other additional separation or post-treatment operations prior to saccharification and fermentation. To overcome the pH mismatch and cytotoxicity issues with commercially availa-ble enzyme mixtures and wild type fermentation hosts, we screened ILs for biocompatibility and employed CO2 to implement an integrated one-pot pretreatment, saccharification and fermentation process using cholinium lysinate ([Ch][Lys]). High ethanol yields (85%) were achieved using wild type yeast (S. cerevisiae) in the presence of [Ch][Lys].


This approach resolves several of the most significant obstacles towards the realization of an efficient, integrated, affordable and scalable IL conversion technology suitable for deployment at a biorefinery and is currently being test-ed for engineered hosts for the production of advanced fuels like methyl ketones. This work is also being extended to evaluate the impact of engineered feedstocks in collaboration with the Feedstocks Division. Development of one-pot high-gravity IL process for concentrated sugar production. Second-generation biofuel production from lignocellulosic biomass is constrained by factors that drive operational costs such as low titer and high water usage. We developed a one-pot high-solid biomass loading process for biofuel production from corn stover, utilizing concentrated sugar stream after saccharification, high titer ethanol production and low water usage (~3 kg per kg corn stover). We examined the effects of biomass loading, ionic liquids loading and the interaction of these effects using response surface methodology, with which an optimal operation condition was obtained. We also developed an integrated fed-batch process to resolve many engineering issues such as the limitation of mass transfer. As a result, a relatively high titer of ethanol (over 40 g/L) was produced with a one-pot approach for the first time. This integrated one-pot process significantly reduces the water usage to around 3 kg per kg corn stover in a single vessel without intervention or cleanup. Our technoeconomic analysis suggests that this integrated process could re-duce annual operation cost by 40%, compared to the conventional IL process. Investigation of the role of water in the dissolution of cellulose in IL/Water mixed solvent systems. A fundamen-tal understanding on how ILs in aqueous environments act on cellulose, particularly at lower IL concentrations with water as a co-solvent, is essential for optimizing pretreatment efficiency, lowering pretreatment cost, and improving IL recyclability. We carried out a systematic experimental investigation to understand the role of water during IL pretreatment of lignocellulosic biomass [36]. [C2C1Im][OAc] at concentrations of 50-80% at 160° C was found to be effective for cellulose solubilization and provides a substrate with enhanced enzymatic digestibility. To understand the role of water as a co-solvent with [C2C1Im][OAc], we investigated the dissolution mechanism of microcrystalline cellulose in different [C2C1Im][OAc]:water ratios at room (300 K) and pretreatment (433 K) temperatures using all at-om molecular dynamics simulations including explicit water molecules, cations and anions. These simulations show that 80:20 ratios of [C2C1Im][OAc]:water as “the tipping point” above which [C2C1Im][OAc]:water mixtures are equally effective on decrystallization of cellulose by disrupting the inter-chain hydrogen bonding interactions. The inclusion of water molecules in the medium increases the diffusivity of cellulose in the IL:water mixture and aids the dissolu-tion of cellulose. Simulations also revealed the resulting decrystallized cellulose from 100% [C2C1Im][OAc] begins to repack in the presence of water but into a less crystalline or more amorphous form. Design, synthesis and assessment of lignin and hemicellulose derived renewable IL mixture for biomass pre-treatment. To replace the conventional imidazolium-based ILs with those derived from renewable resources, we have shown the first synthesis and evaluation of a series of ILs from monomers obtained from lignin and hemicellu-lose [37, 38]. In FY15, we investigated mixtures of lignin and hemicellulose derived products, namely 3,4-dimethoxybenzaldehyde, p-anisaldehyde (both lignin-derived) and furfuraldehyde (hemicellulose-derived), without further separation or product isolation steps to improve the process economics. We employed reductive amination chemistry followed by treatment with phosphoric acid and efficiently converted the byproduct mixture to an IL-mixture. Pretreatment of switchgrass using this IL-mixture followed by saccharification yielded 80% sugar as com-pared to ~90% sugar yield obtained using [C2C1Im][OAc]. These results provide confidence that the reductive amina-tion could be applied efficiently on the byproduct mixtures without further separation/isolation step and the gener-ated IL-mixtures can be used for pretreating biomass. We also addressed two other concerns posed by the reductive amination chemistry: first, reductive amination chemistry requires expensive chemicals; second, reductive amination chemistry can be applied to only compounds bearing aldehyde functional groups. Our newly developed method is capable of transforming monomeric phenolic lignin depolymerized products or oligomeric and even polymeric phe-nolic lignin to tertiary amine-based ILs via Mannich reaction. This route requires cheap solvent, chemicals and con-sists of very simple two-step synthetic steps with minimal purification. Investigation of lignin dissolution and depolymerization in IL. We examined lignin-IL interactions and mechanis-tic aspects of lignin depolymerization during IL-pretreatment (both experimental and theoretical). We leveraged EMSL’s expertise for developing advanced lignin analytics required for this project [39]. To understand the lignin-IL interaction, we used a β-O-4 dimer as model lignin and explored its interaction with two different ILs ([C2C1Im][OAc] and [Chl][Lys]) using 1D and 2D NMR techniques. Our results show multiple H-bonding interactions between the dilignol and IL. For both [C2C1Im][OAc] and [Chl][Lys], both anion and cation takes part in the H-bonding interaction with the dilignol. The Ph-OH group of dilignol was shown to be H-bonded with acetate anion for [C2C1Im][OAc] and with carboxylic group in the lysine anion for [Chl][Lys], whereas the α- and γ-hydroxyl groups are H-bonded with the C2-H of imidazolium cation or hydroxyl group in choline cation for [C2C1Im][OAc] and [Chl][Lys], respectively. Characterization of various lignin streams generated during IL-pretreatment. In order to better understand the fate of lignin in various ILs/processes being developed at JBEI, we used model lignin dimers with different lignin interu-nit linkages and polymeric alkali lignin and screened six different ILs ranging from acidic to neural to basic, at two


different pretreatment temperatures (140 and 160 °C) and for different pretreatment times (1-3h). We analyzed the physical and chemical changes to lignin using SEC, FT-ICR and 2D HSQC NMR techniques. Our results indicate that, among different lignin interunit linkages, the β-O-4 and β-β linkages are prone to degradation during IL-pretreatment and higher pretreatment temperature and prolonged pretreatment time favors the degradation. The results also suggest the β-O-4 and β-β degradation is more favored in a protic or acidic IL such as triethylammonium hydrogensulfate. Lignin streams generated during conventional water-wash and one-pot process using [Chl][Lys] were also compared. For both processes, the liquid stream after IL-pretreatment consists of mainly depolymerized lignin and the solid lignin isolated from the liquid stream contains lower β-O-4, β- β and dibezodioxocin content. Whereas lignin isolated from the residual mass after saccharification contains higher molecular weight lignin with lower β-O-4, β-β and dibezodioxocin content. A comparison with the switchgrass enzymatic mild acidolysis lignin (EMAL) reveal that the lignin isolated from the residual mass consists mainly of recalcitrant lignin backbone, which is more resistant towards depolymerization during IL pretreatment.

2.2.2 Enzyme Optimization Screening GH diversity for ionic liquid tolerant enzymes. In collaboration with the JGI, GLBRC and JBEI’s Tech-nology Division, we are screening approximately 550 enzymes from 12 GH families selected from the CAZy database and a compost microbial community studied by the Microbial Communities group to encompass the diversity among GH families and within each GH family by selecting enzymes from various clads representing mesophiles, thermophiles and halophiles. All genes were codon optimized for the in vitro wheat germ expression system devel-oped at the GLBRC and were synthesized at the JGI. Enzymes were expressed using an in vitro wheat germ expres-sion system developed by Brian Fox’s group at the GLBRC. A subset of enzymes for which higher titer expression was required to allow characterization on IL pretreated biomass were recloned into an E. coli expression vector. En-zymes were screened using the Nanostructure-Initiated Mass Spectrometry (NIMS) assay developed by JBEI’s Tech-nology Division, and a detailed analysis of the functional diversity of GHs secreted by Clostridium thermocellum was completed [40]. To date a subset of 196 of the original ~550 GHs have been expressed in E. coli and screened using the JBEI Suite for Automated Lignocellulosic Saccharification (JSALSA) to determine their Topt, pHopt and activity in four ionic liquids. Results from these assays highlight the difficulties in predicting ionic liquid tolerance from only a small sample of enzymes and the need for large-scale screening efforts. For example, both of the GHs TIL-194 and TIL-188 in the IL [Ch][Glu] specific activity increases as the pH of the IL approaches the pHopt of the enzyme with little to no loss in activity, implying reduced GH activity in [Ch][Glu] is predominately due to a pH mismatch. However, the specific activity of the same two enzymes in [C2C1Im][OAc] decreases as the corresponding pH of the IL increases toward their pHopt. Confounding these results is the fact that the enzyme A5IL97 shows the opposite behavior; its ac-tivity increases with increasing [C2C1Im][OAc] but decreases with increasing [Ch][Glu]. Engineering improved enzyme stability and activity in [C2C1Im][OAc]. We are using directed evolution approaches to engineer a β-glucosidase for enhanced stability and activity in [C2C1Im][OAc]. A high throughput robotics method has been established for screening libraries of mutants of the enzyme generated through error prone PCR. This screening method measures the activity of each mutant in both the presence and absence of [C2C1Im][OAc] and com-pares the ratio of these activities to identify mutants with increased IL tolerance, which are then further screened to measure the concentration of [C2C1Im][OAc] at which the enzyme loses half of its activity (IL50). To date we have gen-erated three mutants with increased IL50: a Phe to Leu and Val to Met double mutant with a 3% increase in IL50 and 80% of the wild type enzyme activity, a Gln to His and Ser to Phe double mutant with a 5% increase in IL50 and 50% retained activity, and a Glu to Gln, Val to Leu, Val to Gly, and Ser to Phe quadruple mutant with a 7% increase in IL50 and 39% wild-type activity retained. The [C2C1Im][OAc] tolerant mutants from this screen are being further character-ized for changes in kinetics, melting temperature, T50, pH profile, and half-life. The most stable active mutant will be subjected to subsequent rounds of mutagenesis and library screening, with the ultimate goal of producing a mutant that is stable and active in 20% [C2C1Im][OAc]. Enzyme recycling using upper critical solution temperature polymers. We are using upper critical solution temper-ature (UCST) polymers to recycle cellulases after saccharification. UCST polymers undergo a temperature induced phase transition: above the transition temperature (Tp) these polymers are soluble; below the Tp the polymers precipi-tate out of solution. Enzymes that are covalently attached to these polymers react with cellulose at their optimal reac-tion temperatures and are recovered by lowering the reaction temperature where the enzyme-polymer conjugates can be separated from the resulting sugars by centrifugation or filtration. Several ureido-based UCST polymers have been synthesized with transition temperatures ranging from 10-30°C. We have also shown these polymers function in the presence of [C2C1Im][OAc], although the Tp of the polymer decreases with increasing IL concentration. Our plan is to covalently link cellulases to these polymers, via a reactive cysteine residue engineered into the c-terminus of the enzyme, using chemical crosslinkers. However, incubating the enzyme with the polymer in the absence of crosslink-er results in nonspecific binding, allowing for efficient recycling of the enzymes from reaction mixtures. Using a 4-nitro phenyl glucopyranoside (pNPG) as a model substrate, a beta-glucosidase-polymer conjugate was recycled four times in the presence of 0%, 10%, and 20% [C2C1Im][OAc]. New ureido based UCST polymers are being synthesized with varying transition temperatures and cellulase mixtures.


Assaying lignin breakdown. We have developed a highly sensitive and quantitative 96-well plate based assay for determining the effects of lignolytic microbial cultures, enzymes, and chemistries on films of high molecular weight insoluble lignin [41]. We demonstrated the use of the thin film assay to rapidly quantify the release of soluble frag-ments and to study changes in lignin surface chemistry using FTIR-ATR spectroscopy. The method was used with organosolv lignin and Fenton chemistry to assay the effects of H2O2 and FeCl2 concentrations. We are also using our film-based method to assay lignases, and a large number of genes coding for putative lignolytic enzymes (~ 150 ex-tracted from the CAZy DB) have been synthesized for us by the JGI. To tune the assay for measuring enzyme activi-ty, a series of tests were conducted with manganese peroxidase using glutathione (GT) as a mediator and H2O2 gener-ated in-situ using glucose/glucose oxidase (Glu/GO). The concentration of GT ranged from 1 mM to 10 mM and a range of Glu/GO concentrations were used. MnP activity in the presence of Glu/GO was verified by monitoring formation of the Mn+3/malonate complex at 270 nm. In all cases, the wells containing the MnP showed less mass loss than the control wells lacking MnP, suggesting MnP adsorbs to the lignin and becomes inactive, blocking low level dissolution that occurs with the control films. We are also studying the effects of four ILs choline lysine, choline ace-tate, choline alpha ketoglutarate and choline glutamate IL on laccase activity. Preliminary results indicate choline acetate is less inhibitory than the three other ILs, with the laccases retaining 39% activity on ABTS at 30% choline ace-tate. The laccase retained only 2%, 0.5% and 9% activity in only 10% choline glutamate, choline alpha ketoglutarate and choline lysine, respectively. Computational studies of protein – ionic liquid interactions. We have continued to use computational methods to understand the performance of enzymes in IL/water mixtures with goals of (1) gaining a fundamental understand-ing of how ILs alter protein stability and enzyme activity, and (2) using this understanding to engineer improved enzyme properties and IL tolerance. We are using a variety of QM (DFT) and MM (EVB/FEP) methods to develop and sample accurate model potential energy surfaces for pure IL solutions and solvated enzymes to compare reac-tion free energy profiles. Thus far we have developed models of [C2C1Im][OAc] and [C2C1Im][OAc]/H2O mixtures. Simulations of binary IL/water mixtures are being used to examine dynamic structure via topological QM ap-proaches. Solution QM studies of the reactions catalyzed by Aa_Cel9a and Tm_Cel5a have provided insights, which are being used to build atomistic MM models of enzyme and solution reactions. MD simulations with these models will be used to compute reaction free energy profiles, which can be decomposed into contributions from residue-active site interactions. This will allow suggestion of useful mutations to test experimentally.

2.2.3 Microbial Communities Mechanisms of microbial ionic liquid tolerance. Previous studies have demonstrated that an MFS-1 pump from Enterobacter lignolyticus and SMR pumps from Bacillus species conferred ionic liquid tolerance to E. coli [42]. During this year, we demonstrated that deletion of a single SMR pump in Bacillus thurigenesis produced an IL-sensitive mu-tant, establishing that individual efflux pumps may be responsible for conferring IL tolerance in a native host. Also, spontaneous mutants of E. coli were isolated with substantially higher IL tolerance than wild-type strains. The source of the increased tolerance was identified as a one-base change in the 5’-UTR region of a native E. coli SMR pump, which promoted increased expression of the native SMR pump. Yeast ionic liquid tolerance was displayed by a broad cross-section of species in a high-throughput culturing assay using the Phaff Yeast Culture Collection at UC-Davis [43]. Thermo/IL tolerant cellulase mixtures. The community approach to understanding cellulases has provided two new types of thermotolerant bacterial cellulases. Previously, we developed Jtherm, an IL/thermo tolerant cellulase cock-tail that consisted of a supernatant from thermophilic cellulolytic community supplemented with purified cellulases [44]. In this year, the active component of Jtherm was purified from a 300 L culture of the cellulolytic community per-formed at the ABPDU and shown to be two complexes with cellulase and xylanase activities containing five multi-domain glycoside hydrolases that was distinct from previously characterized cellulosomes. Time series metagenomic analysis demonstrated that the population producing the Jtherm complex was the primary colonist of the cellulose substrate, but declined in relative abundance over the time course of the cultivation. Cultivation of cellulose-adapted consortia revealed that communities dominated by Thermobispora bispora, a thermophilic actinomycete, had high lev-els of hydrolytic activity in the supernatant on crystalline cellulose. Comparative proteomics in collaboration with EMSL revealed that the level of GH12 in the supernatant was the critical determinant for activity on crystalline cellu-lose, an unexpected result as GH12 is an endoglucanase and the levels of the canonical exoglucanases, GH48 and GH6, as well as the AA10 protein (CBM33) did not correlate with increased activity on crystalline cellulose. Heterol-ogous expression of GH12, GH6 and GH48 demonstrated that the GH12 had the highest specific activity for cellulose hydrolysis of the three glycoside hydrolases. Time course experiments of sugar release with NIMS were consistent with the assignment of Thermobispora GH12 as a mildly processive endoglucanase. Lignin deconstruction and metabolism A study comparing microbial community composition, protein expression and residual biomass was completed for thermophilic communities growing on intact and pretreated switchgrass (AFEX and IL pretreatment) [45]. Two-dimensional NMR studies of the residual biomass indicated that polysaccha-


rides were depolymerized in preference to lignin that was largely unaltered. The genome sequence of Klebsiella sp. BR6L-2, a lignin degrading isolate from the Puerto Rican rain forest, was published [46]. We have developed a meth-od to solubilize the majority (>90%) of residual lignin after enzymatic or acid hydrolysis of the polysaccharides. This lignin is fractionated by size exclusion to provide soluble lignins of defined molecular size. The redox reactivity of these lignin fragments to ferricyanide is inversely correlated with their size, suggesting that high molecular weight soluble lignin is less accessible to oxidants for depolymerization. We are therefore poised to test these size-fractionated in microbial and enzymatic deconstruction of lignin. Bioinformatics. MaxBin, an automated binning tool for recovery of genomes from metagenomes, was implemented this year to recover genomes of uncultivated community members involved in high temperature lignocellulose de-construction. The genomes of uncultivated members of the Deinoccocus-Thermus [47] and the Chlorobi were recon-structed and functions in the community determined by manual annotation of the genomes. For the uncultivated Chlorobi genome, six related genomes were recovered from metagenomic datasets of hot springs samples available through JGI’s IMG/M website, providing a comprehensive genomic survey of an uncultivated lineage ubiquitous in high temperature environments. MaxBin 2.0 was developed to bin metagenomic datasets from multiple samples that are co-assembled. This tool was applied to bin samples obtained from a salt gradient in the San Francisco Bay salt flats and a time series of the 300 L community culture from which the Jtherm complex was isolated. A meta-transcriptomic study of paired switchgrass-adapted solid state cultures grown at 35°C and 55°C demonstrated that GH48 and AA10 enzymes were highly overexpressed in the 55°C culture and are likely responsible for the observed switchgrass deconstruction [48].

2.2.4 Fungal Biotechnology Functional characterization of genetic “parts” to expand A. niger engineering toolset. A. niger has a growing set of genetic tools designed to facilitate genetic engineering and heterologous enzyme production, but still more validated genetic parts would be valuable for driving expression of multiple heterologous enzymes for lignocellulolytic cock-tail development and host engineering. Proteomic analysis of the secretome of A. niger grown under various condi-tions was conducted at EMSL and about 40 promoters, signal peptides, and introns were identified for further analy-sis. These components were inserted into a construct driving the expression of the heterologous thermophilic bacte-rial β-glucosidase A5IL97 and this succeeded in identifying several promising promoters and signal peptides. These parts will be further vetted and used to express both cellulase and lignin degrading enzymes in the coming year. Improving heterologous enzyme production in A. niger. Several efforts this year focused on improving enzyme production in A. niger, including optimizing cultivation conditions, exploring the role of glycosylation on enzyme expression and activity, and targeted host engineering. We also addressed the persistent questions about the ability to express functional bacterial enzymes in a fungal host by expressing 30 of the best JBEI cellulolytic enzymes. All of the enzymes expressed in A. niger had equal or better overall expression levels, thermal and IL stability and enzyme activity, compared to those expressed in E. coli—and they were secreted. Increasing the cultivation temperature for A. niger improves heterologous enzyme production by 60%. We collaborated with JGI on transcriptome analyses of A. niger grown from 30 to 40°C and found that the expression of many genes in the protein secretory pathway were affected. Furthermore, high-resolution micrographs from the EMSL team showed increased branching of fungal hy-phae at higher temperatures. Moving forward, bioreactor studies will be conducted at the Advanced Biofuels Process Demonstration Unit (ABPDU) to monitor and control pH, temperature, etc. in order to develop a bioprocess that maximizes enzyme productivity, and provide further samples for systems biology studies aimed at identifying the genes responsible. In native fungal enzymes, glycosylation is associated with secreted enzyme stability and function, but the effects on normally aglycosylated prokaryotic enzymes is not understood. Therefore, we introduced several artificial N-glycosylation sites into the sequence of the bacterial β-glucosidase A5IL97 and JGI synthesized those gene variants. An interesting revelation was that many variants appear to have greater activity than the unmodified strain. With our EMSL collaborators, we are performing top-down proteomic analyses to determine which variants are gly-cosylated and correlating those results with our enzyme kinetic and thermodynamic characterizations to deepen our understanding of protein glycosylation. With JGI we resequenced the genomes of high enzyme production mutants from our forward mutagenesis screen. Some of the high priority candidates identified in this effort are a sugar trans-porter and two kinases. We are currently knocking out these genes to determine if they are responsible for the en-zyme hyper-production phenotype. Understanding the impacts of position effect variegation on heterologous enzyme production in A. niger. We have observed that random integration of heterologous cellulase genes into A. niger and variable copy numbers can both affect measured enzyme expression. To understand the causes of these effects we sequenced several transformants at the JGI, mapped the position of the integrated genes, and determined copy number. We have found two integration “hot spots” in the genome and have found that copy number is important, but does not always correlate with higher expression. This phenomenon may be due to epigenetic effects, so we established ChIP-seq as a tool in A. niger to examine RNA polymerase occupancy and histone modifications at the transgene sites and the genome at large. These


studies will add another valuable layer to our understanding of protein expression and facilitate the establishment of stable and reliable expression systems in A. niger. Fungal Valorization of Lignin. Technoeconomic analyses have indicated that valorization of lignin will be critical for the success of lignocellulosic biorefineries. Fungi are nature’s lignin degraders, so it was not surprising but grati-fying that we were able to isolate strain of Rhodotorula and Rhodosporidium that grew well on lignin monomers and partially depolymerized lignin from our IL pretreatment processes. These carotenogenic (tetraterpene synthesizing) fungi are logical platforms for terpene production, since they have high flux through the terpene pathway. In collab-oration with the Fuels Synthesis Division, we engineered Rhodosporidium toruloides to produce bisabolene and amor-phadiene utilizing mono-aromatics and lignin-rich hydrolysates as carbon sources, thus demonstrating that fungal valorization of IL-depolymerized lignin streams is feasible. In Years 9 and 10 we will continue this fruitful inter-division collaboration to explore this promising lignin utilizing, terpene synthesizing platform for bioproduct and biofuel production.


Demonstrated that active component of Jtherm is a non-cellulosomal complex with unprecedented structure.

Reconstitute the complex by heterologous expression of its constituent proteins.

Demonstrated genotype to phenotype correlation of ion-ic liquid tolerance with native strains of Bacillus thuri-gensis and Escherichia coli

Mechanisms for regulation of efflux pumps will be investi-gated by molecular and structural biological techniques

Separated ionic-liquid pretreated lignin into soluble, size-defined fractions

Demonstrate microbial and enzymatic deconstruction on these defined fractions of lignin

Advanced fundamental understanding of heterologous enzyme production in fungi by establishing new meth-ods for studying this fundamental biological question. Obtained new parts for driving expression of multiple genes of interest.

Found conditions that increased heterologous expression and have identified a manageable number of promising leads for engineering better expression.

Developed a method to convert lignin and hemicellu-lose into renewable ionic liquids

Develop expanded routes of catalysis that use more complex substrates, and identify alternative reducing agents to lower cost of production

2.4 Major Research & Personnel Changes Beyond the normal and expected turnover of post-doctoral employees, the only significant personnel change was the hiring of Dr. Corinne Scown to be the new Director of Technoeconomic Modeling. Additionally, the milestones for this team will now be included as part of the Deconstruction Division’s annual milestones in Years 9 and 10 (see Ap-pendix E).

2.5 Collaborative Research & Industrial Interactions I N S T I T U T I O N C O L L A B O R A T O R S T O P I C S

BESC Charlie Wyman, Mark Davis, Mike Crowley

Comparison of dilute acid and IL pretreatment; high-throughput screening of biomass; lignin analysis; plant cell wall modeling

GLBRC Tim Donohue, Bruce Dale, Venkatesh Balan, Trey Sato, John Ralph

Comparison of AFEX and IL pretreatment; microbial inhi-bition of biomass hydrolysates; 2D-NMR of plant cell walls, lignin conversion, lignolytic cocktail development

CMS Stuart Nemser Dewatering of ILs Joint Genome In-stitute

Kerrie Barry, Sam Deutsch Gene synthesis, metagenomics, expression and characteri-zation of GH1 enzymes

Environmental Molecular Sciences Laboratory

Robby Robinson, Ljiljana Pasa-Tolic, Scott Baker

Metaproteomics, proteomics, metatranscriptomics, HSQC NMR


I N S T I T U T I O N C O L L A B O R A T O R S T O P I C S

University of Queensland (AU)

Robert Henry Analysis and pretreatment of eucalyptus species for biofuel production

Imperial College (UK)

Tom Welton, Jason Hallett Analysis and testing of new ionic liquids

University of Milan

Fabrizio Adani Ionic liquid pretreatment of Arundo donax

Idaho National Laboratory

Vicki Thompson, David Thompson Mixed and densified feedstocks, enzyme mixture optimiza-tion

ABPDU Todd Pray, Chenlin Li, Deepti Tanjore Scale-up of IL pretreatment and recombinant protein ex-pression

Total Florence Mingardon Conversion of lignin into renewable aromatics

2.6 Impacts of Research The development of the integrated one-pot approaches to biomass conversion represents a significant advance to the field. The realization of renewable, biocompatible ILs that can be tolerated by downstream enzymes and microbes is a potential disruptive technology that would positively impact biorefinery economics and environmental footprint. We are currently partnering with industry and the ABPDU to demonstrate this technology at pre-pilot scales to iden-tify remaining obstacles that will be addressed in Year 9 and beyond. There is one start-up company, Illium Technol-ogies, based on the R&D conducted by the Division, as well as a new CRADA (POET) and a renewal of an existing CRADA (with Total) that has been extended into Phase II based on the successful completion of Phase I. MaxBin has been downloaded more than 1,000 times and is being adopted by users in the external scientific community. The dis-covery of a bacterial non-cellulosomal complex that is very effective at hydrolyzing polysaccharides is novel and provides new insights into the role and mechanisms by which bacteria degrade cellulose in the environment.

2.7 Linkages to Future Plans The one-pot conversion technologies will be used as a new method to collaborate and foster integration with the Feedstocks, Deconstruction, Fuels Synthesis, and Technology Divisions at JBEI. In Year 9 we will develop and im-plement a “Feedstocks to Fuels” HTP capability based on the bionic liquids that require no solid/liquid separation and/or washing in between the steps of pretreatment, saccharification, and fermentation. This will create a unique JBEI capability that can screen hundreds of engineered plants a week and correlate both sugar and advanced biofuel yields to phenotype and genotype. The Deconstruction Division, in partnership with the LBNL ABPDU, will gener-ate hundreds of liters of hydrolysates from wild type and engineered plants for use by the Fuels Synthesis Division. In Year 9, we expect to resequence multiple strains of A. niger from the forward genetics task and will analyze these datasets for genes potentially involved in increased protein expression. Various parameters have been explored to increase heterologous gene expression and we are poised to understand the underlying genetic mechanisms in the future. Valuable genetic parts (e.g., promoters) have been identified to engineer increased production of one or many deconstruction enzymes. Lignin utilizing bacteria and fungi have been isolated that will form the basis of a new thrust in biological lignin valorization. We will continue collaborations with the Fuels Synthesis and Technology Di-visions in the development of enzymes and engineered microbial hosts that can tolerate ILs and synthesize valuable biofuels and renewable chemicals from sugars and lignin, and will aggressively pursue the results obtained in red yeast.

3. FUELS SYNTHESIS DIVISION 3.1 Introduction The primary research goal of JBEI’s Fuels Synthesis Division is to identify challenges and develop approaches to en-able engineering of microorganisms to efficiently convert sugars to advanced biofuels with properties similar to pe-troleum-based fuels. To that end, we are developing fuel synthesis pathways based on the fatty acid, isoprenoid, polyketide, and aromatic amino acid biosynthetic pathways. These pathways are being engineered into one or both of two host organisms: Escherichia coli and Saccharomyces cerevisiae. Because yields and productivities must be high to make production of biofuels economically viable, we are engineering central metabolism in our host strains to deliv-er precursors to the biosynthetic pathways. This requires new computer-aided design software and genetic tools to control expression of the genes, and metabolic models to identify pathway constraints. As the biofuels themselves or


the substrates generated during deconstruction may be toxic to the cell, we need to understand the mechanisms of toxicity and alleviate it.

3.2 Scientific Progress 3.2.1 Biofuel pathways discovery and development The Fuels Synthesis Division, working in conjunction with the Combustion Research Facility at Sandia, has identified the following renewable fuel targets: • Jet fuels: α-pinene, limonene, 1-pentene (precursors to jet fuels), fully reduced α-bisabolene, toluene • Diesel fuel: bisabolane, alkanes, fatty acid ethyl esters, 1-pentene (precursor), methyl ketones, toluene • Gasoline: isopentanol, toluene We have prepared quantities of several of these fuel targets for fuel property tests and confirmed that they have comparable fuel properties to commercial gasoline or diesel fuels. In FY15, we published a comprehensive review of fatty acid-derived and isoprenoid biofuels and bio-based chemicals [49]. Fatty Acid-Derived Fuels. Long, linear hydrocarbons can be readily produced from fatty acids. As such, we have engineered the fatty acid precursor pathways in E. coli and S. cerevisiae. Fatty a cid-producing yeast. We are engineering S. cerevisiae to produce fatty acid-derived fuels by overexpressing ACC1, FAS1 and FAS2, DGAT, and TesA [42]. In prior work, we achieved free fatty acid titers of greater than 400 mg/L. We have also developed approaches to convert the fatty acids into a number of fuels, including methyl ke-tones, fatty acid ethyl esters, fatty alcohols and fatty alkenes. To further improve fatty acid production, we are repro-gramming genes in central metabolism to alter the levels of acetyl-CoA, NADH and NADPH. Expression o f Type 1 FAS in E. co li. We have cloned type 1 fatty acid synthases (FASs) from several organisms and expressed them in E. coli. The advantage of Type 1 FASs over E. coli’s native FAS is the production of fatty acyl-CoAs rather than fatty acyl-ACPs. Fatty acyl-CoAs can be used directly for production of fatty alcohols and fatty acid ethyl esters, whereas fatty acyl-ACPs must be transformed to fatty acyl-ACP, which costs an additional ATP. We have successfully produced approximately 3.5 g/L fatty alcohols and 35 mg/L methyl ketones using the Type 1 FAS [50]. Anteiso -fa tty a cid production. A major disadvantage of fuels derived from biological sources is their undesirable physical properties such as high cloud and pour points, and high viscosity. We developed an Escherichia coli strain that efficiently produces anteiso-branched fatty acids, which can be converted into downstream products with lower cloud and pour points than the mixtures of compounds produced via the native metabolism of the cell. This was achieved through the deletion of metA, tdh, ilvB, and ilvN and the overexpression of thrABC and ilvCD from E. coli, ilvA from Corynebacterium glutamicum, ilvGM from Salmonella typhimurium, as well as bfabH2 and the bkd operon from Bacillus subtilis, which together promote the synthesis of the 2-methylbutyryl-CoA and subsequently use this metabo-lite to initiate fatty acid synthesis. When these genetic manipulations are coupled with those that promote free fatty acid synthesis and accumulation, 20.4% of the free fatty acids produced in the engineered E. coli cells were anteiso-branched, although with a significant decrease in total fatty acid titer. Dynamic regulation of bfabH2 expression helped to increase total fatty acid titers while maintaining a 9.9% proportion of anteiso branched fatty acids [51]. Optimizing fa tty a cid bio synthesis fo r anaerobic conditions. In order to minimize redox cofactor (NADH/NAD+) imbalance under anaerobic conditions, we identified homologs of FabG, an essential reductase involved in fatty acid biosynthesis, that displayed a higher preference for NADH than for NADPH as a cofactor. Four potential NADH-dependent FabG variants were identified through bioinformatic analyses supported by crystallographic structure determination (1.3 to 2.0 Å-resolution), were subjected to in vitro assays, and were overexpressed in fatty acid- and methyl ketone-overproducing E. coli host strains under anaerobic conditions. The best-performing FabG variant led to a 60% higher free fatty acid titer and 75% higher methyl ketone titer relative to the control strains under anaerobic conditions [52]. Diesel-range methyl ketones. In 2014, we made pathway and host engineering modifications to E. coli strains bear-ing a novel pathway for biosynthesis of C11 to C17 methyl ketones [53]. These modifications resulted in a 160-fold in-crease in titer in minimal medium (1% glucose) relative to the best strain reported in 2012 (Figure 2), such that we have attained a yield that is 40% of the maximum theoretical value and a titer of 3.4 g/L in 45 hr fed-batch fermenta-tion. These are the highest titers and yields of methyl ketones (other than acetone) yet reported in engineered or na-tive bacteria. In a related project funded by ARPA-E, we found that the chemolithoautotrophic bacterium Ralstonia eutropha growing on H2 and CO2 could generate comparable titers to E. coli growing on glucose when the same genet-ic manipulations were performed on both bacteria [54]. In FY15, we made efforts to improve methyl ketone produc-


tion in E. coli using a high-throughput NIMS screening technique (with oxime derivatization) coupled with random mutagenesis and by making chromosomal deletions based on metabolic modeling predictions. Ladderanes. Anammox (anaerobic ammonia-oxidizing) bacteria make unusual lipids containing 3 or 5 fused cy-clobutane rings called "ladderanes". Modified versions of these highly energetic biomolecules could potentially serve as a basis for the biosynthesis of aviation or other fuels. The enzymes responsible for ladderane biosynthe-sis are unknown. We completed construction of nine synthetic operons composed of 22 JGI-synthesized can-didate ladderane biosynthesis genes and tested them in vivo in E. coli under anaerobic conditions. In FY15, we carried out in vitro, anaerobic studies with a putative phytoene desaturase (CrtI) homolog from anammox bac-teria and confirmed that it catalyzes nicotinamide cofac-tor-dependent saturation of a double bond in an all-trans polyunsaturated fatty acid [(E,E)-farnesoic acid]. We also confirmed that it does not have CrtI activity, and thus is a novel enzyme. Isoprenoid-derived Fuels. Short, branched alcohols and long, branched and cyclic hydrocarbons can be derived from isoprenoids. We engineered the isoprenoid precur-sor pathways in S. cerevisiae and E. coli. We also engi-neered these two hosts to produce the following isopre-noid-based fuels: α-Bisabo lene. Bisabolane (reduced α-bisabolene; cetane number of 52.6) is a promising diesel and bio-jet fuel candi-date [55]. We engineered the bisabolene-producing E. coli and yeast strains to achieve titers of ~3.5 g/L and ~6 g/L, respectively [56]. The best producing yeast strain was transferred to the Advanced Biofuel Process Demonstration Unit (ABPDU) for larger-scale production. Limonene and α-pinene. We engineered E. coli to produce limonene and α-pinene, potential jet and diesel fuel pre-cursors, by expressing genes encoding codon-optimized geranyl diphosphate (GPP) synthase and Abies grandis limo-nene synthase (for limonene) and pinene synthase for pinene. We improved limonene titers from 50 to 450 mg/L through metabolic engineering efforts in the previous year [57], and we further improved the titer to 600 mg/L by balancing the pathway from the analysis of proteomics data [58]. Initially engineered pathway for pinene production in E. coli showed a titer only about 4 mg/L. To increase flux through the pinene synthesis, various GPPS-PS protein fusions were introduced to pinene producer, and the Abies grandis GPPS-PS fusion produced 32 mg/L of pinene in E. coli [59]. Initially engineered pathway for pinene production in E. coli showed a titer only about 4 mg/L. To increase flux through the pinene synthesis, various GPPS-PS protein fusions were introduced to pinene producer, and the Abies grandis GPPS-PS fusion produced 32 mg/L of pinene in E. coli [59]. Isopentenol. Previously, we engineered E. coli to produce the C5 alcohols (isopentanol, 3-methyl-3-butenol, and 3-methyl-2-butenol) [60]. We leveraged proteomics capabilities at JBEI to achieve improved mevalonate pathway bal-ance and produce isopentenol at high titers (~1.5 g/L, 46% theoretical yield) [61]. The study of the pathway bottle-necks using proteomics and metabolomics data further improved the production titer of isopentenol to 2.3 g/L (over 70% of pathway-dependent theoretical yield and 56% of theoretical max) [62]. New IPP-bypassing pathways for iso-pentenol production were proposed and constructed for more efficient isopentenol production, especially in a less aerated condition. Polyketide-derived fuels. By rationally recombining domains found within polyketide synthases (PKS), short (C3-C6) branched (or unbranched) hydrocarbons can be produced in a predictable manner. Compounds such as the terminal alkenes can be oligomerized into jet and diesel fuels. We have codon-optimized PKS modules known to release ter-minal olefins and alcohols are incorporating these domains into chimeric, multi-modular PKS systems to demon-strate production of compounds such as propene, 1-butene, styrene, 1-hexene, 1-pentanol and 1-hexanol. Toluene. To discover toluene synthase (phenylacetate decarboxylase), we generated anaerobic enrichment cultures from primary sewage sludge that stoichiometrically produced toluene from phenylacetate, partially purified the en-zyme from lysates of the enrichment culture, performed shotgun proteomics of the active fraction after elution through two different chromatographic columns, ranked gene candidates, and have been testing top candidates by

Figure 2. Improvements in methyl ketone production in E. coli.


heterologous expression in E. coli. In vitro studies have shown that the target enzyme is soluble, does not require ca-nonical decarboxylation cofactors, and has roughly equal specific activity for phenylacetate and p-hydroxyphenylacetate but is not the known p-hydroxyphenylacetate decarboxylase (HpdBC). FY15 studies have focused on in vitro studies with a number of toluene synthase candidates, most notably, a novel glycyl radical en-zyme and its cognate activase.

3.2.2 Metabolic Engineering Proteomics-aided metabolic engineering. We analyzed proteomics data together with growth rates, titers, and key metabolites levels from microbial growth experiments. This allowed an in-depth analysis of the pathway under con-sideration and provided guidance on metabolic engineering approaches to improve biofuel production. As an exam-ple, we analyzed the isopentenol production strains based on the correlation of protein levels in each pathway with key metabolite levels, and from this analysis, we engineered the production strain to improve the product yield from 6% to 46% of apparent theoretical yield[61]. Also we developed a novel method for proteomics data mining using principal component analysis (PCA), and applied this method for limonene and bisabolene production strains. The engineering to balance the pathway enzyme expression based on PCA of proteomics data has improved limonene production by 30% and bisabolene production by 40%[58]. Multi-Omics facilitated pathway analysis and engineering. We achieved more advances in metabolomics using hydrophilic liquid interaction chromatography (HILIC) and were able to monitor a large number of important me-tabolites from producing strains. This allowed more in-depth analysis of the pathway using both proteomic and metabolomic data as well as growth and production data, and we established the metabolic pathway designing and engineering procedure to improve biofuel production. We applied this Multi-Omics facilitated approach to the strains producing the three representative isoprenoid targets (C5, C10 and C15) and are currently developing metabol-ic modeling based on these multi-omics data to more accurately understand and predict the pathway behavior in biofuel producing hosts. Functional expression of DXP pathway in yeast. Work completed at JBEI, in addition to a partnership with Amyris Inc., resulted in the development of a partially functional DXP pathway in yeast. Starting from a zero-flux pathway, selection of an appropriate ispG enabled detection of DXP-derived ergosterol at levels below 1%. Following this, modifications to the growth conditions and metabolomics-guided pathway optimization has increased DXP pathway flux to over 33%, close to levels that will support growth in the absence of the mevalonate pathway. Using JGI-synthesized plasmids, we are currently working towards improvement of DXP pathway flux to the point where it can support ergosterol production in the absence of the native mevalonate pathway. Discovery of novel metabolic routes to the DXP pathway (nDXP). Using a dxs knockout strain of E. coli, we have discovered two alternative enzymes, both of which convert ribulose 5-phosphate directly to DXP. We have demon-strated utility of these genes for engineering isoprenoid production in E. coli and further improved flux by fusing the nDXP proteins to DXP reductase (Dxr), the second enzyme in the DXP pathway [63]. Tween-based selection for isoprenoid-based fuels. We discovered that non-ionic surfactants such as Tween 20 (T20) could select for high α-bisabolene production. In addition to screening for improved bisabolene synthase mutants, we found that T20 could also be applied to sustain four-fold higher production titers in a monoculture for an extend-ed period (14 days) by acting as selection for the metabolic product, α-bisabolene [64].

3.2.3 Synthetic Biology DIVA (Design, Implementation, Validation Automation) software. GeneDesign functionality has been integrated into DIVA, which enables DIVA users to reverse-translate protein sequences of interest into their DNA sequence de-signs, as well as to codon-optimize the coding sequences in their designs for expression in their hosts of interest. Over 541 DNA constructs have been submitted to the DIVA PCR Service by Feedstocks, Deconstruction, Fuels Syn-thesis, and Technology Division researchers. The National University of Singapore has adopted DIVA. ICE (Inventory for Composable Elements) repository software. MiSeq sequence validation of DNA constructs (e.g., linear cassettes, plasmids) has been integrated with ICE. This is important because it enables MiSeq sequence valida-tion data results to be stored along with their corresponding reference DNA sequence entries, providing confidence to researchers in the future who would like to see the evidence that a given sequence has been verified to be correct. Users can view detailed sequencing results for a given plasmid entry by clicking a link to open the sequencing data in IGV sequence analysis viewing software. DIVA/ICE/EDD software integration. DIVA, ICE, and EDD (see Section 4.2.6) have been integrated. Namely, a user can specify desired functional characteristics for an enzyme or promoter in DeviceEditor (a visual bioCAD canvas in DIVA), DIVA then queries ICE for enzymes or promoters that meet those specifications, ICE in turn queries EDD for experiment data supporting the assertion that the enzyme or promoter characterization data is consistent with the


user’s specification. For example, a user in DIVA may want a promoter that in S. cerevisiae CEN.PK expresses consti-tutively at a high level, but the user does not know which promoter has been experimentally validated to meet this criteria. With this new functionality, the integration of DIVA, ICE and EDD can automate the selection of this pro-moter for the user, and point the user to the experiment data that supports the automated choice.

3.2.4 Host Engineering Carbon source utilization. We developed S. cerevisiae strains that can utilize xylose. A minimally engineered yeast strain—with modifications including a GRE3 deletion to minimize accumulation of xylitol, a codon-optimized xylose isomerase (XI) from Piromyces and overexpression of the native xylulose kinase (XKS)—was evolved for significant xylose utilization. In collaboration with JGI, we sequenced this evolved strain. The evolved strain (JBEI_ScMO003) contained three SNPs relative to the basal strain. We have confirmed that one of those SNPs, present in a membrane transporter, is primarily responsible for the evolved xylose utilizing phenotype. The discovery has been confirmed using expression and deletion assays in vivo. We have combined this modification with multiple other reported ge-netic modifications (including gene deletions and overexpression) to develop a better utilization strain. The im-proved uptake using the evolved transporter has also been confirmed using 14C labeled xylose uptake assays and membrane vesicle based uptake assays. Tolerance and toxicity studies in E. co li. Solvent-like products, inhibitory pathway intermediates and pretreatment related compounds remain bottlenecks in maximizing the efficiency of microbe-based production of fuels. In the pre-sent year we made several key advances towards different aspects of this bottleneck. Using array based methods we identified several candidates that were involved in isopentenol tolerance. An evaluation of several candidates, span-ning multiple functions categories, led to the discovery of regulators, chaperones as well as transporters that not only improve tolerance to exogenously added biofuel (isopentenol) but also improve production when co-expressed with production pathways [65]. We completed the evaluation of a spontaneous mutant in Limonene tolerance and found the pertinent mutation to be in AhpC, an alkyl hydroperoxidase. We confirmed that it is the oxidized limonene hy-droperoxide responsible for the acute toxicity often observed with limonene, which may have important implica-tions, not only in the production but also the storage of this candidate jet-fuel [66]. In collaboration with Total New Energies, under a joint CRADA, we also conducted a successful directed evolution effort to improve tolerance to α-olefins such as 1-hexene (a biopolymer precursor), using the RND efflux pump AcrAB-TolC [67]. Membrane Capacity Project. Of the tolerance mechanisms discovered, membrane based transporters remain a pow-erful and direct mechanism to improve production. To maximize our ability to utilize these pumps, we have initiated a project to (1) understand the sources of burden associated with membrane protein expression, and (2) develop a host E. coli strains that can accommodate greater expression of a membrane protein of interest. Initial studies using a small set of knockouts generated in collaboration with the JGI using the Cas9/CRISPR based approach, showed that the deletion of certain membrane bound proteins do impact both the ability of the other membrane proteins to be expressed at a greater level, as well the resulting toxicity from the desired protein expression. In order to have a comprehensive understanding of this potentially valuable phenomenon, we altered our strategy and now plan to utilize the entire gene deletion library in E. coli to query this knockout dependent phenomenon at the genome wide level. We are collaborating with other scientists at LBNL and using a bar-coded gene deletion collection in combina-tion with FACS to achieve this goal. Expanding strain engineering tools in S. cerevisia e. To address the need that all pathways may eventually require integration into the genome, an integrated project (across several JBEI divisions) has been initiated to (1) identify chromosomal loci and native promoters that provides the best positions for the integration of a variety of genes and pathway so as to achieve the optimal of expression (e.g., high, low, medium, constitutive); (2) test the efficacy of the-se loci using established pathways for fuel production; and (3) demonstrate the efficiency of the resulting strain for fuel production when using ionic liquid pretreated biomass as carbon source. Using fluorescent proteins, a set of loci was successfully identified. Integration of the pathways is underway, while a joint effort with the Deconstruction Division to generate the IL treated material that can be used for several such proof of concept evaluations was suc-cessfully completed. In collaboration with the gene synthesis team at JGI, we also completed the project to generate a synthetic heterologous gene expression system that uses a series of chimeric transcription factors and inducible, ti-tratable promoters. This manuscript is in preparation. Other completed studies in S. cerevisiae, include the characteri-zation of an evolved yeast strain that can utilize xylose as a sole C source. This study focuses on a novel mutation in the HXT7 transporter in yeast, and in now under review for publication.

3.2.5 Quantitative Metabolic Modeling


Host engineering through flux analysis. We used the Two-Scale 13C Metabolic Flux Analysis (2S-13C MFA, in press) method to measure fluxes for genome-scale models using 13C labeling data, and used this information to increase bio-fuel yields. In particular, we used the flux profiles for a fatty acid producing S. cerevisiae strain bioengineered to ex-press the ATP-dependent citrate lyase (ACL), to infer that the malate synthase (MLS1) reaction was a significant drain of acetyl-coA. This MLS1 activity was unexpected but easily unearthed by the flux analysis. We downregulated the MLS reaction and obtained an increase of 30% in production of fatty acids (Fig. 3). Furthermore, knocking out glycerol production improves fatty acid production by 65% (manuscript in preparation). In parallel, we used Flux Balance Analysis (FBA) and Minimization of Metabolic Adjustment (MoMA) to suggest possible knockout (KO) candidates to increase methyl ketone production in E. coli. One of those suggested KOs involved RPE, a reac-tion converting ribulose 5-phosphate into xylulose 5-phosphate, and not directly involved in fatty acid me-tabolism. Surprisingly, a KO of this reaction improved fatty acid production by 40%, through an unknown mechanism. We are now measuring fluxes through 2S-13C MFA in order to ascertain the mechanism. Finally, in collaboration with EMLS we are producing a unique da-ta set involving the labeling of more than 40 metabolites to constrain fluxes for a genome-scale model of E. coli in what will be the flux measurement backed up by the largest amount of metabolite data so far. Transcriptomics and proteomics data have also been collected to provide independent lines of inference and produce a compre-hensive understanding of metabolism. Data visualization. We are improving the Multiomics Visualization Tool (MvT) to allow for interactive engi-neering. The user can now pick reactions interactively and we are now adding the functionality to be able to KO genes or over/underexpress them, and interactively see the results. We have also explored the possibility of commercializing this tool as a spinoff of JBEI through interview-ing >70 potential users through the I-corps program.


Produced FAEE, alkenes, methyl ketones, isopentanol, limonene, α-pinene, α-bisabolene.

Pathway optimization. Enzymes for highly reduced biofuels. Capture of gas-phase olefins.

Multi-omics facilitated metabolic engineering in isopre-noid pathway.

Expand approach for pathways beyond isoprenoids

Improved methyl ketone-producing, E. coli strain to produce 3.4 g MK/L in 45 hr (fed-batch, glucose) and attain 40% of max. theoretical yield

Further improve performance and scale up production.

Discovery of enzymes that convert xylose to DXP Creation of high-flux xylose-to-DXP pathways. DIVA adopted by the National University of Singapore and the JGI; 1,642 users of j5 at 435 institutions world-wide

Replace outmoded Flex tools including VectorEditor and De-vice Editor with modern javascript counterparts. Implement auto-annotation of DNA sequences in ICE.

Selection developed to detect improvements in isopre-noid production by S. cerevisiae

Further demonstration of this selection for improvement of bisabolene titers in engineered yeast.

Identified a set of genes (e.g. the MdlB pump) that con-fer both tolerance and improved production for the bi-ogasoline candidate Isopentenol

Understand the mechanism via which the improvement in production was obtained.

Confirmed our hypothesis that deletion of certain membrane proteins may impact our capacity to express other membrane proteins of interest

Obtain a comprehensive understanding of this phenomenon and use it for strain engineering.

Used flux analysis (both with 13C labeling data and without it) to increase biofuel production.

Find and test systematic ways to improve yield to a desired percentage of the theoretical maximum.

Figure 3. Fatty acid production for S. cerevisiae. Addition of ATP-dependent citrate lyase (ACL) does not produce an immediate increase in production unless coupled with a downregulation of MLS1 (malate synthase), as un-earthed by 2S-13C MFA. Further increases are obtained by eliminating glycerol production.


K E Y A C C O M P L I S H M E N T S F U R T H E R C H A L L E N G E S

AddedinteractivecapabilitiestotheMultiomicsVisualiza-tionTool(MvT)

Integrate predictive algorithms for reaction KO and downreg-ulation.

EngineeredE.colitoproduceanteiso-branchedfattyacidsandfuelsderivedfromthem.

Improve pathway flux to increase titers and yields of anteiso-branched fatty acid-derived fuels.

3.4 Major Research & Personnel Changes In Synthetic Biology Informatics, software developers Joanna Chen and Manjiri Tapaswi were replaced by Oge Nnadi, Jacob Coble, and Sarah LaFrance.

3.5 Collaborative Research & Industrial Interactions I N S T I T U T I O N C O L L A B O R A T O R S T O P I C S

JGI Deutsch, Simirenko, Oberortner, Tringe

de novo DNA synthesis, DIVA and ICE software platforms, MiSeq sequence validation pipeline, metagenome sequencing

Penn State University Costas Maranas Full E. coli metabolism and atom transition model GLBRC Pfleger Membrane protein economics in E. coli Sandia CRF John Dec Identification and testing of key fuel targets ALS, LBNL Craig Taatjes Combustion chemistry of isopentanol and bisabolane ABENGOA Juan Luis Ramos Funded project to used the new 2S-13C MFA method to improve

production on methyl ketones on a JBEI strain Total Gas and Electric Florence Mingardon Directed evolution of efflux pumps to increase solvent tolerance

and production (Manuscript submitted) EMSL (PNNL) Errol Robinson 2S-13C MFA to improve omics-based flux prediction Amyris Inc. Newman, Zhao Expression of DXP pathway in yeast U. Florida, Arborgen, NREL

Peter, Rothman, Davis, Sykes ARPA-E PETRO: Engineering of terpene biofuels in pine

CRAG (Spain) Rodriguez-Concepcion Complementation of dxs knockout in E. coli ribB mutant

3.6 Impacts of Research Two startups were founded based on technology developed in the Fuel Synthesis Division. TeselaGen Biotechnolo-gy, Inc., a JBEI startup commercializing j5 technology, has seven full time employees in San Francisco, CA and has three established customers - AMGEN, Genomatica, and Dow Agro Sciences. Lygos, another JBEI startup commer-cializing the polyketide technology, has 10 full time employees in Emeryville, CA and is funded through several DOE SBIRs. The Fuel Synthesis Division engineered E. coli and S. cerevisiae to produce several advanced fuels. Over the past year, the titers, rates, and yields of several representative fuels from cellulosic sugars were improved dra-matically. When these fuels are commercialized, they will reduce substantially the amount of green house gas added to the atmosphere without needing a change in engines. The Fuels Synthesis Division also developed synthetic biol-ogy software tools, most recently including DIVA, which has been adopted by the National University of Singapore.

3.7 Linkages to Future Plans We will optimize biofuel pathways for isopentenol and methyl ketones that were developed in Years 1-5, for produc-tion at pilot scale; lay the groundwork to develop polyketide synthases (PKSs) for 1-pentene biosynthesis and phe-nylacetate decarboxylase for toluene biosynthesis; and continue to elucidate novel genes/enzymes involved in pro-duction of ladderanes (as a potential high energy fuel). We will use ‘omics tools and correlation analysis and other mathematical tools to identify pathway imbalances and prioritize engineering targets. This approach can also be easily paired with techniques for high throughput strain construction, and it will allow us to analyze complex meta-


bolic pathways with higher accuracy. Additionally, we will work to improve terpene synthase activity in microbial hosts. We will further demonstrate the capabilities of 2S-13C MFA to rationally direct host engineering in E. coli and S. cerevisiae for different biofuels. We will further develop the MvT tools, in collaboration with the Technology Division, to make 2S-13C MFA a routinely used method. We will use more sophisticated machine learning tools than PCA for the high-throughput omics data. We will replace outmoded Flex (Adobe Flash) software tools including DeviceEdi-tor and VectorEditor with modern javascript counterparts, and further develop the ICE platform to auto-annotate DNA sequences. We will develop hosts that are more tolerant to biomass inhibitors and fuels, and will screen hy-drolysates generated by the Deconstruction Division from engineered and wild type plants produced by the Feed-stocks Division, and that are able to metabolize the variety of sugars that result from biomass deconstruction while minimizing byproduct generation. We will investigate the tolerance and transport system found to improve isopen-tenol production and limonene tolerance, in greater detail. We will develop the project to examine the limits of transport protein expression and develop systems that allow us to examine the concepts of membrane capacity and limits.

4. TECHNOLOGIES DIVISION 4.1 Introduction The mission of the Technology Division is to provide robust analytical tools tailored to biofuels research needs to hasten the development of effective biofuel-production technologies. This is accomplished by implementing high-throughput off-the-shelf systems when possible (e.g., jSALSA); automating, parallelizing, and miniaturizing proce-dures that are currently throughput-limiting; and developing new analytical technologies for enzyme activities, bio-mass, protein, metabolite and cell characterization. The field of biofuels research presents some unique challenges, combining analysis and understanding of highly complex plant materials, validation of a spectrum of pretreatment and sugar-production methods, and the engineering of microbes for fuel production. Imaging and spectroscopic methods are required to quantify changes in plant cell walls in response to mutations/engineering, and pretreatment proto-cols. High-throughput analytical tools are required to test large-parameter spaces for biomass pretreatment and subse-quent enzymatic liberation of sugars including characteriza-tion of heterogeneous biomass substrates. Finally, new tools for design, build and test are required to speed up the process of microbial engineering.

4.2 Scientific Progress 4.2.1 Microfluidics Synthetic biology experiments require optimization of path-ways consisting of many genes and other genetic elements. Given the large number of alternatives available for each ele-ment, optimization of a pathway can require large number of experiments consuming prohibitively expensive amounts of DNA and enzymes. We developed innovative microfluidic platforms [68, 69] for assembling DNA fragments with 10-100-fold lower volumes (compared to microtiter plates) and with integrated region-specific temperature control and on-chip transformation (Fig. 4). Integration of these steps minimizes the loss of reagents and products compared to conventional methods, which require multiple pipetting steps. For assem-bling DNA fragments, we implemented three commonly used DNA assembly protocols in our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plas-mids each. Each DNA plasmid is transformed into E. coli or Saccharomyces cerevisiae using on-chip electroporation and fur-ther sequenced to verify the assembly. We anticipate that the microfluidic platforms being developed at JBEI will integrate DNA assembly, transformation, culturing, and phenotypic screening and enable synthetic biology experi-ments to be performed 10x faster, using 10-100x less reagents, and with significantly greater reproducibility.

Figure 4. Microfluidic chip for DNA assembly, trans-formation using electroporation, and culturing. Left panels show the chip images and the right panels show cartoons for various molecular biology steps being carried out in various parts of the chip.


4.2.2 Array Based Assays Cost effective hydrolysis of biomass into sugars for biofuel production requires high-performance low-cost glycoside hydrolase (GH) cocktails that are active under demanding process conditions. Improving the performance of GH cocktails requires knowledge of many critical parameters including individual enzyme stabilities, optimal reaction conditions, kinetics and specificity of reaction[70]. With this information, rate- and/or yield-limiting reactions that can be potentially improved through substitution, synergistic complementation, or protein engineering. Given the wide range of substrates and methods used for GH characterization, it is difficult to compare results across a myriad of approaches to identify high performance and synergistic combinations of enzymes. This year, in close collabora-tion with GLBRC we further developed our platform for systematic screening of GH activities using automatic bio-mass handling, bioconjugate chemistry, robotic liquid handling, and nanostructure-initiator mass spectrometry (NIMS) and used this to characterize GH enzymes across a range of process relevant conditions [40]. To enable cross experiment comparisons of enzyme activities we standardized methods and defined twelve well-characterized sub-strates spanning the types of glycosidic linkages found in plant cell walls are included in the experimental workflow. This year we also used our NIMS platform to study the time-dependent reactions of GH enzymes for modeling by GLBRC to define a quantitative basis to make functional distinctions among a continuum of naturally evolved reac-tive properties. Related studies investigated the use of Carbohydrate binding modules (CBMs) fusion enzymes to modulate GH performance. Here, again in collaboration with GLBRC, we performed a combinatorial evaluation of the ability of representatives from each of the major CBM families found in C. thermocellum to modulate enzyme function of a single multifunctional enzyme, CelE from C. thermocellum using our NIMS assays. Results show fusion of different CBMs to CelE gave differential enhancement of both rate and yield in hydrolysis of purified polysaccha-ride substrates and biomass. This improvement is correlated with broad specificity and moderate affinity of CBM binding. Numerical analysis of reaction time courses showed that CelE_CBM44 had the fastest rate of hydrolysis in both the hexose and pentose fractions of ionic-liquid pretreated switchgrass, a model bioenergy substrate. Finally, in collaboration with the Fuels Division at JBEI we applied our NIMS technique for characterization of methyl ketone fuel molecules and used this method to screen composition from 3000 E. coli mutants grown in multiwell plates. This approach was able to rapidly determine methyl ketone composition.

4.2.3 Proteomic Analysis Over the past 10 years, the bioenergy field has realized significant achievements that have encouraged many follow on efforts centered on biosynthetic production of fuel-like compounds. Lagging far behind these advancements are analytical methods to characterize and quantify systems of interest to the bioenergy field. In particular, the utiliza-tion of proteomics, while valuable for identifying novel enzymes and diagnosing problems associated with biofuel-producing microbes, is limited by a lack of robustness and limited throughput. The recent adoption of standard flow chromatography (ca. 0.5 mL/min) for targeted proteomics has highlighted the robust nature and increased through-put of this approach for sample analysis. Consequently, we assessed the applicability of standard flow liquid chro-matography for shotgun proteomics using samples from Escherichia coli and Arabidopsis thaliana, organisms common-ly used as model systems for lignocellulosic biofuels research [71]. Employing 120 min gradients with standard flow chromatography, we were able to routinely identify nearly 800 proteins from E. coli samples; while for samples from Arabidopsis, over 1,000 proteins could be reliably identified. An examination of identified peptides indicated that the method was suitable for reproducible applications in shotgun proteomics. Standard flow liquid chromatography for shotgun proteomics provides a robust approach for the analysis of samples for biofuels research. We applied this method to analyze the photoactive Orange Carotenoid Protein (OCP) by X-ray radiolytic labeling and proteomic analysis (X-ray footprinting, XF) to characterize the photoprotective response and study the global tertiary structural changes that occur upon photoactivation [72]. These methods have been adapted to study membrane protein compo-sition with the Host Engineering Directorate. Quantitative comparisons of engineered membrane proteomes to help understand the impact of biofuel toxicity are underway. With the Fuels Synthesis team, we applied targeted proteomics to strains of E. coli that were engineered for the high-yield production C5 alcohols that serve as potential biofuels [62]. Metabolite profiling identified NudB, a promiscu-ous phosphatase, as a likely pathway bottleneck in C5 production. And, by engineering the Shine-Dalgarno sequence of nudB, we increased protein levels by 9-fold, reduced isopentenyl diphosphate (IPP) accumulation by 4-fold, and achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol. Further pathway optimization by tuning mevalonate kinase (MK) production, investigating enzymes from alternative microbes such as Methanosarcina mazei, and expres-sion of a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) resulted in a strain that achieved >2 g/L of C5 alcohols (~70% of pathway-dependent theoretical yield). We also applied targeted proteomics to characterize the mechanism of gem-dimethyl group formation by polykeytide synthase (PKS) modules, with a goal toward engi-neering of novel compounds containing this moiety [73]. The work demonstrated, contrary to the canonical under-standing of reaction order in PKS, that methylation can precede condensation in gem- dimethyl group producing PKS modules. This work is being extended to studies of PKS expressed in S. cerevisiae. With the Feedstocks Division, targeted proteomic methods were applied to help characterize sulfated peptides associated with the rice immune response.


4.2.4 Physical Analysis The Physical Analysis group characterizes plant biomass and microbial samples provided by the Feedstocks, Decon-struction and Fuel Synthesis groups. We developed novel spectroscopic methods to characterize plant cell walls at the microscale [74]. We have now begun to ultrastructurally characterize the 3D organization of plant chloroplasts, which are key to light harvesting thus providing the energy for optimal plant biomass production. We anticipate to use cutting edge cryo-EM tomography as well as novel advanced 3D SEM imaging approaches (FIBSEM) to study the chloroplast ultrastructure and 3D organization at the nano- and meso-scale. In the last year, the focus has been on the 3D imaging of plant cell walls, including antibody affinity tag-labeling of cell wall carbohydrates and proteins. We developed the first computer-assisted design (CAD) model of an Arabidopsis plant cell wall based on 3D cryo-EM tomographic data sets we obtained from vitrified plant sections. We developed computational approaches for mechanical simulation and are now in the position of comparing the mechanical properties (stress/strain profiles) of the determined 3D model with alternative models. We anticipate that this approach, when being applied to genet-ically altered feedstocks will predict their mechanical stability. In order to apply this approach to entire plants, we need to further integrate bioimaging approaches at the nano- and meso-scale, employing multiple imaging modali-ties hat span multiple scales and levels of resolution and integrate this information into a multiscale model. We have begun to integrate fluorescence and other optical microscopy approaches with novel large volume 3D electron mi-croscopy imaging approaches, and found hard X-ray microscopy of heavy-metal stained and resin-embedded sam-ples to be an ideal high-throughput imaging technique that is able to bridge the mesoscale. Its full integration into a sample preparation and imaging workflow promises to solve the challenge to image macromolecular resolution de-tails in the respective cell and tissue context. This will ultimately require the development of a map-based database ideally at the model space that can be queried for quantitative volumetric information.

4.2.5 Structural Biology We continue to pursue crystallographic studies of en-zymes across all divisions within JBEI. Lignin is a combi-natorial polymer comprised of monoaromatic units that are linked together via strong chemical bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to depolymerization presents major ob-stacles to both the production of second-generation biofu-els and the generation of valuable coproducts. Biological degradation of lignin has been relatively well character-ized in fungi, but is less well understood in bacteria. A set of enzymes that catalyze the cleavage of β-aryl ether units typically found in lignin has been reported in the bacte-rium Sphingobium sp. SYK-6. In collaboration with the De-construction Division and GLBRC we solved X-ray crystal structures and biochemically characterized these en-zymes; NAD+-dependent Cα-dehydrogenases LigD, LigO, and LigL; glutathione-dependent β-etherases LigE (Fig. 5) and LigF; and glutathione-dependent lyase LigG. Our re-sults include analysis of cofactor binding sites, substrate binding sites, and catalytic mechanisms. As β-aryl ether bonds account for 50–70% of all inter-unit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has great potential for informing ongoing studies on the valorization of compounds derived from lignin. This work has been submitted for publication. In collaboration with the Feedstocks Division, we continue to pursue a focused study of glycosyl transferases (GTs). There is currently very limited structural information available for any plant GTs involved in plant cell wall biosynthesis, which limits the assignment of function based on sequence. Our goal is to obtain structural information, married to biochemical studies, across the cell wall-synthesizing GT popula-tion to be able to use sequence-analysis methods to predict function. Protein expression of these enzymes has proven challenging because of aggregation and insolubility. In addition to testing fungal and insect cell expression systems we developed a high throughput E. coli expression system to perform large scale tests of vectors, target genes and strains. Also in collaboration with the Feedstocks Division we structurally characterized a protein involved in plant immune response (OsSERK2). This work was recently published [25] and helps lay the groundwork for future stud-ies into plant sustainability. In all cases, crystallographic data collection has leveraged the resources and expertise of the Berkeley Center for Structural Biology. Structure solution and analysis make use of the highly automated PHE-NIX software developed by researchers at LBNL and elsewhere.

4.2.6 Synthetic Biology Informatics The Experiment Data Depot (EDD) has been extended to support enzyme engineering and genetic regulatory com-ponent (e.g., promoter) characterization data. The enzyme characterization data effort has been pursued in coordina-tion with the Deconstruction Division’s Enzyme Optimization group, which conducts multivariate enzyme assays

Figure 5. Cartoon representation of the dimer of LigE, including the N-terminal thioredoxin domain (red), the C-terminal α-helical domain (brown) and the short linker (grey). Bound GSH is shown as yellow spheres.


spanning enzyme variants, domain swaps, and point mutations, at various pH, salt, temperature, buffer, solvent, co-factor, etc. conditions. The goal is to enable researchers to query the EDD for enzymes that meet particular functional specifications (e.g., a hydrolytic enzyme capable of breaking a particular glyco-linkage at a certain temperature and salt concentration above a certain rate after exposure to a given solvent for a particular amount of time). The capacity to do this is an important step towards automating the design of anabolic or catabolic pathways that perform under particular process-relevant conditions. The genetic regulatory component characterization data effort has been pur-sued in coordination with the Fuel Synthesis Division, which conducts fluorescence cytometry assays of promoter variants in various chromosomal (or plasmid) loci, inducer level, strain background, etc. conditions. The goal is to enable researchers to query the EDD for promoters that meet particular functional specifications (e.g., expresses in E. coli DH1 at a very low level in exponential growth phase but at a high level approaching stationary phase). The ca-pacity to do this is an important step towards automating the design of genetic control systems that optimize the ex-pression of metabolic pathways under particular process-relevant conditions.


Developed droplet microfluidic platform for carrying out highly parallel reactions. Invented a patent-pending method for mixing and sorting droplets. Demonstrated DNA assembly, cell transformation, cell culture and phenotypic screening of cells in microfluidic chips.

Automation and end-to-end integration of unit operations such as DNA assembly, cell culture, mass spectrometry-based assays to enable applications such as enzyme evolution and metabolic path assembly.

Automated oxime chemistry and expansion of this effort in collaboration with other BRCs to a diverse panel of representative substrates and enzymes.

Miniaturization of liquid handling using microfluidics to in-crease the number of reactions conditions that can be charac-terized using the small amount of enzyme produced using in vitro expression.

Developed oxime and related methods to support screening of fatty acid derived biofuels including methyl ketones.

Differences in methyl ketones produced depending on growth conditions (multiwell plate vs. shake flask).

Structure determination and CAD model generation of Arabidopsis plant cell wall using cryo-electron tomog-raphy of cryo-sectioned biomass.

Simulation of mechanical cell wall properties and expansion of this approach to feedstock mutants.

Structural and biochemical characterization of the β-aryl etherase pathway from bacterium Sphingobium sp. SYK-6.

Modification of enzymatic activities and specificities in the pathway to create specific lignin-derive products.

Developed robust, reproducible proteomic workflows to quantify native proteins of engineered hosts.

Expand proteomics workflows for additional metabolic engi-neering hosts and enable data visualization.

Further developed the Experiment Data Depot to sup-port enzyme engineering and genetic regulatory com-ponent characterization data.

Extension of the EDD to support proteomics and metabolom-ics workflows.

4.4 Major Research & Personnel Changes In Microfluidic Assays a postdoctoral associate, Dr. Peter Kim, was hired. Another postdoctoral associate, Dr. Todd Duncombe was hired jointly by Microfluidics/Microarray groups. In Proteomics, Melissa Nhan left for graduate school and Dr. Yan Chen was hired to develop proteomics methods. In Structural Biology Thomas Ellinghaus, a Re-search Assistant, was hired to pursue structural studies of thermotolerance in cellulases. In Synthetic Biology Infor-matics, software developer Nat Echols was replaced by Jason Eads; software developer Mark Forrer was hired to support the development of EDD.

4.5 Collaborative Research & Industrial Interactions I N S T I T U T I O N C O L L A B O R A T I O N S T O P I C S

GLBRC Brian Fox Enzyme libraries from JGI gene synthesis and GLBRC in vitro ex-pression. We are also collaborating on characterization of lignin degrading enzymes using biochemical and spectroscopic tech-niques.


I N S T I T U T I O N C O L L A B O R A T I O N S T O P I C S

GLBRC George Phillips, Brian Fox & John Ralph

We continue to collaborate with GLBRC researchers and the JBEI Deconstruction Division to determine the structures and activities of enzymes in the beta-aryl ether lignin degradation pathway.

Autodesk Andrew Hessel Automation of synthetic biology design-build-test cycle using microfluidic chips

Agilent Miniaturized screening platforms for molecular biology

Riffyn Tim Gardner Standardization of experiment data transfer from analytical in-strumentation.

Agilent Inc. Steve Fischer, Chris Miller Integrated Biology: New software tools for enhanced analysis of biofuel processes

Michigan State University-LBNL

Corie Ralston, Cheryl Kerfeld Adapting proteomic methods for X-ray radiolytic labeling (X-ray footprinting) experiments.

4.6 Impacts of Research The work of the microfluidic assays and platforms have made a significant impact in improving the throughput of assays required to screen enzymes and glycans. We also developed integrated microfluidic platforms for automating the process of metabolic engineering that show great potential for accelerating the design-build-test cycle and have attracted significant attention from industrial partners as well as the academic community (C&EN; http://cen.acs.org/articles/93/web/2015/06/Microfluidic-Device-Mixes-Matches-DNA.html). The unique combi-nation of sensitivity, specificity and high-throughput of the NIMS approach was essential in characterizing a library of GH1 enzymes [75]. Both the microfluidic assays and NIMS technology have been critical in collaborations with GLBRC, to characterize lignocellulolytic enzymes [40] [76]. The robust methods developed by the Proteomics group are being used for host engineering work conducted by the Fuels Synthesis Division and for X-ray footprinting ex-periments in collaborations. Short chromatography methods for targeted proteomic assays have enabled large-scale studies involving many strains, conditions, and replicates across all divisions and with collaborators.

4.7 Linkages to Future Plans Microfluidic technology development will focus on further developing the droplet microfluidic chips for two appli-cations: (1) to perform highly-parallel DNA assembly for optimization of metabolic pathways to produce fuels and other chemicals and (2) to screen mutant libraries of biomass degrading enzymes. We will also integrate microfluidic chips to NIMS chips towards creating a completely automated pipeline for building and screening metabolic path-ways. In addition, this approach can be used to increase the number of conditions for characterization of enzyme activities using oxime chemistry and standardized panel of substrates that span key glycosidic bonds to support the development of cocktails in collaboration with JGI and other BRCs. The Proteomics group will continue to collabo-rate with all JBEI Divisions to generate methods for protein quantification. Efforts will focus on developing proteo-mic workflows that can be applied to a wide variety of metabolic engineering hosts. In Physical Analysis we will continue to image plant biomass, microbes for fuel synthesis and microbial communities deconstructing biomass. We will focus on determining the 3D organization of plant cell walls, particularly for genetically engineered feedstocks, and develop integrative approaches for multiscale and mesoscale analysis. In Physical Analysis, we will continue to image plant biomass, microbes for fuel synthesis and microbial communities deconstructing biomass. We will con-tinue to develop integrative approaches for multiscale and mesoscale analysis of genetically engineered feedstocks with a focus on cell walls and ultrastructurally analyze fuel-producing microbes. In Structural Biology, we will con-tinue to collaborate with all JBEI Divisions to generate high-resolution structural information for fundamental un-derstanding of protein function and subsequent engineering. One focus is expression of multiple plant glycosyl transferases with the Feedstocks Division for functional characterization and crystallization trials. Another high pri-ority project is the use of cryo-electron microscopy to study the structures of large molecular complexes involved in fatty acid synthesis. In Synthetic Biology Informatics, we will extend the Experiment Data Depot to support prote-omics and metabolomics workflows, as well as productionize a grassroots JBEI software tool into a web application.


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YEAR 8 ANNUAL REPORT - JBEI · JBEI recorded 28 records of invention (ROI) disclosures, filed 26 patent applications, had 3 patents issued, executed 11 licensing agreements, and initiated